Rapid Report pubs.acs.org/biochemistry
Incorporation of an Unnatural Amino Acid as a Domain-Specific Fluorescence Probe in a Two-Domain Protein Lena K. Ries,† Franz X. Schmid,* and Philipp A. M. Schmidpeter*,‡ Laboratorium für Biochemie und Bayreuther Zentrum für Molekulare Biowissenschaften, Universität Bayreuth, Universitätsstraße 30, D-95447 Bayreuth, Germany S Supporting Information *
absorbance of Tyr and Trp is minimal, and it shows maximal fluorescence at 297 nm9 that can be used as a fluorescence resonance energy transfer (FRET) donor for Trp and to some extent for Tyr. Intramolecular FRET from pCNF to Trp or Tyr has previously been used to monitor structural changes in small, single-domain proteins10−12 as well as in binding studies.13 Here we used a fragment of the chicken signal adapter protein c-CrkII,14,15 which consists of two SH3 domains (SH3N−SH3C), and employed the UAA technology to introduce pCNF into the SH3N domain at position 142. Then we employed its fluorescence as a selective probe for the folding of this domain. The natural residue Phe142 is partially accessible to solvent, which provides space for the extra cyano group of pCNF. Position 142 is close to the two Trp residues in SH3N, which might give rise to FRET between pCNF and these Trp residues (Figure 1A). Using standard experiments such as
ABSTRACT: The biophysical analysis of multidomain proteins often is difficult because of overlapping signals from the individual domains. Previously, the fluorescent unnatural amino acid p-cyanophenylalanine has been used to study the folding of small single-domain proteins. Here we extend its use to a two-domain protein to selectively analyze the folding of a specific domain within a multidomain protein.
T
he analysis of the stability and folding of a protein is a prerequisite for understanding its function.1 In most cases, the intrinsic spectral properties of proteins (e.g., absorbance, fluorescence, or circular dichroism) are appropriate tools for analyzing their stabilities and their folding mechanisms.2 However, with increasing complexity of the system under investigation, these techniques reach their limitations. In oligodomain proteins, it is only rarely possible to monitor specific domains by intrinsic spectral probes, because they are usually distributed over several domains. In some cases, individual domains are stable and can be studied in isolation. However, domain boundaries are difficult to identify, and their choice is often critical for the stability and function of isolated domains.3 Moreover, following this approach, functionally important domain interactions get lost. To generate site-specific reporters, traditionally cysteines are introduced at defined positions, labeled with a chromophore and then used, e.g., for fluorescence experiments. These chromophores often are large molecules with a high risk of perturbing the structure and stability of the protein or of interfering with critical interactions. These inherent problems can be overcome by the use of unnatural amino acids (UAAs) that closely resemble their natural homologues. Incorporation of UAAs using Ambercodon suppression4 allows scientists to generate proteins with spectral or chemical properties tailored to answer specific questions. The allosteric regulation of ion channels,5 photocross-linking studies on G protein-coupled receptors,6 and chemical ligation in vivo7 are only a few examples of the use of UAAs. The UAA technology can also be employed to produce proteins with novel spectral characteristics for the biophysical analysis of proteins. p-Cyanophenylalanine (pCNF)8 is very well suited as unnatural fluorophore because it resembles the natural chromophores Phe and Tyr in size, but not in its spectral properties. pCNF can be excited at 240 nm where the © XXXX American Chemical Society
Figure 1. Incorporation of pCNF. (A) Backbone representation of SH3N−SH3C (Protein Data Bank entry 2L3S).16 The position of pCNF incorporation is shown as red sticks and spheres, and Trp residues are colored green. The graphic was prepared using Pymol.17 (B) Urea-induced unfolding transitions in 0.1 M potassium phosphate (pH 7.4) at 15 °C (excitation at 280 nm, emission at 330 nm). (C) Thermally induced unfolding under equilibrium conditions monitored by the change in the CD signal at 222 nm. Solid lines represent fits according to a two-state model (Table 1). Received: September 5, 2016 Revised: November 18, 2016 Published: November 21, 2016 A
DOI: 10.1021/acs.biochem.6b00898 Biochemistry XXXX, XXX, XXX−XXX
