CHEMPHYSCHEM COMMUNICATIONS DOI: 10.1002/cphc.201400017
IR Probes of Protein Microenvironments: Utility and Potential for Perturbation Ramkrishna Adhikary, Jçrg Zimmermann, Philip E. Dawson, and Floyd E. Romesberg*[a] A variety of IR-active moieties with absorptions that are distinct from those of proteins have been developed as probes of local protein environments, including carbon-deuterium bonds (C D), cyano groups (CN), and azides (N3); however, no systematic analysis of their utility in a protein has been published. Previously, we characterized the N-terminal Src homology 3 domain of the murine adapter protein Crk-II (nSH3) with C D bonds site-selectively incorporated throughout, and showed that it is relatively rigid and electrostatically heterogeneous and that it thermally unfolds under equilibrium conditions via a simple two-state mechanism. We now report the synthesis and characterization of eight variants of nSH3 with CN and/or N3 probes at five of the same positions. In agreement with previous studies, the position-dependent spectra suggest that both probes are predominantly sensitive to hydration, and not to their local electrostatic environments. Importantly, both probes also tend to significantly perturb the protein if they are not incorporated at surface-exposed positions. Thus, unlike C D labels, which are both sensitive to their environment and non-perturbative, CN and N3 probes should be used with caution.
Vibrational spectroscopy has become increasingly popular for the characterization of protein microenvironments. This is due to the development of IR probes that have environmentally sensitive absorptions around 2200 cm 1, which allows for their straightforward observation and characterization in the otherwise prohibitively congested protein IR spectrum. The first such probes to be incorporated into a protein were carbondeuterium (C D) bonds, which were used to replace non-exchangeable C H bonds.[1] Cyano (CN) groups had previously been proposed as probes of electrostatics,[2] and CN-derivatized amino acids have since been incorporated into model peptides[3–10] and proteins.[11–19] More recently, azide (N3) groups have also been examined as probes and have been incorporated into peptides[20] and proteins.[21–28] The CN and N3 probes have the advantage of being strong chromophores; however, unlike C D labels, CN and N3 substituents are extrinsic probes that may introduce interactions that are not present in the [a] Dr. R. Adhikary, Dr. J. Zimmermann, Prof. P. E. Dawson, Prof. F. E. Romesberg Department of Chemistry The Scripps Research Institute 10550 North Torrey Pines Road La Jolla, CA 92037 (USA) E-mail: [email protected] Supporting Information for this article is available on the WWW under http://dx.doi.org/10.1002/cphc.201400017.
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native protein, and sensitivity to these artificial interactions may desensitize them to their native protein environment. Indeed, previous studies have indicated that both probes are predominantly sensitive to hydrogen bonding,[5, 29–34] interactions introduced by the probes themselves. Moreover, as extrinsic probes they might perturb the protein, a possibility exacerbated by the size of the N3 group and by the H-bonding propensity of both CN and N3 groups. For example, Londergan and co-workers demonstrated that cyanylation of cysteine destabilized a model helix, and more so if in the middle than near either terminus.[13] Raleigh and co-workers showed that replacing Met1 or Ile4 of the N-terminal domain of the L9 ribosomal protein with azidohomoalanine destabilized the protein to thermal unfolding by 0.8 and 1.9 kcal mol 1, respectively,[24] while replacing Phe5 with p-cyanophenylalanine, resulted in a slight stabilization to chaotropic denaturation.[35] In addition, Fayer and co-workers demonstrated that the incorporation of two p-cyanophenylalanine probes destabilizes the 35-residue villin headpiece subdomain.[3] Finally, we previously demonstrated that the incorporation of p-cyanophenylalanine at either Tyr67 or Phe82 of cytochrome c destabilizes the protein to denaturation by guanidine hydrochloride.[19] However, no systematic comparison of these probes in a protein has been reported. Src homology 3 (SH3) domains mediate molecular recognition in many different proteins, fold into a conserved structure (Figure 1 a), and have emerged as a paradigm for biological
Figure 1. a) Structure of nSH3 (PDB 1CKA) showing labeled residues. b) Structure of probe-bearing amino acids used in this study.
molecular recognition[36] and protein folding.[37–39] Our recent characterization of the N-terminal SH3 domain of the murine adapter protein Crk-II (nSH3) with C D bonds incorporated at eleven different positions revealed that the protein folds into a relatively rigid structure with different sites experiencing unique electrostatic microenvironments and that thermal deChemPhysChem 2014, 15, 849 – 853
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Table 1. Thermodynamic fit parameters.
