Using tryptophan mutants to probe the structural and functional status

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Using tryptophan mutants to probe the structural and functional status of BsSCO, a copper binding, cytochrome c oxidase assembly protein from Bacillus subtilis Shina Hussain, Diann Andrews, and Bruce Charles Hill Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00833 • Publication Date (Web): 13 Nov 2017 Downloaded from http://pubs.acs.org on November 14, 2017

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Using tryptophan mutants to probe the structural and functional status of BsSCO, a

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copper binding, cytochrome c oxidase assembly protein from Bacillus subtilis

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Shina Hussain1, Diann Andrews1 and Bruce C. Hill*1,2

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1

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Queen’s University, Kingston, ON K7L3N6

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(Email: [email protected])

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Abstract:

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The Synthesis of Cytochrome Oxidase protein from Bacillus subtilis (i.e., BsSCO) binds copper

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with pM affinity, which increases the protein’s melting temperature (i.e., TM) by 20 oC. Here two

Department of Biomedical and Molecular Sciences and 2Protein Function Discovery Group

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native tryptophans (i.e., W36 and W101) are identified as major contributors to BsSCO’s

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structural form and their contributions to stability, intrinsic fluorescence and copper binding

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properties of BsSCO are explored. Single mutations of tryptophan to phenylalanine decrease TM

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by 10 oC and folding free energy by 3-4 kcal/mol. A more severe change to alanine (i.e., W36A

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BsSCO) decreases TM by 20 oC, and stability by 9 kcal/mol. However, these mutants bind copper

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with high affinity and assemble cytochrome c oxidase in vivo. Replacing phenylalanine at a

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position near (~ 5Å) the copper binding site with tryptophan (i.e., F42W) increases the TM of

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apo-BsSCO by 3 oC, but diminishes the effect of copper binding. When both native tryptophans

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are changed to alanine apo-BsSCO is unfolded in vitro, and is not functional in cytochrome c

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oxidase assembly in vivo. A double mutant of BsSCO in which W36A is combined with F42W

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exhibits a form of metastability. Apo-W36A/F42W BsSCO melts at 37 oC, which upon binding

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copper shifts to 65 oC. B. subtilis expressing W36A/F42W BsSCO and grown at 37 oC does not

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assemble cytochrome c oxidase. However, when these cells are shifted to 25 oC cytochrome c

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oxidase activity is recovered. Our results illustrate the subtle relationship between structural

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stability and functional properties of BsSCO in the assembly of cytochrome c oxidase.

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Introduction Cytochrome c oxidase is an integral membrane enzyme that catalyzes electron transfer

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from reduced cytochrome c to oxygen and conserves some of the free energy of this redox

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process via transmembrane charge separation.1 It has been long recognized that the assembly of a

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molecular machine such as cytochrome c oxidase is a complex process in its own right.2 Protein

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subunits and prosthetic groups must be brought together in the proper order, at the correct place

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and time. Two heme and two copper centers within cytochrome c oxidase are responsible for

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catalyzing oxidation of cytochrome c and reduction of oxygen to water. The redox active centers

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are contained in subunits I and II, two large integral membrane subunits that are conserved in

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cytochrome c oxidases from humans to bacteria. Subunit I binds the two hemes (i.e., two A-type

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hemes in mitochondrial and Bacillus oxidases) at protein sites within the membrane-embedded

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portion of subunit I. The two A-type hemes, known as cytochrome a and cytochrome a3, can be

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distinguished by their spectral and reactivity properties. Subunit II consists of a soluble domain,

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which houses one of the copper centers (i.e., CuA), and is linked to two transmembrane α-helical

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segments.1 CuA consists of two copper ions ligated by two histidines, one methionine, a

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backbone carbonyl oxygen and two cysteine residues that bridge between the two coppers.3 The

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bridging cysteines facilitate a highly cooperative structure that acts in a concerted manner to

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shuttle electrons one at a time from cytochrome c to cytochrome a.4, 5 Electrons are passed from

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cytochrome a to the site of oxygen reduction; cytochrome a3 and the second copper center, CuB.6

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Cytochrome a3 and CuB form a binuclear center that acts cooperatively to reduce molecular

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oxygen to water without release of any partially reduced reactive oxygen species. The overall

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redox process culminating in the reduction of oxygen to water is exergonic. Some of the

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available free energy is captured by coupling the redox reaction to the transport of protons across


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the membrane such that the charge transfer of the redox reaction, proton consumption and the

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proton transfer are complimentary in generating an electrochemical gradient.7

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A number of accessory proteins are recognized to aid in assembling the protein subunits

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and prosthetic groups into a functional cytochrome c oxidase unit. The Synthesis of Cytochrome

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c Oxidase, or SCO, protein is one such accessory protein that was originally discovered in yeast

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as being required for cytochrome c oxidase assembly.8 Subsequent work with yeast showed that

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the SCO protein is required for incorporation of copper into the oxidase complex.9 In eukaryotic

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organisms there are two SCO orthologs (i.e., SCO1 and SCO2) that may function at different

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stages of CuA assembly.10 We identified a Bacillus subtilis version of SCO1 (i.e., BsSCO) and

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showed that it is specific for CuA assembly, and not required for CuB assembly.11 Another

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assembly protein, Cox11, has been proposed to be required specifically for CuB assembly.12

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All members of the SCO family of proteins possess a pair of cysteine residues (i.e., -

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CXXXC-) and a histidine about 100 amino acids to the C-terminus. These three residues form

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the conserved CCH-motif and are involved directly in binding copper as inner sphere ligands. In

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addition the cysteines are close enough that formation of the disulfide is energetically facile, but

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is kinetically slow to occur in the native structure.13 The exact role of SCO in the assembly of

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CuA is not known. High-resolution structures of bacterial and mitochondrial SCO proteins14-16

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agree with bioinformatic analysis that places the protein in the thioredoxin super-family and led

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to the proposal of a redox role for SCO.17 SCO could act as a thiol-disulfide exchange protein

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specifically targeted to cytochrome c oxidase subunit II to maintain the CuA-associated cysteine

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residues reduced.18 Copper binding studies show that SCO proteins form 1:1 complexes with

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both Cu(I) and Cu(II) ions and a role for SCO in copper delivery to the CuA site has been

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supported.10, 19 Building on the copper transfer model of SCO functionality, we have observed


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for BsSCO and for SCO1 from yeast a strong preferential affinity for Cu(II) over Cu(I).20, 21 For

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BsSCO we have reported a Cu(II) binding constant of 3.5 pM, and for Cu(I) 10 µM. This

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extreme preference for Cu(II) over Cu(I) defines a redox-driven affinity switch for BsSCO, a

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distinctly different hypothesis than the entatic state model that explains the facile electron

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transfer functionality observed for cupredoxin proteins such as azurin.22 Much of the

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thermodynamic stability of the BsSCO-Cu(II) complex is conferred by a two-step binding

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mechanism, whereas kinetic stability is established by the slow dissociation of Cu(II) from

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BsSCO once the complex is formed.23 We have proposed that these elements could be useful in a

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copper delivery model in which high-affinity Cu(II) binding serves to capture copper in a

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competitive environment, and reduction of Cu(II) to Cu(I) promotes a dramatic increase in the

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dissociation rate of copper from BsSCO, or alternatively by a conformational change in BsSCO

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triggered by docking to its target protein (i.e., apo-CuA).