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denaturant-induced unfolding in equilibrium and protein folding kinetics, we show that in SH3N−SH3C F142pCNF the fluorescence of pCNF can be used to selectively monitor the folding of the SH3N domain, which is possible in the wildtype protein only because the folding reactions of the two domains are kinetically separated. Although the UAA technology is becoming more and more popular, it is still challenging to produce sufficient amounts of UAA-containing protein for biophysical studies. Using sitedirected mutagenesis, we first introduced the Amber stop codon to replace the codon for Phe142 in SH3N−SH3C. Next, we optimized the conditions of pET vector-based protein expression in Escherichia coli BL21(DE3) pLysS cotransformed with pDule2 pCNF RS.8 A modified LB medium-based autoinduction protocol8 together with the SUMO fusion technology resulted in final yields of 1.2 ± 0.2 mg of UAAcontaining protein per liter of bacterial culture (Supplementary Methods and Supplementary Figure S1A). First, we monitored the overall urea-induced unfolding transition of the labeled protein by the intrinsic Trp fluorescence (excitation at 280 nm, emission at 330 nm) and the heat-induced transition by circular dichroism at 222 nm to examine whether the incorporation of pCNF changed the stability of the protein (Figure 1B,C). In isolation (and probably also in the two-domain protein), the two domains show rather similar stabilities in equilibrium transitions (Table 1). Both contain Trp and Tyr residues, and the relative
Figure 2. pCNF fluorescence selectively detects folding of SH3N. (A) Fluorescence spectra of native (0 M urea, solid line) and denatured (8 M urea, dashed line) SH3N−SH3C WT and the F142pCNF variant in 100 mM potassium phosphate (pH 7.4) at 15 °C after excitation at 240 nm. (B) Urea-induced unfolding under equilibrium conditions of the protein variants, with colors and conditions as in panel A. (C) Apparent rate constants (λ) of unfolding (empty symbols) and refolding (filled symbols) of the isolated SH3N and SH3C domains. (D) Chevron-type rate profile for SH3N−SH3C WT and the F142pCNF variant monitored by intrinsic protein fluorescence (excitation at 280 nm and emission at 330 nm) and of the F142pCNF variant selectively monitored by pCNF fluorescence [excitation at 240 nm and emission at 297 nm (red symbols)]. Also see Table 1 and Supplementary Tables S1 and S2.
Table 1. Parameters of Equilibrium Unfolding Transitionsa protein variant Nb
[urea]M (M)
ΔG15 °C (kJ mol−1)
SH3 SH3Cb SH3N−SH3C WTb F142pCNFb F142pCNFc protein variant
4.06 4.43 4.33 4.40 4.04 TM (°C)
18.1 19.9 18.6 18.6 12.9 ΔG15 °C (kJ mol−1)
SH3N−SH3C WT F142pCNF
58.3 58.2
12.9 14.2
m (kJ mol−1 M−1)
ΔHvH
4.46 4.49 4.30 4.23 3.19 (kJ mol−1)
overlap. The spectrum of the denatured protein (Figure 2A, red dashed line) clearly shows the characteristic pCNF signal at 297 nm,13 which provides a direct and reliable test of whether pCNF was incorporated. In the unfolded state, the fluorophore is presumably exposed to solvent and energy transfer from pCNF to Trp or Tyr is probably weak.10 In the native state of the F142pCNF variant (Figure 2A, red line), the fluorescence at 297 nm is completely abolished, but the Trp fluorescence at 330 nm is virtually the same as that observed for the SH3N− SH3C WT protein (without the unnatural fluorophore). These observations immediately lead to two suggestions. First, the pCNF fluorescence strongly differs between the unfolded and folded state of the protein and is thus very well suited to monitoring folding. Second, the Trp fluorescence in the native protein is almost unchanged. This indicates that the strong quenching of the pCNF fluorescence in the folded protein originates only to a small extent from energy transfer to Trp. Other factors are possibly differences in the solvent accessibility of pCNF and/or energy transfer to Tyr residues. Subsequently, we used the change in pCNF fluorescence (Figure 2A) to follow urea-induced unfolding. Figure 2B shows that the increase in pCNF fluorescence at 297 nm can be used as a sensitive probe to determine the stability of the protein without interfering signals from natural chromophores. Also, the small increase in Trp fluorescence after excitation at 240 nm (Figure 2A, black and red lines), which presumably originates from minor FRET from pCNF to Trp, can be used to measure the transition (Supplementary Table S1). Analysis of the pCNF fluorescence (excitation at 240 nm and emission at 297 nm) gives a midpoint of transition of 4.0 M
190 200
a
The midpoint of transition ([urea]M), the Gibbs free energy of unfolding (ΔG), and the cooperativity parameter (m) from the equilibrium unfolding curves (Figure 1B,C) are given according to a two-state model. bExcitation at 280 nm and emission at 330 nm. c Excitation at 240 nm and emission at 297 nm. The midpoint of transition (TM), the Gibbs free energy of unfolding (ΔG), and the van’t Hoff enthalpy (ΔHvH) from thermal unfolding are given. The parameters given for the two-domain proteins are apparent values only.
