Engineering AromaticâAromatic Interactions To Nucleate Folding in

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Engineering Aromatic - Aromatic Interactions to Nucleate Folding in Intrinsically Disordered Regions of Proteins Swati Balakrishnan, and Siddhartha Peddibhotla Sarma Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00437 • Publication Date (Web): 24 Jul 2017 Downloaded from http://pubs.acs.org on August 1, 2017

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Biochemistry

Engineering Aromatic - Aromatic Interactions to Nucleate Folding in Intrinsically Disordered Regions of Proteins.

†

Swati Balakrishnan‡ and Siddhartha P. Sarma∗,‡,§ Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka - 560012, INDIA E-mail: [email protected] Phone: +91 80 2293 3454. Fax: +91 80 23600535

Running header Aromatic - Aromatic Interactions

† Structural data, chemical shift data and files of restraints used for structure calculation of WF - cytb5 and FF - cytb5 have been deposited with the Protein Data Bank (accession numbers 5XE4.pdb and 5XEE.pdb) and BMRB (under the accession numbers 36070 and 36071) respectively. ∗ To whom correspondence should be addressed ‡ Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka - 560012, INDIA § NMR Research Center, Indian Institute of Science, Bangalore, Karnataka - 560012, INDIA

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ACS Paragon Plus Environment

Biochemistry

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Abstract Aromatic interactions are an important force in protein folding as they combine the stability of a hydrophobic interaction with the selectivity of a hydrogen bond. Much of our understanding of aromatic interactions comes from “bioinformatics” based analyses of protein structures and from the contribution of these interactions to stabilizing secondary structure motifs in model peptides. In this study, the structural consequences of aromatic interactions on protein folding have been explored in engineered mutants of the molten globule protein apo-cytochrome b 5 . Structural changes from disorder - to - order due to aromatic interactions in two variants of the protein, viz., WF-cytb5 and FF-cytb5, result in significant long-range secondary and tertiary structure. The results show that 54% and 52% respectively, of the residues in each of these proteins occupy ordered regions as against 26% in apo-cytochrome b 5 . The interaction between the aromatic groups are offset-stacked and edge-to-face for the Trp-Phe and Phe-Phe mutants respectively. Urea denaturation studies indicate that both mutants have a higher Cm and are more stable to chaotropic agents than apo-cytochrome b 5 . The introduction of these aromatic residues also results in “trimer” interactions with existing aromatic groups, reaffirming the selectivity of the aromatic interactions. These studies provide insights into the aromatic interactions that drive disorder - to - order transitions in intrinsically disordered regions of proteins and will aid in de novo protein design beyond small peptide scaffolds.

Abbreviations Standard one- and three-letter amino acid codes; NMR, Nuclear Magnetic Resonace; ANS, 8-Anilino-1-naphthalenesulfonic acid; NOE, Nuclear Overhauser Effect; PDB, Protein Data Bank; BMRB, Biological Magnetic Resonance Bank; SI, Supplementary Information.

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Keywords Aromatic interactions, protein folding, intrinsically disordered regions, NMR spectroscopy

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Introduction The minimum energy conformation of a protein is arrived at through complex interatomic interactions which include hydrophobic, hydrogen - bonding, electrostatic and long - range covalent interactions (disulfide bonds) that result in the “folded” or “native” structure(1 –4 ). Hierarchically, the unfolded polypeptide chain experiences a hydrophobic collapse with the formation of secondary structural elements culminating in a protein with a well - defined three dimensional shape (4 –6 ). While hydrogen bonding interactions between backbone atoms define the protein backbone, the three - dimensional conformation results from specific interactions between the protein side - chains of the constituent amino - acids. Interactions between aromatic side - chains are important for the initial hydrophobic collapse and they play a prominent role in protein folding(7 ). Aromatic residues are predominantly found in protein cores. Nearly 60% of all aromatic side chains in proteins are found to participate in aromatic pairs and networks and 80% of those interactions connect distinct secondary structure elements(7 ). In contrast, surface aromatic residues could play an important role in molecular recognition and intermolecular interactions. Indeed it is thought that aromatic -(8 ), cation - π (9 ) and thiol - π (10 ) interactions could play crucial roles in intermolecular interactions and folding of intrinsically disordered proteins(11 , 12 ). Each aromatic interaction contributes ∼-0.6 to -1.3 kcal / mol to protein stability(13 , 14 ). Interacting ring pairs could be aligned either edge - to - face or offset - stacked, in which the former is driven by electrostatic forces while the latter is stabilized by dispersive interactions(15 ). Aromatic rings are assumed to be interacting if the centroid distance between the rings is between 4.5 - 7Å(7 ). Calculations of the energies of interaction for the aromatic pairs Phe - Phe, Phe - Tyr and Tyr - Tyr in polar and non-polar environments predict that stacked arrangements are strongly favoured in hydrophilic environments, whereas there is only a slight preference of stacking over T - shaped arrangements in hydrophobic environments(15 ). Peptide design strategies have exploited aromatic interactions successfully in order to sta4


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bilize secondary structural elements such as β - hairpins (16 , 17 ), β -strands stabilised by aromatic caps (18 ), the “trp-zip” stablized β -strands (19 ) and α -helices(20 ). Several unnatural aromatic amino acids have also been used to stabilize β - secondary structures in peptides (21 –23 ). Solvent polarity is seen to play a prominent role in folding of β - hairpins and β - turns that are stabilized via aromatic interactions. Similar effects have been seen to stabilize designed α - helices, in aqueous environments, when pairs of phenylalanine residues have been introduced at the ith and i+4th positions(20 ). In proteins, investigations of aromatic interactions have relied on estimating the loss in thermodynamic stability brought about by sequential mutagenesis of aromatic residues (double mutant cycles)(24 ). The long - range effects of introducing aromatic interactions have been examined in detail in small peptides and “mini-proteins”(18 ). However, the influence of these interactions in nucleating tertiary structure in otherwise disordered proteins have not been examined. Introduction of aromatic residues into the interior of a protein presents a difficult proposition due to the existing packing in the protein core. Substitution of aromatic residues for polar residues at the surface could result in aggregation due to an increase in surface hydrophobicity. Thus judicious selection of a model system to study aromatic interactions in an aqueous environment is paramount. Effects of amino acid substitutions on folding and structure are best examined in the backdrop of conformational transitions from disordered to ordered states. The heme - binding protein cytochrome b 5 , in the apo - form, exists as a largely disordered molten globule in solution(25 ) (Figure S-1, SI). This is in sharp contrast to the protoporphyrin IX bound holo - form of the protein(26 –28 ), which incidentally represents one extreme of aromatic - interaction structural space in proteins. The protein is highly soluble in both apo - and holo - forms, and as such presents an exceptional model system in which to test the effects of natural aromatic amino substitutions on structure and folding.