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In the work here our aim is to understand further the linkage between BsSCO’s structure,

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spectroscopic properties and the protein/Cu-ligand interaction. The two native tryptophan

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residues of BsSCO are of particular interest because of their spectroscopic and energetic

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contributions to biophysical attributes of BsSCO. In thioredoxin family proteins tryptophan

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fluorescence is a sensitive reporter of the status of the redox active thiol pair24, and tryptophan

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fluorescence has been used to assess copper binding by SCO proteins.20 In BsSCO the two

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native tryptophans are a substantial distance (i.e., 15 and 22 Å) from the metal binding site

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defined by the conserved thiol pair. Our initial motivation was to see if we could improve the

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responsivity of the tryptophan signal within BsSCO to the thiol/disulfide state, or to copper

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binding by moving the trypophans closer to the site of action. We find here that the two

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tryptophans of BsSCO are computed to have a marked contribution to the stability of the overall


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protein fold. We have, therefore, made a series of tryptophan mutations to explore the structural

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stability of the protein matrix of BsSCO. We have changed each of the two native tryptophans to

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phenylalanine or alanine, singly or in pairs. We have converted a phenylalanine in native BsSCO

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to tryptophan at a site closer to the reactive thiol-pair in BsSCO (i.e., < 5 Å) and at a position that

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corresponds to one of the ‘reporter’ tryptophan residues in thioredoxin. We have assessed these

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mutants for spectroscopic changes by circular dichroism and intrinsic fluorescence, stability

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changes by thermal-induced melting, and isothermal, urea-induced unfolding and functional

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status by their ability to assemble active cytochrome c oxidase in vivo. Wild-type BsSCO binds

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copper with pM affinity in a binding reaction that occurs without any discernible change in the

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overall structure of the protein, even though the resistance to thermal melting increases by close

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to 20 oC. Mutation of either tryptophan to phenylalanine decreases the thermal melting by 10 oC,

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and yet copper binding still occurs with high affinity and induces a 20 oC increase in melting

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temperature. Double mutants of BsSCO in which both tryptophans are changed to phenylalanine

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or alanine are unfolded under native conditions and copper binding does not induce overall

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refolding in vitro. However, the double mutants in which tryptophan is replaced with

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phenylalanine are able to function in vivo, whereas in the double alanine replacement activity is

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not recovered in vivo. Our results illustrate the close coupling of BsSCO’s remarkable copper-

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induced, stability shift, and its ability to function in a CuA assembly role. We propose that the

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inherent stability of the apo-BsSCO fold serves to protect the copper-ligating cysteines from the

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side reaction of oxidation to the inactive, disulfide form.

21


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Materials and Methods PROTEIN EXPRESSION AND PURIFICATION. The soluble domain of wild-type and mutant

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versions of BsSCO were expressed in E. coli DH5α and purified as described previously.25

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Briefly, BsSCO is expressed in fusion with glutathione-S-transferase and the hybrid protein is

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captured from the cell extract on a glutathione Sepharose column (Glutathione Sepharose 4B, GE

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Healthcare). The fusion protein is cleaved on the column by treating with thrombin (Sigma, 100

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units per 100 mg of fusion protein) for 16 h. Thrombin is removed from the cleaved BsSCO by

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passage over Benzamidine Sepharose (Benzamidine Sepharose 4 Fast Flow, GE Healthcare).

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The purity of the preparations were assessed by SDS-polyacrylamide gel electrophoresis. The

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yield of purified BsSCO was in the range of 5-10 mg per liter of liquid culture. The redox state of

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the two thiols on BsSCO was assessed using 4,4’-dithiodipyridine.26 Expression levels of native

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and mutant BsSCO in Bacillus were determined by western blots of membrane extracts using

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anti-BsSCO antibodies as described previously.27

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SPECTROSCOPY. Absorbance spectra were obtained on a HP-8453 diode array

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spectrophotometer equipped with a temperature controlled cuvette holder. Fluorescence spectra

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from 300 nm to 450 nm were measured on a Cary Eclipse spectrometer using a 2.5 nm excitation

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bandpass at excitation wavelengths of either 280 nm or 295 nm, and a 2.5 nm emission bandpass.

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Fluorescence intensities were measured by integrating the area under the emission envelope after

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correcting for any background signal from the buffer alone. To compare intensities between

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different samples the observed intensities were corrected for the inner filter effect using the

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absorbance of the sample at the wavelength of excitation and the absorbance at 330 nm.28 CD

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spectra were collected using an Applied Photophysics Chirascan spectrometer with a 1 nm

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bandpass. Far UV-CD spectra were analyzed for secondary structural content using the CDNN


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program with the 23 basis protein spectral set.29 Far UV spectra were collected in a 0.1 mm

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cuvette, whereas a 1 cm cuvette was used for near UV and visible region spectra.

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CD-MONITORED THERMAL-INDUCED DENATURATION. To characterize BsSCO’s thermal

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stability UV-CD spectra were measured as a function of temperature. Spectra were scanned from

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260-190 nm at 2 oC intervals. The temperature was increased at a rate of 1oC per minute and the

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temperature was measured with a probe immersed in the sample. Sample concentrations were in

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the range from 0.15 to 0.2 mg/ml (i.e., 7.5-10 µM) and the cuvette path length was 1 mm.

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Temperature response curves were assembled by averaging the CD response over the entire

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wavelength range. The inflection point (i.e., apparent TM) of the response was determined by

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fitting a sigmoidal function to the data using OriginPro software. ISOTHERMAL UREA DENATURATION. Equilibrium measurements of BsSCO stability were

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done by measuring intrinsic fluorescence as a function of urea concentration. Samples were

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prepared in the range of 0.1 mg/ml by dilution into buffer containing 6.5 mM sodium phosphate

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pH 7.0 at the desired urea concentration. The samples were brought to equilibrium by incubation

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at room temperature for 1 h. Stock urea concentrations were prepared according to the method

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outlined by Shirley.30 Urea denaturation profiles were made by plotting the wavelength of the

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emission maximum as a function of the urea concentration and scaling this between zero and

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one, where zero denotes the value for native and one for fully denatured BsSCO. The

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fluorescence signal over the unfolding transition is fit to the following function, 31, 32

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f = (λN + mN [D]) + (λU + mU [D])exp((∆G-meq[D])/RT) / 1+exp((∆G-meq[D])/RT)

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where λN and λU are the relative wavelength maxima of the native and unfolded states of BsSCO

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respectively, mN and mU are the pre- and post-transition responses of native and unfolded states to


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denaturant concentration, respectively. ∆G is the unfolding free energy and meq is the

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equilibrium m-value. ASSAY FOR SCO FUNCTION IN VIVO. All of the mutants of BsSCO that were purified from

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the E.coli expression system and examined here in vitro were also expressed in native form in

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Bacillus subtilis. Expression of native BsSCO was accomplished by cloning the intact gene for

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BsSCO, ypmQ, and its mutant forms into the plasmid pHP13.33 pHP13 has a low copy number

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in Bacillus and has been used successfully for expression of membrane proteins in Bacillus.34 In

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keeping with the low copy number of pHP13 we observe modest overexpression of BsSCO by

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western blots of membrane extracts.27 To test for function of these proteins the pHP13 constructs

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were transformed into a strain of B.subtilis in which the chromosomal copy of ypmQ is deleted.11

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The cytochrome c oxidase activity assay was performed by a modification of the method

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described by Mueller and Tabor.35 Harvested Bacillus cells that had been grown overnight at 37

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o

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Bacillus suspension (i.e., 10 µL) was applied to filter paper saturated with a 50 mM solution of

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N,N,N’,N’-tetramethyl-p-phenylenediamine (i.e., TMPD). In this assay a blue color is generated

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corresponding to Wurster’s blue radical cation formed from the one-electron oxidation of TMPD

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by cytochrome c oxidase.

C were suspended in a volume of buffer adjusted for the cell wet weight. A small aliquot of the

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FOLDX CALCULATIONS. The FoldX force field was run as a plug-in to the Yasara

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molecular graphics suite.36, 37 The program was used to calculate changes in the equilibrium free

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energies of mutant versions of BsSCO relative to its wild-type structure (i.e., ∆∆G’s).

21 22


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Results

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SPECTRAL AND STABILITY CHARACTERISTICS OF WILD-TYPE BSSCO

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Wild-type BsSCO has intrinsic fluorescence due to the presence of six tyrosine and two

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tryptophan residues in its primary sequence. Reduced, apo-BsSCO’s intrinsic fluorescence

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emission spectrum is dominated by the contribution of the tryptophan residues.28 The two

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tryptophan residues are at positions 36 and 101 in the sequence of soluble, recombinant BsSCO

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and are located approximately 22 Å and 15

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Å, respectively, from the copper binding

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site (see Figure 1).

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The emission spectrum of reduced,

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apo-BsSCO has maximal emission at 329

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nm with a shoulder centered at 308 nm over

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and above the Raman scattering from the

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aqueous solvent (Figure 2a). The shoulder

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at 308 nm disappears when the protein is

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denatured. The emission intensity declines

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by 30% when the reduced, dithiol form of

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BsSCO is oxidized to its disulfide form,

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cystine (see Figure 2 and Table 1). The

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mechanism of cystine quenching has been

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proposed to be due to excited-state electron transfer.38

Figure 1 Structure of BsSCO shown as a ribbon diagram (PDB code 1XZO). The side chains of Trp 36 and Trp 101 are shown in blue and their distances to the putative copper binding site are indicated. This image was generated using PyMOL.