contributions of the individual domains to the fluorescence of the two-domain construct cannot be separated. This leads to the observation of an equilibrium unfolding transition with apparent two-state character. Both the SH3N−SH3C WT protein and the pCNF-labeled variant show apparent transition midpoints of [urea]M = 4.4 M and TM = 58 °C, which indicates that the overall stability of the two-domain protein and thus presumably also its structure were not altered by the incorporation of the UAA. Figure 2A shows the influence of the pCNF residue on the fluorescence of the protein. The first observation is that in the unlabeled protein Tyr shows a very low background fluorescence after excitation at 240 nm, which is important because the emission spectra of pCNF and Tyr show a certain B
DOI: 10.1021/acs.biochem.6b00898 Biochemistry XXXX, XXX, XXX−XXX
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Rapid Report
urea, compared to 4.4 M urea from the intrinsic fluorescence [excitation at 280 nm and emission at 330 nm (Table 1 and Supplementary Table S1)]. The value from selectively detecting pCNF is the same as for the isolated SH3N domain ([urea]M = 4.06 M) (Tabe 1 and Supplementary Figure S1B), while from the intrinsic protein fluorescence, the results agree with the two-domain protein SH3N−SH3C WT (Table 1). These results provide the first indications that pCNF, incorporated at a proper position, is a domain-specific fluorescence probe in the SH3N−SH3C two-domain protein. In equilibrium unfolding experiments, the SH3N−SH3C twodomain protein shows only one transition (Figures 1B and 2B). Therefore, quantitative interpretation of the results according to a two-state model is limited. However, the two domains, SH3N and SH3C, show different folding kinetics in isolation as well as in the two-domain protein18 and thus can be analyzed separately in kinetic folding experiments (Figure 2C). Figure 2C shows that SH3C (squares) displays unfolding and refolding rates 5−10-fold higher than those of SH3N (circles), depending on the urea concentration. An additional slow phase with a small amplitude is observed for both domains (triangles) and probably originates from prolyl isomerizations.18 To test whether pCNF emission provides a domain-specific probe, we followed the folding kinetics of SH3N−SH3C F142pCNF by the pCNF-specific fluorescence at 297 nm. In reference experiments, we also followed the change in the intrinsic protein fluorescence upon unfolding and refolding (Figure 2D). As for denaturation experiments in equilibrium, the measurements of the intrinsic protein fluorescence showed that the incorporation of pCNF also did not affect the folding kinetics of the domains (Figure 2D and Supplementary Table S2). The apparent rate constants λ of folding and unfolding of SH3N− SH3C WT and the F142pCNF variant show the same dependence on the urea concentration (Figure 2D, black and gray symbols). In these experiments, the fast folding SH3C domain can be resolved only after stopped-flow mixing, whereas the slow folding domain SH3N also can be monitored after manual mixing. The unfolding and refolding kinetics of the F142pCNF variant were also followed by the change in pCNF fluorescence at 297 nm after excitation at 240 nm (Figures 2D and 3). The apparent rate constants of unfolding and refolding (red symbols in Figure 2D) match with the rate constants for SH3N very well, while kinetics for SH3C could not be detected even after stopped-flow mixing (Figure 3A,B). The direct comparison of the unfolding (Figure 3A) and refolding (Figure 3B) reactions monitored by pCNF fluorescence (red traces) or by intrinsic protein fluorescence (black traces) shows that in the first few seconds the signal change recorded for pCNF gives kinetics slower than those for the intrinsic protein fluorescence because the fast unfolding and refolding reactions of the SH3C domain are not monitored. In all experiments, unfolding and refolding, the pCNF fluorescence could be analyzed using a single-exponential function, while for the intrinsic protein fluorescence, two exponential terms were necessary to analyze the traces. The apparent rate constants determined from the pCNF traces match those for the SH3N domain. The comparison between the folding rates measured by pCNF fluorescence (Figure 2D) and those obtained for the isolated domains (Figure 2C) shows that pCNF fluorescence reports selectively the folding kinetics of the SH3N domain and that the kinetics are not affected by the presence of the SH3C domain. This corroborates our results from the equilibrium transitions that pCNF at position 142 is a
Figure 3. Kinetics of unfolding and refolding. Kinetics of (A) unfolding in 7 M urea and (B) refolding in 0.7 M urea of 2 μM SH3N− SH3C F142pCNF [in 100 mM potassium phosphate (pH 7.4) at 15 °C] measured after stopped-flow mixing [excitation at 240 nm and emission at 300 nm (red lines)] compared to the same kinetics monitored by the intrinsic protein fluorescence [excitation at 280 nm and emission above 320 nm (black lines)]. (C) Initial (squares) and final (circles) values of the folding kinetics shown for SH3N−SH3C F142pCNF (see Figure 2D) and comparison with the equilibrium unfolding (red circles, from Figure 2B), both selectively monitored by pCNF fluorescence (excitation at 240 nm and emission at 297 nm).