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In this study, we have introduced an aromatic interaction via substitution of the heme binding axial histidine residues with phenylalanine and phenylalanine - tryptophan pairs. The consequence of these substitutions on structure, stability and folding have been examined by biophysical and solution NMR spectroscopic methods. The results of these studies are described below.

Experimental Protein mutagenesis, cloning and expression Aromatic amino acid substitutions for the axial histidine residues were introduced in the wild-type rat cytochrome b 5 gene encoded in the pET21(a) plasmid via site - directed mutagenesis, resulting in H43W H67F cytb5 and H43F H67F cytb5 mutant genes. Mutagenesis was confirmed by sequencing. The wild-type and mutant proteins encoded for in the corresponding genes are henceforth referred to as apo-cytb5, WF-cytb5 and FF-cytb5 respectively. The proteins were expressed and purified using procedures described for cytochrome b 5 (29 , 30 ) and cytochrome b 5 based fusion proteins(31 ). Proteins samples of apo -, WF - and FF - cytb5 at natural isotopic abundance were produced for biophysical studies. Isotopically enriched samples (15 N -,

13

C,

15

N - and 2 H,

13

C,

15

N - ILV methyl protonated) (32 –34 ) of

apo - cytb5, WF - cytb5 and FF - cytb5 were produced for solution NMR structural studies. Mass spectrometry The identity of the mutant proteins was confirmed by comparing the calculated and experimentally determined masses using mass spectrometry. Mass spectra were acquired by electrospray ionization mass spectrometry (HCT Ultra ETD II ion trap mass spectrometer, Bruker Daltonics, Germany) connected to an Agilent 1100 series HPLC system. The samples were injected into the spectrometer using a gradient elution through a reverse phase C18 column. A binary solvent system (solvent A: 0.1% trifluoroacetic acid in water, solvent B: 6


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0.1% trifluoroacetic acid in acetonitrile) and a flow rate of 1mL / min was used. ANS binding by fluorescence spectroscopy The relative binding of the fluorescent dye ANS (35 ), to apo - cytb5, WF - cytb5 and FF - cytb5 was measured by Fluorescence spectroscopy. Fluorescence data was acquired on a JASCO FP-6300 spectrofluorimeter. All measurements were made using protein samples of 5µ M concentration prepared in 20mM Phosphate buffer (pH 7.0) containing 20mM NaCl, 15mM arginine, 15mM glutamate and 5mM proline. The ANS concentration was maintained at 50 µM diluted from a stock solution of 1 mM. The excitation wavelength was 388nm, while the emission wavelength was scanned from 450-500nm. The bandwidth of excitation and emission were 5nm and 10nm respectively, and the final spectra were averaged over three scans. Maltose binding protein (5µM) at pH 3 in 10mM Citrate-glycine-HEPES buffer was used as a positive control(36 ). Urea induced chemical denaturation studies Denaturation of apo -, WF - and FF - cytb5 as a function of urea concentration was monitored by intrinsic tryptophan fluorescence spectroscopy(37 ). Protein samples of 5µM concentration in 20mM Tris-HCl buffer with 20mM NaCl, 15mM arginine, 15mM glutamate and 5mM proline, pH 7 were incubated overnight with urea at 25◦ C. Preliminary denaturation studies were carried out by varying urea concentrations from 0 to 7.2M in alternate increments of 1.2M and 0.8M. The final studies were carried by varying urea concentrations from 0.0M to 1.0M in 0.1M increments and thereafter from 1.0M to 6.0M in 0.2M increments. Reversibility of unfolding was checked by diluting out the urea and comparing the intensity and red shift of the tryptophan fluorescence emission against samples incubated with urea at the concentrations used in the unfolding reaction. Fluorescence data were acquired on a JASCO FP-6300 spectrofluorimeter. The excitation wavelength was set at 280nm, and emission spectra were scanned from 300-400nm and bandwidths of 5nm and 10nm were used during excitation and emission respectively. The final spectra were averaged over 3 scans.

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The fluorescence emission baselines for the folded and unfolded states were fit for each protein, and the fraction of unfolded protein at each urea concentration (Fu ) was calculated. A plot of Fu vs urea concentration was used to determine the Cm values for apo -, FF - and WF - cytb5. NMR Spectroscopy All NMR data were acquired on purified samples of apo -, WF - and FF - cytb5 prepared in 20mM potassium phosphate buffer (pH 7.0) (90% H2 O / 10%D2 O), containing 20mM NaCl, 25mM arginine, 25mM glutamate, and 10mM proline. Protein samples ranged in concentration from 0.3mM to 0.8mM. Data Acquisition Double and triple resonance experiments were acquired in order to obtain sequence specific assignments, distance restraints, measurement of coupling constants and

15

N R 1 and R 2 re-

laxation rates. All spectra were acquired on either a Varian (Agilent) 600 MHz spectrometer equipped with a 5mm triple resonance cold probe fitted with a single (Z-axis) pulsed field gradient accessory, or a Bruker Avance 700 MHz spectrometer also equipped with a cryoprobe fitted with a Z-axis only pulsed field gradient accessory. All NMR data were acquired at 10◦ C, as the FF - cytb5 sample tended to degrade over time at higher temperatures. Solvent suppression was achieved in one-dimensional and two - dimensional homonuclear spectra(38 ) with the excitation sculpting solvent suppression scheme(39 ). In two - and three - dimensional double - and triple - resonance NMR experiments(40 ), solvent suppression was carried out using pulse programs that incorporate water flip-back pulses, or flip back pulses along with coherence selection via pulse field gradients (38 , 41 ). Nitrogen - 15 T 1 , T 2 and heteronuclear NOE experiments (42 ) were acquired on a 600 MHz NMR spectrometer. All spectra were referenced to an external DSS standard and recorded in phase sensitive mode. The list of all NMR experiments and their data acquisition parameters are provided in Table S-1, SI. Data processing and analysis 8