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1.0

(a)

0.2 ∆A

(i)

RFI 0.8

(b)

(ii)

0.6

0.1 0.4 (iii)

0.2

0

0 320 1

400

360

440

Wavelength (nm)

300

400

500

600

Wavelength (nm)

2 3 4 5 6 7 8 9 10

Figure 2. Spectroscopic properties of wild type BsSCO. (a) Relative fluorescence intensity (RFI) spectra of (i) reduced apo-BsSCO, (ii) oxidized BsSCO and (iii) BsSCO-Cu(II). The fluorescence intensities have been corrected for the inner filter effect, normalized for concentration and the value of the peak intensity of reduced, apo-BsSCO assigned a value of 1.0. The concentration of copper in the sample of BsSCO-Cu(II) is 15 µM as CuCl2 and the buffer for each sample is 50 mM sodium phosphate pH 7.0. (b) Absorbance spectra of apo- and Cu(II)-bound BsSCO. The solid line is the spectrum of apo-BsSCO (10.1 µM). The dashed line is the same sample plus 15 µM CuCl2. The concentration obtained for BsSCO-Cu(II) is 10.2 µM using an extinction coefficient of 4.87 mM-1cm-1 at 352 nm.

11

The binding of Cu(II) leads to loss of approximately 70 % of the fluorescence intensity from

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reduced, apo-BsSCO (Figure 2a and Table 1) and we propose that much of this quenching is due

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to energy transfer mediated by the spectral overlap of tryptophan emission with the copper

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absorbance spectrum centered at 352 nm (Figure 2b). Titrations of apo-BsSCO with copper

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reveal such a tight interaction that binding affinity is difficult to assess quantitatively39, but is

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estimated to be in the pM range by competition experiments 21, 40 and by the effect of copper

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binding on thermal denaturation.20

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Table 1. Intrinsic fluorescence properties of BsSCO BsSCO mutants

TRP positions 36 101 42 Wild type + + W36F + W101F + W36A + F42W + + + W36F/F42W + + W101F/F42W + + W36A/F42W + + W36F/W101F/F42W +

7 8 9 10 11 12 13 14 15 16

λmax em (nm)a apo ox Cu2+ 330 331 330 328 329 323 332 331 330 331 332 331 330 330 329 330 330 328 332 330 329 330 332 330 334 334 328

Relative fluorescence intensityb Mut/WT Ox/red Cu/red 1 0.70 0.30 0.75 0.55 0.24 1.35 0.86 0.23 0.53 0.70 0.40 0.76 0.68 0.43 0.34 0.42 0.36 0.75 0.88 0.42 0.41 0.55 0.24 0.33 0.90 0.55

2 3 4 5 a

Emis sion maxima for three different forms of each species of BsSCO are shown. Apo-BsSCO has both cysteine residues reduced and is copper free, ox-BsSCO has both cysteines oxidized and is copper free and Cu2+-BsSCO has both cysteines reduced and is saturated with Cu2+. bRelative fluorescence intensity is shown for three comparisons. For the ratio Mut/WT the intrinsic fluorescence intensity was determined by integration of the emission spectrum after correction for the buffer blank. This value is corrected for the inner filter effect of the absorbance at the excitation wavelength and the absorbance at the emission maximum. The intensity is compared on the basis of the tryptophan concentration. For the ratios of Ox/red and Cu/red these values are comparisons between forms of the same mutant of BsSCO. 6

Circular dichroism in the far ultraviolet region (i.e., UV-CD) is useful for assessing the

17

secondary structural elements that compose a protein’s overall structure and to characterize

18

changes that might occur upon ligand binding41, protein/protein interactions42, or during

19

unfolding43. The UV-CD spectrum of wild-type, reduced apo-BsSCO at 20 oC (see Figure 3a ) is

20

characteristic of folded BsSCO with a minimum at 224 nm, a shoulder at 209 nm and a peak at

21

195 nm, and is consistent with the complement of secondary structural components reported

22

previously for the protein in solution and that are observed in the crystal structure.16


Biochemistry

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20

195 nm

Ellipticity (mdegrees)


(a)

10

0

-10 180

209 nm 224 nm

200

220

240

20 (b)

10 0

0

10

Cu:BsSCO

1.0

260

300

400

500

600

700

Wavelength (nm)

277 nm │ 287 nm │

20

}

0.5

-10

Wavelength (nm) Ellipticity (mdegrees)

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│ │ 263 269 nm

(c)

296 nm │

BsSCO

Cu-apo apo-BsSCO

0 260

280

300

320

Wavelength (nm) 1 2 3 4 5 6 7 8 9 10 11

Figure 3. The effect of copper binding on the circular dichroism of wild-type BsSCO. (a) The effect of copper on the far UV-CD spectrum of BsSCO. The concentration of BsSCO is 67.5 µM in 5 mM sodium phosphate buffer pH 7.0 (apo, ───). Copper was added to a final concentration of 70.0 µM (─ ─ ─). The path length is 0.1 mm and the temperature 25 oC. (b) The effect of copper on the near UV and visible CD of BsSCO. The concentration of apo-BsSCO (───) was 53 µM and the spectra were measured at 25 oC in a 1 cm path length cuvette. Copper was added to give final concentrations of 26 µM (─ ─ ─) and 52 µM (─ • ─ • ─) of CuCl2. (c) Near UV spectra of apo-BsSCO (───) and BsSCO-Cu(II) (─ • ─ • ─). The difference spectrum of the copper complex minus apo is shown on the same scale (- - - -). The experimental conditions are identical to those listed above in (b).

12

The addition of copper to form the high affinity BsSCO-Cu(II) species results in little change in

13

the far UV-CD spectrum (Figure 3a) leading to the conclusion that copper binding to BsSCO


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does not give rise to any large-scale conformational change of the protein. To probe this

2

conclusion further we examined the visible and near UV-CD spectra for apo- and copper-bound

3

BsSCO. The visible CD for a protein such as BsSCO will arise from the copper chromophore44,

4

whereas the near UV-CD (i.e., 260-320 nm) is composed of contributions from the aromatic

5

amino acids; phenylalanine, tyrosine and tryptophan, and is sensitive to tertiary structural

6

changes.45 Figure 3b shows the CD signal from the near-UV and across the visible region for

7

apo- and metallated-BsSCO. As the copper complex is formed a strong signal arises in the visible

8

region with peaks 458 nm and 342 nm, troughs at 570 nm and 386 nm, and zero-crossing points

9

at 527 nm, 419 nm and 365 nm. This signature is characteristic for the BsSCO-Cu(II)

10

chromophore.23 However, in the near UV region from 260-320 nm there is hardly any change

11

coincident with metal binding. A closer inspection of the near UV-region for apo- and copper-

12

bound BsSCO is shown in Figure 3c. The apo-protein exhibits a composite spectrum of positive

13

ellipticity centered at 277 nm that arises from overlapping contributions from the eleven

14

phenylalanines, six tyrosines and two tryptophans of the BsSCO sequence. There are shoulders at

15

287 nm and 296 nm that are consistent with distinct electronic transitions from tryptophan,

16

whereas the additional fine structure at 263 nm and 269 nm arises from electronic transitions of

17

phenylalanine.45 Addition of one equivalent of copper to BsSCO causes loss of the signals at 263

18

nm and 269 nm. The difference spectrum in Figure 3c shows there are no other changes to the

19

near UV-CD spectrum. This result is entirely consistent with the conclusion above that copper

20

binding causes no large scale rearrangement in the protein secondary, or tertiary structure for

21

BsSCO. The change in the near UV-CD at 263 nm and 269 nm is assigned to a change in the

22

local environment of one or more of the phenylalanine residues. The crystal structure of BsSCO


Biochemistry

14 1

shows that one of the set of phenylalanine residues (i.e., F42) is adjacent to the putative copper

2

binding site and is the likely source of the changes observed here. Despite the lack of a copper-induced structural change in BsSCO, copper binding does

3 4

effect drastically the stability of BsSCO. This stability shift is illustrated by changes in the

5

response of the protein’s far UV-CD spectrum to increased temperature (Figure 4). As reduced,

6

apo-BsSCO is heated the protein initially resists unfolding and maintains the spectral properties

7

of the native, folded state (see Figure 4a). Thermal-induced unfolding commences at about 45

8

o

9

melting transition is complete just above 60 oC, and an isodichroic point at 215 nm is maintained

C, and the peaks at 224 nm and 195 nm collapse and are replaced by a minimum at 203 nm. The

10

throughout the process consistent with two-state unfolding previously observed for apo-

11

BsSCO.46

20 (a)

8 (b)

10

4



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 38

0

o

20-85 C -10

200

220

240

260

0

BsSCO-Cu(II)

-4 apo-BsSCO -8 20

Wavelength (nm)

80

T (oC)

12 13 14 15

60

40

Figure 4. Effect of copper binding on the thermal stability of wild-type BsSCO. (a) Temperature dependence of apo-BsSCO. The temperature was increased from 20 to 85 oC at a rate of 1 oC per m. Spectra were taken at 2 oC intervals and scanned from 260-180 nm. The concentration of apo-


Page 15 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

15 1 2 3 4 5

BsSCO was 6.75 µM in a 50 mM sodium phosphate buffer pH 7.0 and the spectra were measured in a 1 mm path length cuvette fitted with a probe to monitor the temperature of the sample. (b) CD response versus T for apo-BsSCO (○) and for the BsSCO-Cu2+ (●) complex. The conditions are the same as those described in panel (b) above. For the copper complex the concentration of copper is 7.0 µM as CuCl2.