C D labeled[a] (CN)Phe143 (CN)Phe153 (CN)Phe186 (N3)Phe143 (N3)Phe153 (N3)Phe186 Aha159 Aha181 (d3)Met159
Tm [8C]
DH8 [kcal mol 1]
DS8 [cal mol 1 K 1]
DG8(25 8C) [kcal mol 1]
55.6 0.1 46.9 0.5 29.2 0.3 55.3 0.4 52.6 1.3 34.6 1.5 52.8 0.8 34.2 0.5 52.8 0.7 47.0 0.7
37.2 0.5 30.0 1.0 45.5 6.8 42.6 4.4 34.1 3.0 36.1 2.3 35.7 1.1 26.8 1.0 36.9 3.7 35.0 0.2
113 2 94 4 150 24 130 14 105 12 117 12 110 5 87 5 113 13 109 2
3.4 0.1 2.1 0.1 0.6 0.1 3.9 0.4 2.9 0.3 1.1 0.1 3.0 0.1 0.8 0.1 3.2 0.4 2.4 0.1
[a] Common transition observed with C D bonds incorporated at nine different sites within nSH3.[40]
naturation induces a global two-state unfolding transition (Tm = 55.6 0.1 8C and DG8 = 3.4 0.1 kcal mol 1 at 25 8C, Table 1).[40] To explore the relative utility of CN and N3 probes, we used tert-butyloxycarbonyl (Boc)-solid phase peptide synthesis to synthesize eight nSH3 variants with CN or N3 probes site selectively incorporated at five of the positions characterized previously with C D bonds (Figure 1 a and Supporting Information). IR spectra of 10 mm samples of each folded protein were acquired at either 6 or 24 8C (depending on the stability of the protein), and for comparison, spectra of thermally unfolded protein were acquired at 93 8C. To facilitate comparison of the folded and unfolded states, spectra were fit with a minimum number of pseudo-Voigt functions. (CN)Phe and (N3)Phe (Figure 1 b) have been used extensively as probe-bearing mimics of Phe or Tyr.[3–8, 11, 17, 19, 25–27] We first synthesized and characterized nSH3 with these modified amino acids replacing Phe143, Phe153, or Tyr186 [denoted as (CN)Phe143, (CN)Phe153, (CN)Phe186; and (N3)Phe143, (N3)Phe153, (N3)Phe186, respectively; Figure 2]. At 93 8C, each CN-labeled protein showed a similar spectrum consisting of one absorption band that was well fit with a single pseudoVoigt function, and that was virtually identical to that of the Boc-protected (CN)Phe (see the Supporting Information), suggesting, as expected, that each residue is solvent exposed in the unfolded protein. The same conclusion was drawn based
on the absorptions of C D bonds incorporated at the same positions.[40] The low-temperature spectra of the CN-labeled proteins also showed a single absorption band that was well fit with a single pseudo-Voigt function. Previously, the requirement of multiple functions to fit the spectra of (CN)Phe incorporated into a model peptide was taken as evidence of conformational heterogeneity,[8] and thus the quality of the fits using a single pseudo-Voigt function suggests that the CN probes experience a single, well-defined environment in the folded state. Similarly, no evidence for conformational heterogeneity was observed with C D bonds incorporated at the same positions.[40] As with the C D probes, and in contrast to the high-temperature spectra, the low-temperature spectra varied with the probe’s position within the protein. Relative to 93 8C, the frequency of the (CN)Phe186 absorption at 24 8C is blue-shifted by ~ 4 cm 1. The absorption frequencies of (CN)Phe143 and (CN)Phe153 at 6 8C are also blue-shifted relative to their high-temperature spectra, but less so (1.6 and 3.1 cm 1, respectively). Blueshifts with decreasing temperature were also observed with Boc-protected (CN)Phe in the same solvent (see the Supporting Information), and previously by others with nitriles in aqueous solution. These blueshifts are thought to result from increased H bonding with water at low temperatures, which strengthens the C N bond by reducing its antibonding character.[29, 30] Moreover, solvent studies have convincingly demonstrated that the absorption frequency is predominantly sensitive to H-bond formation, and that H bonding induces a blueshift.