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All data processing and analysis was carried out on an Apple Macintosh system running Mac OSX 10.6.8. All 2D and 3D NMR data were processed using NMRPipe / NMRDraw software(43 ). The time domain data in both the direct and indirectly acquired dimensions were apodized with either a 90◦ phase-shifted square sine bell or by multiplication with a Gaussian function with a 10Hz line broadening parameter and zero-filled once before Fourier transformation and baseline correction. Spectra were analyzed using CCPNMR (version 2.1) suite of programs(44 ). The nitrogen-15 R 1 and R 2 rates and 1 H - 15 N NOE values were determined by fitting peak heights using the algorithms bundled in CCPNMR. Hydrogen / Deuterium exchange studies To a 0.5mM sample of WF - cytb5, 5mM trehalose was added and the sample was lyophillized. The sample was then re-dissolved in 99.96% D2 O and 1 H - 15 N HSQC spectra were acquired at intervals of 30 min, over a period of 72 hours. Trehalose was added as a stabiliser to protect the protein degradation during lyophillization(45 ). Structural restraints Secondary Structure and Dihedral Angle Restraints Assignment of secondary structure, as a function of sequence, was made on the basis of calculated CSI values. The CSI values were calculated using the Hα ,

13

Cα ,

13

Cβ and

13

C′

secondary chemical shifts(46 ). 3D HNHA data was used to calculate the 3 J H N H α coupling constants using which the backbone dihedral angle φ was calculated using the Karplus equation. Backbone dihedral angles φ and ψ were also predicted using DANGLE (47 )(provided with the CCPNMR program suite), using HN , Hα ,

13

Cα ,

13

Cβ and

13

C′ chemical shifts as

input data. Dihedral angle restraints with an angular variation of ± 30◦ was input as restraints for structure calculations. Distance restraints and tertiary structure The intensities of unambiguously assigned peaks from the 2D 1 H - 1 H,

15

N edited - and

13

C

edited NOESY-HSQC experiments were used to calculate inter - proton distance restraints(48 ).

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The upper distance bounds were set to 6Å, 4.5Å, 3.5Å and 2.5Å for very weak, weak, medium and strong NOEs. A lower distance bound of 1.8Å was enforced for all NOE restraints. Hydrogen bond restraints were derived from the assignment of secondary structure for alphahelices, and from the presence of NOE correlations for beta -sheets. The restraints limit the distance between the oxygen acceptor from the proton and nitrogen donors to 3.5Å and 2.5Å respectively. Calculation of rotational correlation time (τ c ). The rotational correlation time(49 ) was calculated using equation (1) below: 1 τc = 2ωN

r

6 T1 −7 T2

where ω N is the Larmor precession frequency of the

15

(1)

N nucleus. The T1 and T2 values here

were calculated for each amide nitrogen, with values outside the standard deviation of the said calculated values being excluded. Calculation of solution structure Three dimensional structures were calculated using NMR derived dihedral angle, distance and hydrogen bond restraints. The torsion angle dynamics protocol of CYANA version 3.0(50 ) was used. The program calculated 50 low energy conformers with no distance violations > 0.2Å or dihedral angle violations > 5◦ from 250 random conformers subjected to 20000 annealing steps. An ensemble of 50 low energy conformers were subjected to structural refinement with water using CNS(51 ). The refinement was carried out with relaxation of upper and lower distance bounds by 10%. The lowest energy conformer was chosen as the representative conformer. Structures were validated using the PSVS server(52 ). All structures were analyzed using MOLMOL(53 ) or PyMOL (54 ). Hydrogen bond and ring angle calculations were carried out using algorithms provided in MOLMOL and PyMOL respectively. Ring centroid distances were calculated using the Protein Interaction Calculator

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Biochemistry

(55 ). Calculation of accessible surface area. Accessible surface area was calculated using the program naccess_2.1.1(56 ), with probe size 1.4Å. The accessible surface areas of the mutant proteins were compared with those of wild - type holo - and apo - cytb5.

Results and discussion Expression and purification of cytb5 mutants FF - cytb5 and WF - cytb5 are expressed to high levels and the yields of purified proteins are in the range of 40 - 50 mg / L. Mass spectral analysis indicates that the mutational changes have been introduced with high fidelity (Figures S - 2A, B SI). Biophysical data indicates structural changes ANS is known to have a high affinity to the molten globule state(35 ), which allows greater access to the protein hydrophobic core. The values of the fluorescence emission (at 475nm) of ANS upon binding to apo - cytb5 and FF - and WF - mutants are shown in Figure - 1. The data shows that the mutant proteins have a lower surface hydrophobicity despite the introduction of aromatic residues. The chemical denaturation data showing the variation in fluorescence and fraction of unfolded protein (Fu ) with increasing urea concentration are shown in Figure - 2. All three proteins show reversible unfolding in urea. Apo cytb5 and WF - cytb5 show transition curves consistent with multiple unfolding transitions, while the transition curve for FF - cytb5 may be fit reasonably to a two-state transition model. No attempt was made to calculate ∆G values for the unfolding reaction, due to the occurrence of multiple transitions. The calculated Cm values show that higher urea concentrations, 2.50M and 2.85M respectively, are required to unfold WF - and FF - cytb5 as compared to a urea concentration of 2.25M that 11


Biochemistry

70

Fluorescence upon binding ANS (AU)

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60

55.6±0.6 48.6±0.2

47.7±0.2

FF - cytb5

WF - cytb5

50

40

30

20

10

0

Apo - cytb5

Figure 1: Comparison of fluorescence emission of ANS (λem 425 nm ) when bound to apo - cytb5, FF - cytb5 and WF - cytb5. It is seen that ANS binds better to apo - cytb5, indicating a greater number of exposed hydrophobic residues.