6

The curve fit to the unfolding data has an inflection point at 53 oC that is read as the apparent

7

melting temperature, or TM (Figure 4b). The apparent TM measured here is in good agreement

8

with the value reported previously by differential scanning calorimetry.20 The UV-CD response

9

of the BsSCO-Cu(II) complex to temperature is also depicted in Figure 4b. The temperature-

10

induced change in the UV-CD spectrum of BsSCO-Cu(II) (not shown) proceeds in a manner

11

highly similar to apo-BsSCO. The characteristic spectrum of native BsSCO-Cu(II) (i.e.,

12

minimum at 224 nm, shoulder at 209 nm and peak at 195 nm) is replaced by a spectrum with a

13

single minimum at 203 nm as the temperature increases. Again, there is an isodichroic point at

14

215 nm that is maintained over the duration of the transition indicating that the entire process can

15

be described as the admixture of two spectral forms. The detection of only two spectral forms

16

during BsSCO-Cu(II) unfolding is consistent with the lack of spectral perturbation of the UV-CD

17

spectrum of apo-BsSCO by copper binding (see Figure 3). The key distinction for the unfolding

18

transition of BsSCO-Cu(II) compared to apo-BsSCO is that the melting curve is shifted to higher

19

temperatures (Figure 4b). The melting curve for wild-type BsSCO-Cu(II) is composed of two

20

transitions with apparent TM values of 53 oC and 71 oC. We propose that the transition that

21

occurs at low temperature arises from contributions from a small amount (i.e., < 10 %) of

22

oxidized protein in the sample that does not bind copper13, and also from some dissociation of

23

BsSCO-Cu(II) over the time of the applied temperature ramp. We assign the higher temperature

24

transition to unfolding of the BsSCO-Cu(II) complex, a result that is in keeping with melting

25

temperatures obtained by calorimetry.20


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 38

16 1 2 3

CALCULATED ALANINE SCAN OF BSSCO FINDS TRPS AS MAJOR STABILITY DETERMINANTS In order to probe the connection between the stability of BsSCO and the high affinity the

4

protein exhibits for binding copper we make use of an empirical force field (i.e., FoldEF)

5

implemented in the FoldX program to calculate changes in the free energy of a folded protein’s

6

structure in response to mutations introduced into the protein sequence (i.e., ∆∆G).36 As a

7

prerequisite for introducing specific amino acid changes into BsSCO, and to determine which

8

residues contribute prominently to overall stability, we used FoldX to perform an alanine scan of

9

the sequence. FoldX replaces each amino acid in turn with alanine, and computes the ∆∆G for

10

that version of mutant BsSCO. The results of this calculation are displayed versus residue

11

number in Figure 5 where positive ∆∆G values are destabilizing to the overall BsSCO structure,

12

and those that produce negative ∆∆G’s are considered stabilizing. The vast majority of changes

13

to alanine calculated for BsSCO are destabilizing with very few single residue changes predicted

14

to result in a stabilizing free energy change. The two tryptophan residues (i.e., W36 and W101)

15

in the native structure have the largest contribution when changed to alanine and produce

16

destabilizing ∆∆G’s near to, or greater than 6 kcal/mol. Although such calculations are never

17

exact47, in the present case FoldX predicts that the two tryptophans of BsSCO represent good

18

candidates to probe the coupling of the stability of the protein fold to ligand binding.


Page 17 of 38

17

W101 W36

6

∆∆G (kcal/mol)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

(a)

F42

4 2 0 C45 C49

-2 40

H135

80

120

160

Residue number (b) BsSCO sequence 10

20

30

40

50

QQIKDPL NYEVEPFTFQ NQDGKNVSLE SLKGEVWLAD FIFTNCETIC PPMTAHMTDL 60

70

80

90

100

110

QKKLKAENID VRIISFSVDP ENDKPKQLKK FAANYPLSFD NWDFLTGYSQ SEIEEFALKS 120

130

140

150

160

170

FKAIVKKPEG EDQVIHQSSF YLVGPDGKVL KDYNGVENTP YDDIISDVKS ASTLK

1 2 3 4 5 6

Figure 5. Alanine scan of the BsSCO sequence calculated using FOLDX. (a) Free energy change calculated as a result of changing each residue one at a time to alanine plotted against residue number. The bars for tryptophan 36 and tryptophan 101 are shown as solid. (b) The sequence of the soluble domain of BsSCO. The three copper ligating residues (i.e., C45, C49 and H135) are underlined and in bold. The residue positions subjected to mutagenesis here are in bold.

7


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 38

18 1 2

SINGLE SITE TRYPTOPHAN MUTANTS OF BSSCO The two tryptophan residues, W36 and W101, of wild-type BsSCO are located about 22

3

Å and 15 Å, respectively, from the presumptive copper binding site (see Figure 1). We anticipate

4

that changing the tryptophan complement of the protein will change BsSCO’s intrinsic

5

fluorescence. Single mutants of BsSCO in which each tryptophan is changed to phenylalanine

6

(i.e., W36F and W101F) have been constructed, purified and characterized. Table 1 summarizes

7

the fluorescence properties of these mutants of BsSCO, along with other mutants made for the

8

work here, compared to wild-type BsSCO. The emission intensity for reduced, apo-W36F

9

BsSCO is 75 % of that relative to wild-type when normalized for the tryptophan concentration,

10

whereas the W101F mutant has a relative intensity of 134 % that of wild-type. The emission

11

maximum for W36F apo-BsSCO is slightly blue-shifted relative to wild-type apo-BsSCO

12

indicating a slightly more hydrophobic environment for the remaining W101 residue. The

13

reverse is found for W101F apo-BsSCO which has a slightly red-shifted emission maximum

14

indicating a more solvent exposed site for W36. We conclude, therefore, that within wild-type,

15

reduced apo-BsSCO that the W36 residue makes a larger contribution to the overall fluorescence

16

than does W101. Furthermore, the contributions from the two tryptophan residues in the mutants

17

are close to being additive compared to wild-type BsSCO, and this implies that the substitution

18

of phenylalanine at either position has not induced much change to the core structure of BsSCO

19

in keeping with the view that the W to F change is a relatively conservative substitution.48 In

20

addition the UV-CD spectrum for the two tryptophan mutants is essentially unchanged in form

21

and intensity relative to wild-type apo-BsSCO (see below).

22 23

Reduced, apo-BsSCO can react in one of two ways to produce species that both probably participate in its functional cycle. Reduced, apo-BsSCO is capable of binding one equivalent of


Page 19 of 38

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Biochemistry

19 1

copper with high affinity, or it can be oxidized such that the dithiol site becomes disulfide. The

2

disulfide form, or oxidized BsSCO does not bind copper.25 In terms of fluorescence quenching,

3

all else being equal, it is anticipated that when the more distant of the two tryptophan residues

4

(i.e., W36) is removed that the contribution from the closer, remaining tryptophan (i.e., W101)

5

should be enhanced making the mutant more sensitive to quenching via thiol oxidation or copper

6

binding. The values in Table 1 are consistent with this prediction as the quenching of the

7

intrinsic fluorescence for W36F BsSCO by copper binding is enhanced relative to that for wild-

8

type BsSCO-Cu(II). The effect of oxidation to the disulfide is also enhanced for W36F BsSCO

9

compared to quenching for oxidized, wild-type BsSCO. Somewhat surprisingly, W101F BsSCO

10

has slightly more pronounced sensitivity to copper binding, being quenched by 78.5 % within the

11

BsSCO-Cu(II) complex. In contrast, the effect of formation of the disulfide is less evident in

12

W101F than with wild-type, oxidized BsSCO (Table 1).