[31–33, 41] In addition, the data are inconsistent with electrostatics as the origin of the position-dependent spectra, because the more buried positions of (CN)Phe143 and (CN)Phe153 would be expected to provide a more hydrophobic environment, and thus based on a literature precedent induce a greater blueshift than the surface-exposed and more polar environment of (CN)Phe186.[5, 41, 42] Thus, we assign the site-specific blueshifts to increased nitrile hydration. Correspondingly, the larger blueshift observed for (CN)Phe186 suggests that it is the most hydrated in the folded state, which is consistent with its surfaceexposed position in the nSH3 structure (Figure 1). The smaller blueshifts observed for (CN)Phe143 and (CN)Phe153 suggest that in the folded state they are less hydrated, consistent with their more buried positions.
Figure 2. Low-temperature (upper panels) and high-temperature (lower panels) spectra of (CN)Phe- and (N3)Phe-labeled nSH3. Data is shown as circles, best fit as bold lines, and individual pseudo-Voigt profiles as thin lines, respectively.
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CHEMPHYSCHEM COMMUNICATIONS At 93 8C, each N3-labeled protein shows similar asymmetric absorption bands, which require a major high-frequency and minor low-frequency pseudo-Voigt function for fitting, and which are similar to that of (N3)Phe under identical conditions, but red-shifted by 3.0 to 4.9 cm 1 (Figure 2 and Supporting Information). Based on solvent studies such redshifts are expected to result from reduced H bonding,[33, 34] suggesting that the N3 probes are not fully solvent exposed in the unfolded state. As the C D and CN absorptions at the same positions in the unfolded protein are virtually identical to those of the free amino acid, the protected environment must be unique to the azide probes. The N3 absorption band of (N3)Phe shows a 1.5 cm 1 blueshift upon cooling from 93 to 6 8C, but remains well fit by two pseudo-Voigt functions (see the Supporting Information). While the low-temperature spectra of the N3-labeled proteins are similar to the high-temperature spectra (Figure 2), the minor low-frequency absorption of (N3)Phe153 is more intense. Comparison to (N3)Phe at the same temperature reveals that the major bands are red-shifted by 4.7, 7.2, and 9.1 cm 1, respectively, for (N3)Phe186, (N3)Phe153, and (N3)Phe143 (the minor bands are also red-shifted, see the Supporting Information). Again, such redshifts are suggestive of reduced H bonding, and thus the data suggest that the azide moiety of (N3)Phe143 undergoes the most folding-dependent desolvation, followed by (N3)Phe153, and then (N3)Phe186. These conclusions are also consistent with the C D absorption data[40] and with the structure of nSH3. A more detailed interpretation of the N3 absorptions is complicated by a dependence on the structural details of the H bonds[22] and by the possible presence of Fermi resonances.[34, 43, 44] To characterize the effects of the (CN)Phe and (N3)Phe probes on protein stability and to explore their use as probes of protein folding, we collected spectra at 5 8C intervals between the low-temperature (folded) and high-temperature (unfolded) states (see the Supporting Information). The spectra and thermal denaturation curves of each site probed after heating were indistinguishable from those acquired without heating, suggesting that the unfolding is reversible (see the Supporting Information). All spectra at intermediate temperatures were well fit by a superposition of the low- and hightemperature spectra, and the resulting fractional concentrations were fit to two-state transitions (Figure 3 a) to determine the residue-specific values of Tm, DH8, DS8, and DG8 at 25 8C (Table 1). In most cases, a moderate to significant destabilization was observed upon the introduction of either a CN or N3 probe. The destabilization is