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100

110

200

(B)

(A) 100

190

90

90

(C)

180 170

80

160

80 70

70

150 140

60

60 apo - cytb5

50

0

1

2

3

4

5

6

50

130 120

FF - cytb5 0

1

2

3

4

5

6

WFcytb5 0

7

1

2

3

4

5

6

7

Urea Concentration (M)

Fraction of unfolded protein (Fu)

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Biochemistry

Fluorescence intensity (AU)

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1.2 (D)

1.0 (E)

1.0 (F)

0.8

0.8

1.0 0.8 0.6

0.6

0.6 Cm= 2.25M

0.4

Cm= 2.85M

0.4

0.0

apo - cytb5 0.0

1.0

2.0

3.0

4.0

5.0

Cm= 2.5M

0.2

0.2

0.2

0.4

0.0

FF - cytb5 0.0 1.0 2.0 3.0 4.0 5.0 6.0

0.0

WFcytb5 0.0 1.0 2.0 3.0 4.0 5.0 6.0

Urea Concentration (M)

Figure 2: Chemical denaturation with Urea and calculation of Cm . (A) (B) and (C) show the variation in fluorescence intensity with increasing urea concentration for Apo -, FF - and WF - cytb5 respectively. Apo - and WF - cytb5 are seen to show multiple transitions upon unfolding while FF - cytb5 shows a curve indicative of a single transition. (D) (E) and (F) show the fraction of unfolded protein (Fu ), calculated from the fluorescence intensity data, plotted against increasing urea concentration. The Cm was calculated, and is seen to be 2.25M, 2.85M and 2.5M for Apo -, FF - and WF - cytb5 respectively.

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is required to unfold apo - cytb5. The Cm values agree with the values calculated from the preliminary data as well (Figure S - 3, SI). The increase in Cm indicates greater stability of the folded forms of WF - and FF - cytb5 proteins. These data corroborate the conclusions drawn from the ANS binding, that the structural changes are stabilizing in nature. Solution structures of WF - and FF - cytb5 proteins The one-dimensional proton NMR spectra of apo-cytb5, WF- and FF-cytb5 (Figure - 3) show characteristic resonance lines at 11.0 and -1.3 ppm. These resonance lines are observed in the NMR spectra of all cytochrome b 5 variants,(25 , 29 , 30 , 32 , 57 , 58 ) irrespective of the oxidation state or species variation. Thus, structural changes have accrued on the apo-cytb5 molten globule scaffold. Sequence-specific assignments Two - dimensional spectra of WF - cytb5 and FF - cytb5 also show well-dispersed and resolved peaks in the amide region (7-10ppm), indicating that both mutants are folded. The sequence specifically assigned 1 H -

15

N HSQC spectra of WF - and FF - cytb5 are shown in

Figures - 4 and S - 4 (SI). Sequence specific assignments for residues at the sites of mutation (Leu40 - Trp43 and Asp64 - Phe67 ) are shown in Figure - 5. The assignments were obtained through analysis of triple resonance data sets that correlate backbone and side - chain proton (Hβ ), carbon(Cβ ) and nitrogen nuclei. Side-chain aliphatic and aromatic proton and carbon assignments were obtained through analysis of C(CO)NH and H(CCO)NH - TOCSY and homo- and hetero-nuclear NMR experiments. In all 92.6% of HN, 92.6% of N, 98% of

13

Cα ,

94.6% of 13 Cβ and 98% of 1 Hα atoms were assigned. Analysis of similar spectra in the case of FF - cytb5 yielded 88.4% HN, 88.4% N, 96 % of 13 Cα , 93.5% of 13 Cβ and 90% of 1 Hα assignments for backbone and side chain nuclei. Peaks corresponding to multiple conformers of the protein observed in the HSQC were traced in the triple resonance backbone experiments (Figure S - 5, SI). Unambiguous assignments have been obtained for the major conformers of WF - and FF - cytb5. On the other hand it has been difficult to obtain resonance assignments for several of the minor peaks due to their low population and/or because of spectral 14


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A)

12

10

8

6

4

2

0

-2

ppm

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8

6

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2

0

-2

ppm

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-2

ppm

B)

12

C)

12

Figure 3: One-dimensional proton NMR spectrum of 15 N-labeled apo-cytb5 (A), WF-cytb5 (B) and 15 N labeled FF-cytb5 (C). The resolved downfield and upfield shifted lines at 11.0 and -1.3 ppm are assigned to D86 HN and I80 Hδ1 methyl protons respectively.These resonance are observed in the NMR spectra of all cytochrome b 5 variants. The presence of these resonances in the spectra of these three proteins indicates that structural changes are built on the apo - cytb5 scaffold.

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Biochemistry

46G 146G 56G

55G 156G

89S

12T 82E

80I

110

81G

122S

8V 27V 22S 95S 37T 97T 40L 31H 4S 14E 87D 11Y

86D 13L

66G 45G 59T

24S 115

78Y 32K 91I 42E 41E 49V

120

88R

-,-,-,-

79I

83L 25T 30H

34Y

29L

125

7D 60E 54A 9K 36L 26W 92A 109K

Nitrogen Chemical Shift (ppm)

(A)

130 98L

84H

11.0

10.5

10.0

9.5

9.0 8.5 8.0 Proton Chemical Shift (ppm)

,

(B)

7.5

77K 61N

65V

75S 57D 10Y

67F 19H

21D

35D 16I 50L 62F 23K 33V 93K 53Q 43W 52E 20K 51R 64D 5D 71A 28I 70D 96E 108V

119 15E 197T

110Y 90K

121 39F 122

6K 123 124

58A 8.6

120

38K

158A

8.8

6.5

76K

72R

11Y

7.0

Nitrogen Chemical Shift (ppm)

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

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125

8.4 8.2 8.0 Proton Chemical Shift (ppm)

7.8

Figure 4: (A)1 H - 15 N HSQC showing the complete sequence specific assignment of the for the major conformer of WF - cytb5. Also observed in the spectrum are the peaks corresponding to a minor conformation (numbered as 100 + n, where n is residue number in the major conformation). Inset (B) shows assigned peaks in the centre of the spectrum.