13

An underlying assumption in these types of mutation studies is that the overall fold of

14

BsSCO is not changed, nor its stability altered by introduction of such conservative changes. We

15

address this assumption directly by assessing the spectral and stability properties of the mutant

16

forms of BsSCO. The UV-CD spectrum of W36F apo-BsSCO shows a spectrum with a

17

minimum at 224 nm, a shoulder at 209 nm and a peak at 195 (Figure 6a) that is indistinguishable

18

from wild-type, apo-BsSCO, consistent with the view that the basic fold and complement of

19

secondary structural elements is largely unchanged in W36F apo-BsSCO.


Biochemistry

20

20

0


16

(b)

∆CD

(a)

8 (ii)

-0.5

(i)

0

-8 180

200

220

240

260

20

Wavelength (nm)


(c)

10 0

-10

-1 40

60

300 400 500 600 Wavelength (nm)

80

T (oC)

700

20 (d)

10

0 260

280

300

Wavelength (nm)

320

Ellipticity (mM-1cm-1)


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 38

(e)

260

280

300

320

Wavelength (nm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Figure 6. Spectral properties and thermal stability of W36F BsSCO assessed by UV-CD versus temperature. (a)The effect of copper binding on the far UV-CD spectrum of W36F BsSCO. The concentration of W36F apo-BsSCO (───) was 55.8 µM in 5 mM sodium phosphate pH 7.0 buffer. One equivalent of copper (i.e., 60 µM CuCl2) was added to give the copper complex (─ ─ ─). Spectra were measured in 0.1 mm cuvette at 20 oC. The difference spectrum of the copper complex minus apo-protein is shown (─● ─● ─). (b) The response of the UV-CD signal to temperature. The temperature was increased from 25 to 85 oC at a rate 1oC per minute and spectra were recorded at 2 oC intervals. The concentration of BsSCO was 5.6 µM in 50 mM sodium phosphate buffer pH 7.0, the path length was 1 mm and a probe was immersed in the sample to record the temperature. Data set (i) (■) is for W36F apo-BsSCO and (ii) is for the copper complex. The solid line through the apo-protein data is a single phase sigmoidal function with an inflection point, or TM at 43 oC. The dashed line is a two phase sigmoidal function fit to data set (ii) for the copper complex of W36F and yields apparent TM’s of 44 oC and 67 oC. (c) Near UV and visible CD for W36F BsSCO. The apo-protein is shown as the solid line (───) and the copper complex spectrum as the dashed line (─ ─ ─). The sample conditions are the same as those for panel (a) except that the spectra are measured in 1 cm cuvette. The spectra shown are the average of four scans. (d) Near UV spectra for apo- (───) and copper complex (─ ─ ─) of W36F BsSCO. The spectra shown are the average of 16 scans. The difference spectrum of the copper complex minus apo is shown as (─ ● ─ ● ─). The sample conditions are the same as those for panel (c). (e) Comparison of near UV intensities for wild-type apo-BsSCO and two single TRP mutants. The Y-axis is plotted as ellipticity normalized for the protein concentration. The different forms of apo-BsSCO are indicated as follows, wild-type (───), W36F BsSCO (─ ─ ─), and W101F (─●─●─).


Page 21 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

21 1

Formation of the copper complex with W36F BsSCO does cause a small, but discernible change

2

to the UV-CD spectrum. As the temperature increases the UV-CD spectrum for folded W36F

3

BsSCO is replaced by a spectrum with a minimum at 203 nm and during the transition an

4

isodichroic point is maintained at 215 nm. A single transition is observed in the response of the

5

UV-CD signal to temperature (Figure 6b) with an apparent TM of 43 oC for apo-BsSCO that is

6

about 10 oC less than observed for wild-type apo-BsSCO (Table 2). Despite the induced

7

instability of W36F apo-BsSCO the mutant exhibits a tight binding profile and 1:1 stoichiometry

8

in titrations with Cu(II). Thermal-induced unfolding monitored by UV-CD spectroscopy reveals

9

that melting of W36F BsSCO-Cu(II) is shifted to higher temperature than the apo-protein (Figure

10

6b). As seen previously for wild-type BsSCO, W36F BsSCO-Cu(II) melting is a two-component

11

process with the first phase proceeding at an apparent TM of 44 oC, and the second phase with an

12

apparent TM of 67 oC. The first phase is assigned to a combination of some oxidized protein (i.e.,

13

10 %) in the sample and the copper complex dissociating over the time of the run. A similar

14

pattern is observed for the thermally-induced unfolding of W101F BsSCO with melting for the

15

apo-protein at 43 oC and for the Cu(II) complex at 66.5 oC. Despite the substantial destabilization

16

of the apo-protein in both of these mutants the high affinity copper binding function has

17

remained intact and moreover the shift induced in apparent TM is more pronounced than that

18

observed upon Cu(II) binding to wild-type BsSCO (Table 2). The implication is that the

19

contribution of the tryptophan residues at these positions to the stability of the copper complex of

20

BsSCO is less than their contributions to stability of the apo-protein, and that the protein-copper

21

complex compensates for lost stability of the apo-protein.

22 23

Formation of the copper complex with W36F BsSCO generates a visible CD spectrum (see Figure 6c) that has the same peak positions, trough positions, zero-crossing points and


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 38

22 1

intensities as observed for the copper complex of wild-type BsSCO. The major features of the

2

near UV-CD observed for wild-type BsSCO are observed for W36F apo-BsSCO (see Figure 6d).

3

The positive ellipticity band is centered at 277 nm with shoulders at 288 nm and 298 nm and fine

4

structure at 263 nm and 269 nm. The overall intensity of the near UV-CD signal for W36F

5

BsSCO is reduced by less than 10 % relative to wild-type when normalized for the protein

6

concentration (see Figure 6e). Addition of copper to W36F BsSCO induces loss of signals at 263

7

nm and 269 nm with little change elsewhere indicating that changes in structure upon copper

8

binding are limited to local effects near the copper binding site. In contrast the near UV-CD

9

spectrum of W101F BsSCO is quite distinct from wild-type and W36F versions of BsSCO (see

10

Figure 6e). The overall intensity of W101F BsSCO at 278 nm is reduced by 40 % relative to

11

wild-type and the shoulder at 296 nm is lost, whereas the fine structure at 263 nm and 269 nm is

12

retained. Thus much of the overall intensity of the near UV-CD spectrum of wild-type apo-

13

BsSCO and the shoulder at 296 nm can be assigned to the W101 chromophore. Copper binding

14

to W101F BsSCO is accompanied by loss of the fine structure in the near UV spectrum at 263

15

nm and 269 nm, as found above for wild-type and W36F BsSCO-Cu(II) and assigned to the

16

change in the environment of phenylalanine near the copper binding site.

17

ISOTHERMAL DENATURATION OF BSSCO

18

In order to better estimate the effect of these mutations on the equilibrium energetics of

19

BsSCO’s stability we performed isothermal chemical denaturation using urea as the denaturant

20

monitored by intrinsic fluorescence. The denaturation process is monitored by a shift in the

21

fluorescence emission maximum as the protein unfolds. As an example the urea-induced

22

unfolding of wild-type, reduced, apo-BsSCO is shown in Figure 7a.


Page 23 of 38

23

1.0 Normalized Fluorescence Shift

1.0 Fluorescence Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

(a) 0.8 0.6 0.4 0.2

(b)

0.8 0.6 0.4 0.2 0

0 300

350 400 Wavelength (nm)

450

0

1

2

3 5 4 [Urea] (M)

6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Figure 7. Thermodynamic analysis of wild-type apo-BsSCO stability as measured by iso-thermal urea denaturation. (a) Normalized fluorescence emission at increasing concentrations of urea. Samples were prepared by dilution into the appropriate urea containing buffer (6.5 mM sodium phosphate pH 7.0) at a protein concentration of 9.8 µM and allowed to incubate for 20 h at 25 o C. The concentrations of urea are indicated as follows, 0 M (───), 0.5 M (─ ─ ─), 1.0 M (• • •), 1.5 M (─ • ─ • ─), 2.0 M (─••─••─), 2.2 M (- - - -), 2.4 M (- • - • -), 2.6 M (───), 2.8 M (─ ─ ─), 3.0 M (• • •), 3.2 M (─ • ─ • ─), 3.4 M (─••─••─), 3.6 M (- - - -), 3.8 M (- • - • -), 4.0 M (───), 4.5 M (─ ─ ─), 5.0 M (• • •), and 6.0 M (─ • ─ • ─). (b) The shift in the fluorescence emission maximum versus urea concentration. The position of the emission maximum is normalized on a scale of 0 to 1. The response of wild-type apo-BsSCO (■) is compared to W36F BsSCO (□). The curves are fit to the data according to the formula described in Materials and Methods. The equilibrium parameters derived from these fits are as follows, for wild-type ∆Gurea = 10 kcal/mol, meq = 3.50 kcal/mol M-1 and the M1/2 = 2.86 M and for the W36F mutant ∆Gurea = 5.86 kcal/mol, meq = 3.82 kcal/mol M-1 and M1/2 = 1.52 M.