selective probe for the SH3N domain in the SH3N−SH3C protein and does not monitor the fast folding reaction of the SH3C domain, which is complete within 1 s. Unfortunately, the emission wavelength of 297 nm is at the detection limit of the photomultiplier of the stopped-flow instrument. Although specific, the signal change of the pCNF fluorescence is significantly smaller than for the intrinsic fluorescence. These two properties together explain the poor signal-to-noise ratios of the folding traces (Figure 3A,B). However, we were able to obtain the same results after manual mixing with a strong increase in sensitivity (Supplementary Figure S1B). Besides the rate constants, the amplitudes of the reactions also contain important information. Kinetic unfolding and refolding experiments always start with the same state of the protein, respectively, and thus, the initial fluorescence value should be constant for each of these experiments. Compared to an equilibrium transition, they should start at the extrapolated baselines for the native and denatured protein, respectively (Figure 3C, squares). The amplitudes of the reactions are sensitive to the final urea concentration. The largest amplitudes should be observed in the baseline regions (highest urea concentrations for unfolding experiments and lowest urea concentration for refolding experiments). Thus, the final values of the kinetic experiments (the difference between the initial value and the amplitude) should trace the equilibrium transition. The direct comparison of this initial-final-value analysis with the equilibrium transition (Figure 3C) shows that indeed the results from kinetic experiments measuring the pCNF fluorescence [that monitor selectively SH3N (Figures 2D and 3A,B)] agree with the equilibrium transition (Figure 2B). This observation corroborates our previous assumption that pCNF can be used to selectively monitor only one domain in the SH3N−SH3C protein in equilibrium and in kinetic folding C
DOI: 10.1021/acs.biochem.6b00898 Biochemistry XXXX, XXX, XXX−XXX
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A practical approach (Creighton, T. E., Ed.) pp 261−298, Oxford University Press, Oxford, U.K. (3) Schmidpeter, P. A., Ries, L. K., Theer, T., and Schmid, F. X. (2015) Long-Range Energetic Changes Triggered by a Proline Switch in the Signal Adapter Protein c-CrkII. J. Mol. Biol. 427, 3908−3920. (4) Noren, C. J., Anthony-Cahill, S. J., Griffith, M. C., and Schultz, P. G. (1989) A general method for site-specific incorporation of unnatural amino acids into proteins. Science 244, 182−188. (5) Pless, S. A., Galpin, J. D., Niciforovic, A. P., Kurata, H. T., and Ahern, C. A. (2013) Hydrogen bonds as molecular timers for slow inactivation in voltage-gated potassium channels. eLife 2, e01289. (6) Grunbeck, A., Huber, T., Sachdev, P., and Sakmar, T. P. (2011) Mapping the ligand-binding site on a G protein-coupled receptor (GPCR) using genetically encoded photocrosslinkers. Biochemistry 50, 3411−3413. (7) Plass, T., Milles, S., Koehler, C., Szymanski, J., Mueller, R., Wiessler, M., Schultz, C., and Lemke, E. A. (2012) Amino acids for Diels-Alder reactions in living cells. Angew. Chem., Int. Ed. 51, 4166− 4170. (8) Peeler, J. C., and Mehl, R. A. (2012) Site-specific incorporation of unnatural amino acids as probes for protein conformational changes, Methods in molecular. Methods Mol. Biol. 794, 125−134. (9) Serrano, A. L., Troxler, T., Tucker, M. J., and Gai, F. (2010) Photophysics of a Fluorescent Non-natural Amino Acid: pCyanophenylalanine. Chem. Phys. Lett. 487, 303−306. (10) Miyake-Stoner, S. J., Miller, A. M., Hammill, J. T., Peeler, J. C., Hess, K. R., Mehl, R. A., and Brewer, S. H. (2009) Probing protein folding using site-specifically encoded unnatural amino acids as FRET donors with tryptophan. Biochemistry 48, 5953−5962. (11) Taskent-Sezgin, H., Chung, J., Patsalo, V., Miyake-Stoner, S. J., Miller, A. M., Brewer, S. H., Mehl, R. A., Green, D. F., Raleigh, D. P., and