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8.27

7.95

F1

Gly66

Phe67 F3 = 119.70 ppm

8.23

6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 F2 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78

Val65

F3 = 110.82 ppm

7.28

Asp64

F3 = 119.28 ppm

7.50

Trp43 F3 = 122.05 ppm

8.53

Glu42 F3 = 117.30ppm

F2

6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78

Glu41 F3 = 117.77 ppm

Leu40 F3 = 117.15 ppm

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Biochemistry

F3 = 122.51 ppm

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8.36

7.95

F1 F1 = Proton Chemical Shift (ppm) F2 = Carbon Chemical Shift (ppm) F3 = Nitrogen Chemical Shift (ppm)

Figure 5: Overlay of the strip plots from the HNCACB(Cα in green, Cβ in pink) and CBCACONH (in blue) experiments for residues Leu40 - Trp43 and Asp64 - Phe67 . The sequential correlations are connected by arrows.

17


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

crowding and overlap. Given the limited data for the minor conformers, the structural data derived for WF- and FF - cytb5 are relevant for the major conformers observed in solution. Secondary structure The secondary structure of the WF - and FF - cytb5 proteins were calculated from the 1 Hα , 13

Cα ,

13

Cβ and

13

C′ secondary chemical shifts using the CSI protocol, backbone 3 J H N H α

coupling constants, short and medium-range NOEs assigned in the 2D 1 H - 1 H NOESY and 3D

15

N edited NOESY NMR data. These data are summarized in Figures - 6, 7, 8 and

Table - 1.

The secondary structure elements in FF - cytb5 are seen to be identical to

Table 1: Secondary structures of WF -, FF -, Apo - and Holo - cytb5 as a function of sequence. Protein residue numbers Secondary structure element WF-cytb5 FF-cytb5 Holo-cytb5 Apo-cytb5 PDB ID This study This study 1cyo.pdb 1I87.pdb α-helices α1* Ser4 -Asp7 α1 Leu13 -Lys18 Leu13 -Lys18 Leu13 -Lys18 Leu13 -Ile16 36 42 38 42 36 42 α2 Leu -Glu Leu -Glu Leu -Glu Thr37 -Leu40 α3 Glu47 -Glu52 Glu47 -Glu52 Glu47 -Glu52 α4 Glu60 -G66 Glu60 -Gly66 Glu60 -G66 69 77 69 77 α5 Thr -Lys Thr -Lys Thr69 -Lys77 α6 Asp86 -Ile91 Asp86 -Ile91 Asp86 -Ile91 Total no. of helices 7 6 6 2 β-strands β1 Lys9 -Tyr11 Lys9 -Tyr11 Lys9 -Tyr11 β2 Trp26 -Leu29 Trp26 -Leu29 Trp26 -Leu29 Trp26 -Ile28 β3 Lys32 -Asp35 Lys32 -Asp35 Lys32 -Asp35 Lys32 -Asp35 β4 Gly55 -Ala58 80 83 80 83 β5 Ile -Leu Ile -Leu Ile80 -Leu83 Gly81 -Leu83 Total no. of β-strands 4 4 5 4 % of residues in 54.1% 52.1% 64.9% 25.5% secondary structure WF - cytb5, except for the absence of the α1* - helix. This is in contrast to the absence of secondary structure elements in apocytb5. It should also be noted that the secondary structure distribution in FF -cytb5 and WF -cytb5 show resemblance to that in holo -cytb5 18


Page 18 of 43

0

-1

F)

0

1

CSI

E)

5

10

-1.3

7.5

D)

0

Δδ(1Hα)

-8

1.3

-1.5

8

1.5 Δδ(13Cβ) 0

-1.5 -6

1.5 Δδ(13Cα) 0

6

C)

dαN(i,i+2)

dNN(i,i+2)

dαN(i,i+3) dαN(i,i+4)

dαN dNN dβN

Kex

A) B)

3JHNH α

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

10 20 30 40 50 60 70 80 90 AEQSDKDVKYY T L EE I QKHKDSKS TWV I L HHKVYD L T K F L EEWPGGEEV LREQAGGDA T ENF EDVGF S TDARE L SKKY I I GE L HPDDRSK I AKPSE T L

Page 19 of 43

Figure 6: Secondary structure assignment from NMR data of WF - cytb5. (A) H/D exchange data, where filled circles identify the residues whose backbone amide protons are protected for 10 hours or longer. (B) Short and medium range NOE connectivities plotted against residue number. The short range sequential NOE connectivities d αN , d β N and d N N NOEs are represented as bars with thicknesses varying with NOE intensity. The medium range NOEs (d αN (i,i+3), d αN (i,i+4), d N N (i,i+2), d αN (i,i+2)) are shown as lines connecting the two residues. (C) Secondary chemical shift values of ∆δ 13 Cα , ∆δ 13 Cβ and ∆δ 1 Hα , plotted against residue number. (D) Residue wise 3 J H N H α coupling constant values obtained from the 3D HNHA experiment. (E) Chemical shift index (CSI) plot for WF - cytb5 as a function of sequence. Regions of the protein that have α-helix, β-strand or are in random coil configuration loops are designated numerically as -1, 1 and 0 respectively. (F) Assigned secondary structure based on NMR data shown above. 19 ACS Paragon Plus Environment

D)

E)

CSI

0

1 0 -1

5

10

C)

7.5

0 -1.4

Δδ(1Hα)

Δδ(13C’)

-8 6 1.5 0 -1.5 -6 5 1.5 0 -1.5 -5 1.4

Δδ(13Cβ)

Δδ(13Cα)

8

1.5 0 -1.5

B)

dαN(i,i+2)

dαN(i,i+3) dαN(i,i+4) dNN(i,i+2)

90 80 70 60 50 40 30 20 10

dαN dNN dβN

α

A)

3JHNH

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 43

A EQS D K D V K Y Y T L E E I QK H K D S K S TWV I L HH K V Y D L T K F L E E F PGGE E V LR EQAGGD A T E N F E D VG F S T D A R E L S K K Y I I GE L H P DDR S K I A K P S E T L

Biochemistry

Figure 7: Secondary structure assignment from NMR data of FF - cytb5. (A) Short and medium range NOE connectivities plotted against residue number. The short range sequential NOE connectivities d αN , d β N and d N N NOEs are represented as bars with thicknesses varying with NOE intensity. The medium range NOEs (d αN (i,i+3), d αN (i,i+4), d N N (i,i+2), d αN (i,i+2)) are shown as lines connecting the two residues. (B) Secondary chemical shift values of ∆δ 13 Cα , ∆δ 13 Cβ and ∆δ 1 Hα , plotted against residue number. (C) Residue wise 3 J H N H αcoupling constant values obtained from the 3D HNHA experiment. (D) Chemical shift index (CSI) plot for FF - cytb5 as a function of sequence. Regions of the protein that have α-helix, β-strand or are in random coil configuration loops are designated numerically as -1, 1 and 0 respectively. (E) Assigned secondary structure based on NMR data shown above. 20