16

The emission spectra are normalized to the same intensity. The emission maximum of native

17

apo-BsSCO is at 330 nm and shifts to 356 nm in its urea-denatured state. The position of the

18

emission maximum is used to indicate the progress of denaturation (Figure 7b), and

19

thermodynamic analysis of the denaturation transition allows determination of the equilibrium

20

free energy for the stability of wild-type BsSCO (i.e., ∆Gurea) along with the equilibrium m-value

21

(meq).31, 32 The free energy measured for stability of wild-type, apo-BsSCO is about 10 kcal/mol


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 38

24 1

and this falls to 5.68 kcal/mol in W36F apo-BsSCO in keeping with the 10 oC decrease in

2

melting temperature for this mutant. The change in free energy of the folded protein introduced

3

by the W36F mutation has also been calculated using the FoldX’s program36 and is presented for

4

comparison in Table 2. We have also expressed each of these mutants in their full integral

5

membrane form in a strain of Bacillus subtilis lacking native BsSCO where the cytochrome c

6

oxidase activity can be assessed, and hence SCO’s functional status.11, 19 Results of this activity

7

assay are scored for each mutant in Table 2 and each of these single tryptophan mutants is found

8

to be functional in CuA assembly.

9

Table 2. Measurements of BsSCO stability TM (oC)a BsSCO mutants Apo Cu 52.7 71.7 ±0.15 ±0.81 pH 9.0 48.8 68.5 W36F 42.8 68.4 ±0.73 ±1.83 W101F 43.5 66.0 c W36F/W101F ─ ─ W36A 32.5 63.8 W101Ad n.d. n.d. W36A/W101Ac ─ ─ F42W 57.0 71.1 pH 9.0 52.1 64.5 W36F/F42W 46.5 62.6 W101F/F42W 45.6 60.2 W36A/F42W 37.4 66.0 W36F/W101F/F42W 34.7 56.0 Wild type

Ureab (M) Apo 2.91 ±0.31

∆Gourea (kcal/mol) Apo 9.93±0.14

meq (kcal/mol)/M Apo 3.51±0.13

∆∆GofoldX TMPD (kcal/mol) assay Apo 0 +

1.5

5.68

3.82

0.73

+

1.77 ─ 0.75 n.d. ─ 3.04

7.04 ─ 1.0 n.d. ─ 12.8

3.93 ─ 1.92 n.d. ─ 4.24

4.71 5.05 5.64 6.60 10.7 0.39

+ + + + ‒ +

1.72 1.81 0.8 n.d.

5.79 5.75 1.90 n.d.

3.37 3.17 3.74 n.d.

1.12 5.10 6.03 5.83

+ + ‒ +

10 11 12 13 14 15 16

a

The apparent melting temperature (i.e., TM) is taken from the inflection point of the response of the change in UV-CD spectrum as a function of temperature. Where there is a standard deviation reported these are from the average of four separate determinations. Otherwise the TM’s are the average of two determinations. bFor the urea denaturation experiments the values for wild-type BsSCO are the mean of five determinations. And for the mutants these are the average of two determinations. cThese double mutants were denatured in vitro and so unfolding studies were


Page 25 of 38

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Biochemistry

25 1 2

not conducted. dFor W101A BsSCO we were not able to purify sufficient amounts for these experiments.

3

SUBSTITUTING ALANINE FOR TRYPTOPHAN

4

The sensitivity of BsSCO’s stability to these single amino acid changes to phenylalanine

5

at the two tryptophan sites prompted us to try the more drastic substitution of alanine for

6

tryptophan. The UV-CD spectra of W36A apo-BsSCO has the same form (i.e., a trough at 224

7

nm, shoulder at 209 nm and peak at 195 nm) as wild-type apo- BsSCO and copper binding

8

induces little spectral change suggesting that the basic fold of BsSCO is retained in the W36A

9

mutant. In addition the fluorescence emission properties for W36A BsSCO are hardly different

10

from wild-type BsSCO (see Table 1). However, the melting temperature of W36A apo-BsSCO

11

drops to 32.5 oC, now more than 20 oC below wild-type (see Table 2). But the addition of copper

12

to W36A BsSCO still reflects a tight binding interaction with a more than 30 oC increase in TM to

13

63.8 oC. Isothermal urea denaturation reveals an approximate loss in stability of 9.0 kcal/mol

14

(i.e., ∆∆G) for the W36A mutation of BsSCO consistent with the greater effect on TM and the

15

FoldX-calculated stability change (see Table 2). The W101A mutant is unstable in the extreme to

16

the extent that when expressed in recombinant form in E. coli we are unable to purify the protein

17

in sufficient quantities to complete these studies. However, when W101A BsSCO is expressed in

18

its native form in B. subtilis a positive cytochrome c oxidase phenotype is found, implying that

19

W101A BsSCO is able to function in vivo.

20

ADDING A TRYPTOPHAN CLOSER TO THE COPPER/DISULFIDE SITE

21

The final single mutant we examine here is one in which a tryptophan is added to wild-

22

type BsSCO. The site chosen for addition of tryptophan is residue 42 that is occupied by

23

phenylalanine (i.e., F42) in wild-type BsSCO. The residue F42 in BsSCO aligns with a ‘reporter’


Biochemistry

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Page 26 of 38

26 1

tryptophan in the sequence of E. coli thioredoxin (i.e., EcTRX), the tryptophan in this position is

2

strongly quenched when the redox active thiol pair in EcTRX is oxidized.24 The F42W mutation

3

places the tryptophan in close proximity (< 5 Å) to the copper-binding thiol pair (i.e., -C45-ETI-

4

C49-) of BsSCO. Under our standard conditions in vitro (i.e., 50 mM sodium phosphate pH 7.0)

5

F42W BsSCO exhibits the tight-binding phenotype with copper as observed for wild-type

6

BsSCO. However, the endpoint of the copper titration with F42W BsSCO corresponds to a

7

stoichiometry of 0.5 Cu: BsSCO. This result implies that copper binding is to a dimeric form of

8

F42W BsSCO, or that copper binding induces formation of a dimer. However, we find no

9

evidence of a stable dimer for apo- or copper-bound forms of F42W BsSCO by analytical

10

ultracentrifugation, or size-exclusion chromatography. We do find that the stoichiometry of

11

copper binding to F42W BsSCO increases as the concentration of F42W BsSCO is lowered, or

12

with increasing pH of the buffer medium. A stoichiometry near one for the copper complex is

13

obtained for F42W BsSCO at pH 9.0. We have, therefore, characterized the effect of copper

14

binding on the TM of F42W BsSCO at pH 7.0 and pH 9.0 to compare with wild-type BsSCO (see

15

Table 2). The melting temperatures for apo- and copper bound, wild-type BsSCO are decreased

16

by about 5 oC when the pH is shifted from 7.0 to 9.0. At pH 9.0, F42W BsSCO is about 3 oC

17

more stable than apo-, wild-type BsSCO, but copper-bound F42W BsSCO is less stable by 4 oC

18

than copper-bound, wild-type BsSCO. This implies that introducing a tryptophan residue at

19

position 42 is more disruptive to stability of the copper complex than it is to the stability of the

20

apo-protein. The fluorescence properties for apo- and copper-bound F42W BsSCO are compared

21

to wild-type BsSCO in Table 1. Addition of one more tryptophan in F42W BsSCO does make

22

the protein more fluorescent than wild-type apo-BsSCO per protein molecule, but when

23

normalized for the amount of tryptophan, F42W apo-BsSCO is less fluorescent than wild-type


Page 27 of 38

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Biochemistry

27 1

apo-BsSCO. This means that emission from W42 is quenched to a greater extent than either of

2

the native tryptophans. The W42 residue is in a relatively solvent-exposed position and a shift in

3

the overall emission to the red is anticipated. The lack of an apparent shift is consistent with the

4

highly quenched intensity from this residue.