Carrico, I. (2009) Interpretation of p-cyanophenylalanine fluorescence in proteins in terms of solvent exposure and contribution of side-chain quenchers: a combined fluorescence, IR and molecular dynamics study. Biochemistry 48, 9040−9046. (12) Glasscock, J. M., Zhu, Y., Chowdhury, P., Tang, J., and Gai, F. (2008) Using an amino acid fluorescence resonance energy transfer pair to probe protein unfolding: application to the villin headpiece subdomain and the LysM domain. Biochemistry 47, 11070−11076. (13) Tucker, M. J., Oyola, R., and Gai, F. (2006) A novel fluorescent probe for protein binding and folding studies: p-cyano-phenylalanine. Biopolymers 83, 571−576. (14) Feller, S. M., Posern, G., Voss, J., Kardinal, C., Sakkab, D., Zheng, J., and Knudsen, B. S. (1998) Physiological signals and oncogenesis mediated through Crk family adapter proteins. J. Cell. Physiol. 177, 535−552. (15) Birge, R. B., Kalodimos, C., Inagaki, F., and Tanaka, S. (2009) Crk and CrkL adaptor proteins: networks for physiological and pathological signaling. Cell Commun. Signaling 7, 13. (16) Sarkar, P., Saleh, T., Tzeng, S. R., Birge, R. B., and Kalodimos, C. G. (2011) Structural basis for regulation of the Crk signaling protein by a proline switch. Nat. Chem. Biol. 7, 51−57. (17) DeLano, W. L. (2003) The PyMOL molecular graphics system, DeLano Scientific, LLC, San Carlos, CA. (18) Schmidpeter, P. A., and Schmid, F. X. (2014) Molecular determinants of a regulatory prolyl isomerization in the signal adapter protein c-CrkII. ACS Chem. Biol. 9, 1145−1152.
experiments and thus can be used as a domain-specific fluorescence tool in a multidomain protein. In conclusion, our work shows that the unnatural amino acid pCNF can be incorporated into a multidomain protein with high efficiency using an optimized expression protocol. Analysis of the stability and kinetic folding revealed that the incorporation of the unnatural amino acid pCNF did not affect the stability and folding and, thus, the structure of the protein. A strong increase in pCNF fluorescence was observed upon unfolding of the protein. Because the Trp and Tyr fluorescence is hardly affected by the presence of pCNF, it is probably not FRET that causes this increase. More likely, the solvent accessibility of pCNF is the major determinant to render it a selective probe. Here we showed in a proof-ofprinciple study that the pCNF signal can be used to analyze a single domain in the context of a two-domain protein without interfering signals from natural chromophores or from the other domain. We hypothesize that, if positioned correctly, the fluorescence of pCNF is a promising tool for detecting domain interactions that cannot be monitored by the intrinsic protein fluorescence. Our results for a well-characterized two-domain protein point to a potential use of fluorescent UAAs for studies of proteins in submolecular detail.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.6b00898. Detailed methods, additional figures, and summarizing tables (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Philipp A. M. Schmidpeter: 0000-0003-2871-9706 Present Addresses
† L.K.R.: Rudolf Virchow Center for Experimental Biomedicine, Josef-Schneider-Straße 2, D-97080 Würzburg, Germany. ‡ P.A.M.S.: Department of Anesthesiology, Weill Cornell Medicine, 1300 York Ave., New York, NY 10065.
Author Contributions
P.A.M.S. and F.X.S. designed the research. L.K.R. and P.A.M.S. performed the experiments. All authors analyzed the data and wrote the paper. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank R. Mehl for providing us with the pCNF expression system (pDule2 pCNF RS) and J. Koch for many discussions.
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REFERENCES
(1) Baldwin, R. L. (2007) Energetics of protein folding. J. Mol. Biol. 371, 283−301. (2) Schmid, F. X. (1997) Optical spectroscopy to characterize protein conformation and conformational changes. In Protein structure: D
DOI: 10.1021/acs.biochem.6b00898 Biochemistry XXXX, XXX, XXX−XXX