α-6 β-5 α-5 α-4 β-4 α-3 α-2 β-3 β-2 α-1 β-1

Holo - cytb5

Apo - cytb5

FF - cytb5

WF - cytb5

α-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

Biochemistry

10 20 30 40 50 H H 60 70 80 90 A E Q S D K D V K Y Y T L E E I Q K H K D S K S TWV I L H H K V Y D L T K F L E EWP GG E E V LR E Q A GGD A T E N F E D V G F S T D A R E L S K K Y I I G E L H P D D R S K I A K P S E T L F

Page 21 of 43

Figure 8: Comparison of secondary structures of WF -, FF -, apo - and holo - cytb5’s. Regions of the proteins that differ in secondary structure from the apo - cytb5 are marked in boxes (red broken lines). The areas in blue dashed boxes show the differences between WF - cytb5 and FF - cytb5.

21


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

(27 , 28 ). 15

N backbone dynamics

The T1 , T2 and heteronuclear NOE values were measured for WF - cytb5 (Figures S - 6A and B, SI) and for FF - cytb5 (data not shown). From the average values of T1 and T2 and using equation 1, the rotational correlation times for WF - and FF - cytb5 were calculated to be 8.1 ns and 10.2 ns respectively. These values are lower than that calculated for apo cytb5 (11.5 ns) using previously published data (59 ), indicating that WF - and FF - cytb5 are monomeric in solution. Structural restraints NOE restraints 2D - 1 H - 1 H NOESY,

13

C and

15

N - edited NOESY-HSQC spectra were manually assigned

to identify through space correlations between pairs of dipolar coupled protons. NOEs‘ that define the β - sheet topology are indicated shown in Figure - 9. This β - sheet sheet topology closely resembles the sheet topology in holo - cytb5. A total of 1282 and 1163 unambiguous NOE restraints were assigned to WF - cytb5 and FF - cytb5 respectively. Dihedral angle restraints A total of 34 and 44 3 J H N H α coupling constants were determined for WF - and FF - cytb5 from the 3D HNHA data, as for many of the residues the HA cross-peaks were weak or absent. The backbone dihedral angles for these residues were predicted using the DANGLE subroutine in CCPNmr. In all 177 and 179 backbone dihedral (φ, ψ) angles were used as input for structure calculation. Hydrogen bonds Hydrogen - deuterium exchange studies were performed on a sample of WF - cytb5 that was lyophillized in the presence of the protein stabilizer trehalose. In the absence of trehalose the protein was not stable to lyophillization. The protein FF - cytb5 was unstable to

22


Page 22 of 43

8.90 8.89 9.53

N 27H

F3 = 121.41

8.54 H O

N H

Asp35

H

H

N H O

H H

Ile80

N

Lys9

O

H

O

H

H

N

N

Gly81

N

Trp26

O

H

Tyr34

O H H

O

H O H

H

Tyr10 O

N

N

A)

H

N H

Glu82 H

H

O

H

Tyr11

O

Val33

N

H

O

H

H N

Leu83

O

O

H

N

N

Lys32

H

H

Val27

N

Ile28

O

H

F2 (ppm) N

12

11

10

9

8

7

6

5

4

3

2

1

-1

0

F3 = 125.73 H

F1 = Proton Chemical Shift (ppm) F2 = Proton Chemical Shift (ppm) F3 = Nitrogen Chemical Shift (ppm)

F3 = 114.82

α 81H

8.95

α 35H

8.58

α 82H

F3 = 125.70

B)

F1(ppm)

N 9H

7.49

N 35H

N 33H

α 34H α 28H

F3 = 122.66 -2

9.11

N 11H

α 10H

N 33H

α 32H

F3 = 110.20

Val33

F3 = 118.26

Lys9 Val27

F3 = 122.71

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

Tyr34 Ile80 Gly81 Glu82 Leu83

Page 23 of 43

Figure 9: The topology of the β - sheet in WF - cytb5. (A) A schematic of the five stranded β - sheet that is made up of two parallel strands and three anti - parallel β - strands. The dashed lines in magenta and blue indicate the observed HN - HN and Hα - HN NOEs that define the strand registry. Dashed parallel lines indicate inter - strand hydrogen bonds that stabilize the β - sheet. (B) Strip - plots from the 3D NOESY - HSQC spectrum that show the key NOE correlations, on the basis of which the β - sheet topology has been constructed.

23


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

lyophillization even in the presence of trehalose. Forty-eight of the 98 residues in WF-cytb5 were protected against H/D exchange after 12 hours (cf. Figure - 6). These nuclei lie in structured regions of the molecule and hence must be involved in hydrogen bond interactions (60 ). Tertiary structures of WF- and FF-cytb5 The three-dimensional structures of WF - cytb5 and FF - cytb5 were calculated using experimentally determined NOE, backbone dihedral angle and hydrogen bond restraints. The number of each type of restraint along with the structural statistics obtained for the final ensemble of structure is given in Table - 2.

24


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Page 25 of 43

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Biochemistry

Table 2: NMR restraints and structural refinement statistics Restraints & statistics NOE - based restraints Intra - residue ( |i-j| ) = 0 Sequential ( |i-j| ) = 1 Medium - range (1 5◦ Distance violations > 0.1Å van der Waals violations Ramachandran map statistics (Procheck %) Most favored regions Additionally allowed regions Generously allowed regions Total allowed regions Disallowed regions rmsd from mean structure coordinate (Å) Backbone (N,Cα , C’) residues - 6 to 92 Average heavy atom