5

In addition, the near UV-CD spectrum of F42W apo-BsSCO is devoid of the fine

6

structure seen at 263 nm and 269 nm for wild-type apo-BsSCO (see Figure 8). This finding

7

supports the assignment of this signal to electronic transitions from Phe42 that is near the copper

8

binding site. Moreover, formation of the copper complex with F42W BsSCO induces a shift in

9

the near UV-CD spectrum with a loss in intensity at 277 nm and an increase at 295 nm consistent

10

with an effect on the local environment of the tryptophan now present at residue 42. 11 12 13 14 15 16 17 18 19 20 21 22

Figure 8. The effect of copper complex formation on the near UV-CD of F42W BsSCO. The apo-protein is dissolved in 5 mM sodium phosphate pH 9.0 at a concentration of 35 µM (───). One equivalent of CuCl2 is added to make the copper complex (─ ─ ─). These spectra are the average of 16 scans. The difference spectrum between the copper complex minus the apo-protein is shown as (─ • ─ •─).

23 24 25 26 27


Biochemistry

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Page 28 of 38

28 1 2

DOUBLE MUTANTS OF BSSCO- MOVING TRYPTOPHAN TO A NEW POSITION Since F42W BsSCO has added stability over wild-type BsSCO it is of interest to see if

3

this stabilizing effect would carry over to the previous single tryptophan mutants in which one of

4

the wild-type tryptophans is replaced with either phenylalanine, or alanine. The double mutants

5

replacing a wild-type tryptophan with phenylalanine, and a wild-type phenylalanine with

6

tryptophan has the overall effect of moving a tryptophan residue within an overall isomeric

7

structure. Both of the double mutants, W36F/F42W and W101F/F42W, are slightly more stable

8

in the apo-form and less stable in the copper-bound forms than corresponding W to F single

9

mutants (see Table 2). Thus, again the F42W change seems to contribute to the stability of apo-

10

BsSCO, but is disruptive to the copper complex. In the near UV-CD spectra the fine structure at

11

269 and 273 nm has returned in both mutants and this is consistent with the extra phenylalanine

12

residue in each of these constructs. However, the formation of the copper complex for each of

13

these mutants does not affect the phenylalanine signature spectral components in the near UV,

14

but does result in a shift of the tryptophan features consistent with the presence of tryptophan at

15

residue 42 near the copper binding site (see Figure 9). 16 17 18 19 20 21 22 23 24 25 26

Figure 9. The near UV-CD spectrum of the double mutant W101F/F42W BsSCO. The protein is dissolved in 50 mM sodium phosphate pH 7.0 at a concentration of 43.2 µM. The copper complex is formed by addition of one equivalent of CuCl2. The spectra are indicated as follows, apo W101/F42W BsSCO (───), copper complex (─ ─ ─) and Cu-apo difference spectrum (─ • ─ • ─).

27


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Biochemistry

29 1 2 3 4

Adding the tryptophan at residue 42 in place of phenylalanine also adds stability to the

5

single mutant in which tryptophan 36 is replaced by alanine. This double mutant (i.e.

6

W36A/F42W BsSCO) has TM about 5 oC higher than the single mutant W36A BsSCO in the

7

apo- form and about 3oC more stable in the copper-bound form. A curious feature of

8

W36A/F42W BsSCO is that when expressed in B. subtilis it is not able to complement the

9

deletion of native BsSCO (see Figure 10), even though the expression level of W36A/F42W

10

BsSCO is indistinguishable from wild-type.

11 12 13 14 15 16 17 18 19 20

Figure 10. Assay of BsSCO’s activity to affect assembly of the CuA center of cytochrome c oxidase. TMPD oxidase activity is depicted for B. subtilis cells expressing two mutant forms of BsSCO. The cells are grown over night at 37 oC, harvested, suspended in buffer and applied to TMPD-saturated filter paper. The cells expressing F42W BsSCO generate a blue color on the filter paper in manner identical to cells expressing wild-type BsSCO. In contrast cells expressing the double mutant do not express TMPD oxidase activity. Aliquots of both cultures were shifted from 37 oC to 25 oC for 2 hours. These cells were then harvested, suspended in buffer and applied to TMPD saturated filter paper. The TMPD assays are conducted at room temperature in each case.


Biochemistry

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Page 30 of 38

30 1

Therefore, the strain of B. subtilis expressing W36A/F42W BsSCO in place of wild-type BsSCO

2

is cytochrome c oxidase deficient when grown under standard conditions at 37 oC. This is very

3

close to the TM seen for W36A/F42W BsSCO in vitro and when these cultures are allowed to

4

incubate at 25 oC for a few hours a cytochrome c oxidase positive phenotype is restored.

5

A TRIPLE MUTANT OF BSSCO WITH A SINGLE TRYPTOPHAN ADJACENT TO THE METAL BINDING SITE.

6

One aim of our tryptophan construction was to generate a form of BsSCO with a single

7

tryptophan present much closer to the copper binding site than the native tryptophans and

8

thereby generate a more sensitive reporter of copper binding. The triple mutant

9

W36F/W101F/F42W of apo-BsSCO has relative fluorescence of 33 % of wild-type apo-BsSCO

10

when normalized for the tryptophan concentration (see Table 1). Thus, the tryptophan at position

11

42 does appear to be heavily quenched in apo-BsSCO relative to the native tryptophan sites as

12

implied above. However, the emission maximum of the reduced, apo- triple mutant is at 334 nm

13

and the UV-CD spectrum is distinctly different from wild-type apo-BsSCO indicating that this

14

mutant is not fully folded. In addition both thermal induced melting and urea-induced unfolding

15

do not exhibit two-state behavior as seen with the other BsSCO species examined here. An

16

apparent TM can assigned to the apo-protein and formation of the copper complex does shift the

17

apparent TM by more than 20 oC (see Table 2). In addition the emission maximum of

18

W36F/W101F/F42W BsSCO shifts to 328 nm upon copper binding and this is accompanied by a

19

small, but measureable change in the UV-CD spectrum. These data indicate that the apo-triple

20

mutant is partially unfolded and copper binding drives return to a more native structure. Copper

21

binding to the triple mutant also diminishes the fluorescence intensity by 45 %, compared to 70

22

% quenching within the copper complex of wild-type BsSCO (see Table 2). However, the

23

absorbance spectrum of the copper complex of the BsSCO triple mutant is also diminished from


Page 31 of 38

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Biochemistry

31 1

a ∆ε352 nm of 4.87 mM-1cm-1 in wild-type BsSCO to 2.50 mM-1cm-1 in the triple mutant. If much

2

of the quenching in the copper complex is due to Förster-type energy transfer the diminished

3

extinction coefficient for the triple mutant would lead to a loss in spectral overlap, and a loss in

4

efficiency of energy transfer. In contrast the sensitivity of the intrinsic fluorescence to oxidation

5

of the dithiol in reduced BsSCO to the disulfide in oxidized BsSCO is lost. The fluorescence

6

intensity is the same in oxidized and reduced W36F/W101F/F42W BsSCO as compared to 30 %

7

quenching in wild-type BsSCO.

8 9 10

Discussion

11

The binding of copper by BsSCO is an ultra-high affinity interaction and yet is

12

accompanied by a limited change in the protein’s secondary and tertiary structural elements. In

13

contrast to this mode of protein/ligand interaction between BsSCO and copper are the

14

intrinsically disordered proteins that undergo large scale rearrangements from a disordered to an

15

ordered structure upon ligand binding.49 Intrinsically disordered protein transitions are proposed

16

to play controlling roles in eukaryotic signaling pathways.50 Such transitions are also reported in

17

prokaryotic proteins as a disorder to order transition has been invoked to account for the metal-

18

binding regulated activity of the diphtheria toxin repressor protein.51 We suppose in such ligand

19

binding interactions that, from the perspective of the ligand, there would be an accompanying

20

change in binding affinity whereby in the disordered state protein/ligand affinity would be weak

21

and ligand affinity would increase in the ordered state. However, in the case of BsSCO we

22

observe high affinity ligand binding, but no change in protein structure. So, how is such a high-


Biochemistry

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Page 32 of 38

32 1

affinity interaction energetically founded? A plausible explanation for a highly energetically

2

favorable ligand/protein interaction can be found in a ligand-triggered change in the protein’s

3

dynamics. The protein’s average structure may remain largely unchanged upon ligand binding

4

but the degree of flexibility of the structure is reduced. Such a highly localized disordered to

5

ordered transition has been observed with NMR-based structures for apo- and metallated

6

constructs derived from human SCO1 where nickel binding hardly effects the structure of the

7

protein, but does alter the overall volume and molecular dynamics of the protein loops that

8

include the inner sphere ligands (i.e., 2 Cys and 1 His).52 Although the affinity of the interaction

9

considered for the human SCO1 constructs have not been reported. We propose that such protein

10

dynamical changes could underlie the energetic consequences we observe here for high-affinity

11

copper binding to BsSCO.

12

In any of the disorder to ordered transitions that might mediate protein/ligand interactions

13

the binding interaction does not have necessarily to be of the ultra-high affinity type. It is often

14

the case that in the transition to an ordered state the emphasis is placed on new interactions that

15

are being taken up in forming the protein/ligand complex. However, the new interactions that are

16

formed in the protein/ligand complex may be balanced energetically by modes of interactions

17

present in the disordered state that are lost in transition, particularly entropic contributions. In the

18

case of the ultra-tight binding interaction exhibited by BsSCO for copper we propose it is the

19

case that not much is lost at all from the protein’s internal structural stability when binding

20

copper and that what is gained energetically arises from the new interactions that are mediated

21

by the ligand itself. One must recall that the shift in overall stability upon ligand binding giving

22

rise to a 20-25 oC increase in TM observed here, or the resistance to urea denaturation reported

23

previously for the copper/BsSCO complex46 could be explained by the addition of 5-10 kcal/mol


Page 33 of 38

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Biochemistry

33 1

of stability to the already stable native, apo-protein. This energy content could be found in the

2

coordination complex with copper that mediates the interaction of the inner sphere ligands

3

supplied by BsSCO.

4

The binding of copper by BsSCO is accompanied by little overall protein structural

5

change and is probably well characterized by a lock and key type of interaction. In this mode of

6

binding, copper is guided by the pre-existing BsSCO structure to its ultimate binding site. In this

7

case there is a spectroscopically distinguishable intermediate that precedes the equilibrium

8

BsSCO-Cu complex.23 53 We imagined that one might be able to observe copper-triggered

9

folding of a protein such as BsSCO that has such a large equilibrium energetic difference

10

between the apo- and ligand-bound forms in a manner similar to that observed for electron

11

transfer triggered folding of cytochrome c.54 It is also known that the presence of copper during

12

the refolding of apo-azurin speeds attainment of the metallated protein.55 We have characterized

13

previously the folding kinetics of BsSCO as a two-state protein both for the oxidized and reduced

14

forms of BsSCO.46 However, addition of copper to the urea-denatured protein gives rise to

15

immediate thiol oxidation and formation of the disulfide containing form of BsSCO. This is not

16

surprising as copper is a known catalyst for cysteine oxidation. We have argued that the thiols

17

within folded reduced BsSCO are protected from copper mediated oxidation.53 The highly

18

ordered structure of BsSCO that precisely directs high affinity copper binding also protects the

19

protein from an unwanted reactivity, namely thiol oxidation. The exception to this view of

20

BsSCO is provided here by the triple mutant (i.e., W36F/W101F/F42W BsSCO) which has

21

spectral features consistent with partial-unfolding of apo-BsSCO, and copper binding drives a

22

shift to a more folded structure. But on balance we suggest that the interaction of BsSCO with

23

copper is an example of a paradigm opposite to that underlying the intrinsically disordered


Biochemistry

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Page 34 of 38

34 1

protein hypothesis. It is argued that an advantageous feature of an intrinsically disordered

2

protein, along with the capacity to switch from inactive to active for its native functionality, is

3

the possibility that the disordered state may be capable of other useful activities, or interactions

4

with multiple ligands.56 This is a benign view of the functional status of a disordered protein. We

5

would recall that proteins are complex molecules and their precise function is dictated by their

6

precise structure. At the same time this precise, native and stable structure prevents other

7

reactivities of the protein. And one description of the disordered state is that many structures are

8

accessible to the protein. The manifold structures of IDP’s implies that many reactivities are

9

accessible to the disordered protein, and it is possible that some of these could be detrimental to

10

the cell. There seem to be proteins that have evolved to sustain this dangerous lifestyle, but there

11

are surely others, such as BsSCO, for which a highly structured form is more suitable.

12 13

Acknowledgements. This work was supported by operating (no. 153113) and equipment (no.

14

358903) grants from NSERC to BCH and by an Infrastructure Award (no. 2356) from CFI to the

15

Protein Function Discovery Research Group. We are grateful to Mr. Kim Munro of the Protein

16

Function Discovery facility for help with analytical ultracentrifugation.

17 18

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Biochemistry

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[40] Hill, B. C., and Andrews, D. (2012) Differential affinity of BsSCO for Cu(II) and Cu(I) suggests a redox role in copper transfer to the Cu(A) center of cytochrome c oxidase, Biochim Biophys Acta 1817, 948-954. [41] Crawley, S. W., de la Roche, M. A., Lee, S. F., Li, Z., Chitayat, S., Smith, S. P., and Cote, G. P. (2006) Identification and characterization of an 8-kDa light chain associated with Dictyostelium discoideum MyoB, a class I myosin, J Biol Chem 281, 6307-6315. [42] Dogan, J., Schmidt, T., Mu, X., Engstrom, A., and Jemth, P. (2012) Fast association and slow transitions in the interaction between two intrinsically disordered protein domains, J Biol Chem 287, 34316-34324. [43] Kelly, S. M., Jess, T. J., and Price, N. C. (2005) How to study proteins by circular dichroism, Biochim Biophys Acta 1751, 119-139. [44] Stanyon, H. F., Cong, X., Chen, Y., Shahidullah, N., Rossetti, G., Dreyer, J., Papamokos, G., Carloni, P., and Viles, J. H. (2014) Developing predictive rules for coordination geometry from visible circular dichroism of copper(II) and nickel(II) ions in histidine and amide main-chain complexes, Febs j 281, 3945-3954. [45] Pain, R. (2005) Determining the CD spectrum of a protein, Current protocols in protein science Chapter 7, Unit 7.6. [46] Lai, M., Yam, K. C., Andrews, D., and Hill, B. C. (2012) Copper binding traps the folded state of the SCO protein from Bacillus subtilis, Biochim. Biophys. Acta 1824, 292-302. [47] Steinbrecher, T., Zhu, C., Wang, L., Abel, R., Negron, C., Pearlman, D., Feyfant, E., Duan, J., and Sherman, W. (2017) Predicting the Effect of Amino Acid Single-Point Mutations on Protein Stability-Large-Scale Validation of MD-Based Relative Free Energy Calculations, J Mol Biol 429, 948-963. [48] Brisbois, C. A., and Lee, J. C. (2016) Apolipoprotein C-III Nanodiscs Studied by Site-Specific Tryptophan Fluorescence, Biochemistry 55, 4939-4948. [49] Wright, P. E., and Dyson, H. J. (2009) Linking folding and binding, Current opinion in structural biology 19, 31-38. [50] Wright, P. E., and Dyson, H. J. (2015) Intrinsically disordered proteins in cellular signalling and regulation, Nature reviews. Molecular cell biology 16, 18-29. [51] Twigg, P. D., Parthasarathy, G., Guerrero, L., Logan, T. M., and Caspar, D. L. (2001) Disordered to ordered folding in the regulation of diphtheria toxin repressor activity, Proceedings of the National Academy of Sciences of the United States of America 98, 11259-11264. [52] Banci, L., Bertini, I., Calderone, V., Ciofi-Baffoni, S., Mangani, S., Martinelli, M., Palumaa, P., and Wang, S. (2006) A hint for the function of human Sco1 from different structures, Proceedings of the National Academy of Sciences of the United States of America 103, 8595-8600. [53] Bennett, B., and Hill, B. C. (2011) Avoiding premature oxidation during the binding of Cu(II) to a dithiolate site in BsSCO. A rapid freeze-quench EPR study, FEBS Lett 585, 861-864. [54] Kumar, R. (2016) Analysis of the pH-dependent thermodynamic stability, local motions, and microsecond folding kinetics of carbonmonoxycytochrome c, Archives of biochemistry and biophysics 606, 16-25. [55] Wittung-Stafshede, P. (2004) Role of cofactors in folding of the blue-copper protein azurin, Inorg. Chem 43, 7926-7933. [56] Hsu, W. L., Oldfield, C. J., Xue, B., Meng, J., Huang, F., Romero, P., Uversky, V. N., and Dunker, A. K. (2013) Exploring the binding diversity of intrinsically disordered proteins involved in one-tomany binding, Protein Sci 22, 258-273.

46


Biochemistry

Copper shifts thermal stability of BsSCO by 30 oC without structural change Ellipticity (mdegrees)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

12

-0.5

195 nm +1 eq Cu

T1M = 32.5 oC

-1.0

8

CD

apo

T2M = 67 oC

-1.5

4 Cu-apo

0

-2.0

-4

209 nm

-8 180

+Cu

-2.5

224 nm

-3.0

200

220

240

Wavelength (nm)

260

20

40

60

80

T oC


Page 38 of 38

Using tryptophan mutants to probe the structural and functional status

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