WF - cytb5

FF - cytb5

252 335 316 379 13 1282 40 177 1499 15 200 20

236 309 244 374 12 1163 40 179 1382 14 200 20

0 0 0

0 0 0

77.90 20.80 1.30 100 0

83.60 14.80 1.60 100 0

0.58 ± 0.09 1.19 ± 0.11

1.01 ± 0.21 1.59 ± 0.21

A backbone superposition of the 20 lowest energy structures for WF - cytb5 (panel A) and FF - cytb5 (panel B) are shown in Figure - 10. Ribbon representation of the lowest energy conformer for the respective proteins are shown in panels C and D. The rmsd values for the ensemble of structures, calculated by superposing the structures on the N, Cα , and C’ backbone atoms of residues Lys6 - Ala92 , are 0.58 ± 0.09 Å and 1.01 ± 0.21 Å for WF - cytb5 and FF - cytb5 respectively. A high percentage of backbone dihedral angles are found to be in the allowed ( 97%) and generously allowed regions ( 2%) of the Ramachandran map(61 ). The high number of restraints (Table - 2), low rmsd and the absence of backbone dihedral 25


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

(A)

(B)

(C)

(D)

(E)

Figure 10: Tertiary structures WF - cytb5 and FF - cytb5. Panels (A) and (B) show the supersposition of the ensemble of the 20 lowest energy structures for WF - and FF cytb5 respectively. The structures have been superposed on backbone N, Cα and C’ atoms of residues 6 - 92. Panels (C) and (D) show the representative lowest energy conformers for WF - cytb5 and FF - cytb5 in ribbon representation. Panel (E) shows the superposition of the lowest energy structures of WF - cytb5 (green) and FF - cytb5 (red).

26


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Biochemistry

angle violations indicate that the ensembles of structures are of good stereochemical quality. The tertiary structure of WF-cytb5 shows a 4-stranded mixed β-sheet formed from the β1-β4 strands mentioned above and a four-helix bundle, comprising of the helices α3-α6 (Table - 1) resting on top of the βsheet. FF-cytb5 also shows the same topology with the 4-helix bundle resting on the mixed 4-strand β-sheet. The structures of both mutants align well (Figure 10E), with an rms deviation of 0.8Å when the lowest energy structures are superposed on backbone N, Cα , and C’ atoms . Differences are observed in the alignment of the E42 -E47 loop which is probably due to the steric requirements of the larger tryptophan aromatic group.

27


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

Comparison of structures of apo-, WF- and FF- and holo-cytb5’s Apo-cytb5 is largely a disordered molten globule when compared with the holo form of the protein. The residual structure consists only of the α1 and α2 helices and 3 short β-strands, i.e., β1, β2 and β5 that form an anti-parallel β-sheet. Introduction of an aromatic interaction also triggers a hydrophobic collapse that results in significant conformational changes of residues that were previously disordered in the apoprotein as listed in Table - 1. The conformational changes are manifest in the form of four α-helices and one β-strand, that result in the formation of the four helix bundle. Furthermore, an extension of the beta-sheet at the base through the addition of a parallel β-strand, i.e., β1 is seen, along with the lengthening of the 3 existing β-strands (β2, β3 and β5). There is measurable transition from order to disorder upon introduction of amino acid based aromatic interactions in the apo-protein. These changes are similar to the ones observed upon heme sequestration.WF- and FF-cytb5 differ from holo-cytb5 in the absence of the β4 strand, the residues of which are conserved in all cytochrome b 5 ’s. In holo - cytb5, Ala58 (in β4) is part of the cluster of hydrophobic / aliphatic residues that line the heme-binding pocket. In WF - and FF - cytb5, while Phe and Trp participate in strong aromatic interactions that result in formation of secondary structure elements similar to that induced by the heme, it is clear that the combined hydrophobic surface area of Phe and Trp are insufficient to identically replicate the effects of heme. WF- and FF-cytb5 have 28 and 19 additional hydrogen bonds in comparison with apo-cytb5 that contribute to overall stability, as seen in the increased Cm values. The total accessible surface area (ASA) of WF-cytb5 (5996.1 Å2 ) and FF-cytb5 (6842.5 Å2 ) are significantly lower than that of apo-cytb5 (7814.3 Å2 ). The ASA values for the residues in the original heme-binding site are provided for reference (Figure S-7, SI). The lowered ASA values of these residues in the mutants compared to apo-cytb5 are further evidence of the folding induced by aromatic interactions. Aromatic interactions The offset - stacked interaction between Trp43 and Phe67 results in the shielding of the 28


Page 28 of 43

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Biochemistry

Trp43 Hζ and Phe67 Hβ methylene protons, both of which show upfield shifts Table S - 2 A and B, SI. This is consistent with previously studied interactions (16 ). The presence of an aromatic interaction is confirmed by the presence of NOE correlations between Trp43 /Phe43 and Phe67 (Figure - 11). The calculated structures show that the centroids of two aromatic rings are at a distance of 5.0 ± 0.4 Å and 4.9 ± 0.1 Åfor WF cytb5 and FF - cytb5 respectively. These distances are well within the upper - limit of the 7 Å distance defined for aromatic interactions. However the orientation of the rings is seen to be different for the two aromatic pairs. The two Phe rings in FF - cytb5 are in an edgeto-face orientation, while the Phe - Trp pair in WF - cytb5 is seen to be in an offset-stacked orientation (Figure - 12). Both orientations indicate an energetically favourable interaction. In all the conformers the distance between the Phe43 Cα and Phe67 Cα is 8.8 ± 1.1Å, which is predicted to favour the T - shaped structure seen here(15 ). A majority of aromatic interactions are known to occur between buried residues, where a buried residue is defined as one whose accessible surface area(ASA) is < 20%. Surface accessibility calculations on WF - cytb5 and FF - cytb5 show that the aromatic residues that were introduced have a higher residue ASA, which is consistent with the surface exposed location of His43 and His67 in apocytb5. In the case of WF - cytb5 we see a relative accessible surface area of 42% for both Trp and Phe. In FF - cytb5 Phe43 is buried (RSA 7%), while Phe67 is exposed (RSA 37%). The aromatic interaction occurs despite the exposed nature of these residues, a phenomenon previously observed in small peptides(16 , 17 ). Analysis of protein structures has shown that aromatic interactions extended beyond the canonical “dimer” interaction, i.e., trimer, tetramer etc(8 ). The introduction of these aromatic residues is also seen to lead to the formation of aromatic networks in the mutants (Figure - 13, Table 3).

Wild-type cytb5 has a total of 7 aromatic residues: Tyr10 , Tyr11 ,

Trp26 , Tyr34 , Phe39 , Phe62 and Tyr78 . In holo-cytb5 two aromatic interactions are seen (Tyr11 - Trp26 , Phe39 - Tyr78 ) in addition to the interactions between heme and aromatic residues. The Tyr11 - Trp26 interaction is conserved in the apo - cytb5, whereas the Phe39

29


Biochemistry

(A)

Trp43

Hε1 Hε3 2.9 5.3

HN

3.6 5.6

Hδ1

Hε1 Hζ

Phe67

Hβ3

6.0

HN Trp43Hε3 Phe67Hβ3

2

Phe43He* Phe67Hζ

Trp43Hε3 Phe67HN

(B)

*

3 4 3

4 A

4 5 5

P

5 6 6

Proton Chemical Shift (ppm)

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 30 of 43

*

6

p

7 e *

7

8 d

e

7

e G

(H

8

7

7

Proton Chemical Shift (ppm)

Figure 11: Orientation of the aromatic rings in WF - cytb5 and their interactions. (A) Offset - stacked orientation of the rings. The red dashed lines connect pairs of protons for which NOE correlations are observed and the corresponding distances in the calculated structures. The ideal offset - stacked ring orientation is shown schematically to the right. (B) Stripplots showing selected NOE correlations between the aromatic rings in WF - cytb5 (left and center) and FF - cytb5 (right).

30


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Biochemistry

(A)

(B) Phe43

Trp43

Phe67

Phe67

(C)

(D)

Trp43

Phe43

Phe67

Phe67

Figure 12: Orientation of the aromatic rings in WF - cytb5 and FF - cytb5. (A and B) orientation of the rings in the ensemble of 20 lowest energy NMR structures of WF - cytb5 and FF - cytb5 respectively. (C and D) Orientation of the aromatic rings in the lowest energy structures of WF - cytb5 and FF - cytb5. It can be seen that in WF - cytb5 the rings are in an offset stacked interaction, while in FF - cytb5, they are in a T-shaped edge-to-face interaction.

31


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 ACS Paragon Plus Environment

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Biochemistry

Table 3: Distance and angular data that define aromatic interactions. Protein

Residue 1

Residue 2

Ring angle (◦ )

Centroid distance Å

Holo - cytb5

a

Tyr11 Phe39

Trp26 Tyr78

80.0 40.0

5.0 4.8

Apo - cytb5

b

Tyr11 Phe39

Trp26 Tyr78

54.0 63.0

5.2 7.1

FF - cytb5

Tyr11 Phe39 Phe39 Phe39 Phe39 Phe43

Trp26 Trp43 Phe62 Phe67 Tyr78 Phe67

55.0 23.0 115.0 137.0 22.0 65.0

4.9 5.1 4.7 6.7 5.6 4.9

± ± ± ± ± ±

0.2 0.1 0.9 0.3 0.2 0.1

WF - cytb5

Tyr11 Phe39 Phe39 Trp43

Trp26 Phe67 Tyr78 Phe67

70.0 58.0 57.0 22.0

5.1 4.4 5.5 5.0

± ± ± ±

0.2 0.5 0.5 0.4

a

Values for Holo - cytb5 were calculated using the crystal structure ( PDB ID - 1CYO.pdb) Values for Apo - cytb5 were calculated from the lowest energy solution NMR structure (PDB ID 1I87.pdb). b

33


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

- Tyr78 interaction now has a centroid separation of ∼ 7 Å, making it extremely weak or non-existent. The Tyr11 - Trp26 interaction is seen to be conserved in both mutants, and is strongly corroborated by the abundance of NOEs between the two residues. In FF - cytb5 we see a “symmetric trimer” (8 ) interaction between Phe39 - Phe43 - Phe67 . While the Phe43 - Phe67 and Phe39 -Phe67 pairs are in edge - to - face interactions, the Phe39 - Phe43 pair is offset - stacked. Phe39 also interacts with Phe62 and Tyr78 in edge - to - face and offset - stacked orientations respectively, completing the network. The interaction of Phe39 with Phe62 is long -range (centroid distance of ∼ 7 Å) and probably very weak. Tyr10 and Tyr34 are not within range of any aromatic interaction. The offset-stacked arrangement of Trp43 and Phe67 in WF - cytb5 leads to the formation of a "ladder trimer" ,instead of a symmetric trimer, with Phe39 . Thus Phe39 interacts only with Phe67 in an edge - to - face interaction. The bulkier Trp43 residue forces the Phe62 out of interaction range, resulting in Phe39 interacting only with Tyr78 , also in an edge-to-face orientation. Therefore Tyr10 , Tyr34 and Phe62 are not seen to participate in the aromatic network.

Outline Aromatic interactions have been widely studied for their role in the folding and structural stability of proteins. We have presented here a study on the effects of aromatic interactions in proteins using apo - cytochrome b 5 as a model system. The structural effects of introducing pairs of aromatic residues, in the proteins WF - cytb5 and FF - cytb5, have been studied using solution NMR spectroscopy and other biophysical methods. The propensity for paired interactions between aromatic amino acids has been structurally verified in the two proteins. High resolution structural data convincingly show that the aromatic interactions nucleate long-range secondary and tertiary structure well beyond the mutational site of interaction. Structural effects manifest as a distinct change from disorder to order, in which non-canonical “trimer” aromatic interactions are also witnessed. Large structural changes are phenomena that cannot generally be observed in studies that involve designed peptides of small size. 34


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Biochemistry

The aromatic interactions increase the overall stability and decrease the hydrodynamic radii of the mutant proteins. Evidence for the this comes from chemical denaturation studies and from

15

N - NMR relaxometry. These observations could in principle provide insights into

the incorporation of aromatic interactions into protein design and furthermore, to provide insight into intermolecular interactions, that are driven by aromatic interactions.

Acknowledgement The authors thank the Department of Science and Technology and the Department of Biotechnology, Government of India for the NMR and Mass Spectrometric Facilities at the Indian Institute of Science. Swati Balakrishnan is the recipient of the Council for Scientific and Industrial Research (Government of India) Senior Research Fellowship.

Supporting Information Available The following file(s) are available free of charge. • Supp1: 7 figures and 2 tables This material is available free of charge via the Internet at http://pubs.acs.org/.

35


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Graphical TOC Entry H43 H67

H43W,H67F

H43F,H67F

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