generally smaller for the probes at positions predicted to be solvent exposed, and larger at positions predicted to be buried within the core of the protein. For example, no destabilization was observed with (CN)Phe186, but (CN)Phe153 and (N3)Phe153 were destabilized by 2.8 and 2.3 kcal mol 1, respectively, at 25 8C. Interestingly, although an unfolding transition was not observed when Tyr186 was labeled with C D bonds, due to the similar solvent-exposed environment of the side chain in the folded and unfolded states,[40] the inclusion of a CN or N3 probe at this position introduces interactions that are sensitive to the unfolding transition. 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 3. a) Overlay of the unfolding titration curves for labeled proteins (see Table 1 for Tm values). Each data point was determined in triplicate (standard deviation is shown) and fit to residue-specific, two-state transitions. b) For comparison, analogous data for C D probes at nine different positions within nSH3 are shown, revealing a common unfolding transition.[40]
Azidohomoalanine (Aha, Figure 1 b) has been used as an probe-bearing mimic of Met.[21–24] nSH3 contains a single Met residue, Met181, which like Tyr186 is solvent exposed in the crystal structure.[45] The high-temperature spectrum of the protein with Aha replacing Met181 (Aha181) is well fit with a single pseudo-Voigt function, while the 24 8C spectra required three pseudo-Voigt functions for fitting (Figure 4). The
Figure 4. Low-temperature (upper panels) and high-temperature (lower panels) spectra of Aha159, Aha181, and (d3)Met159. Data is shown as circles, best fit as bold lines, and individual pseudo-Voigt profiles as thin lines, respectively.
frequency of the most intense low-temperature absorption was similar to that of the high-temperature absorption, and both were ~ 2.7 cm 1 red-shifted relative to the free amino acid. This suggests that the azide probe is not fully solvent exposed and that it is similarly hydrated in both the folded or unfolded state. Substitution at this solvent-exposed position does not significantly affect the stability of the folded protein (Table 1 and Figure 3). Due to their similar hydrophobicity, Leu and Met are often considered interchangeable.[46–48] Therefore, we synthesized the variant with Leu159 replaced with Aha (Aha159) to explore the use of Aha to probe the nSH3 hydrophobic core. We also synthesized and characterized the variant with (methyl-d3) methionine at the same position [(d3)Met159]. The CD3 symmetric absorption band of (d3)Met159 was well fit by a single pseudoVoigt function at both 93 and 24 8C (Figure 4). In the folded ChemPhysChem 2014, 15, 849 – 853
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CHEMPHYSCHEM COMMUNICATIONS state, the absorption band was narrower and 10.6 cm 1 redshifted relative to the unfolded state. (d3)Met159 showed a DG8 of unfolding of 2.4 0.1 kcal mol 1 at 25 8C, indicating that the substitution of Met for Leu destabilizes the protein by 1.0 kcal mol 1 (Table 1), despite their similar volume and hydrophobicity. The high-temperature spectrum of Aha159 shows a single absorption band that is well fit by a single pseudo-Voigt function and is virtually identical to that observed with Aha181. However, at 6 8C, Aha159 exhibits two clearly resolved absorption bands (Figure 4). These bands each required a pseudoVoigt function for fitting, and they are each red-shifted relative to the corresponding absorptions of the free amino acid (by 5 and 30 cm 1 for the major and the minor band, respectively), suggestive of a strongly hydrophobic environment that effectively precludes hydration, which is consistent with its position in the hydrophobic core of the protein (Figure 1 a). The N3 probe at this buried position significantly destabilized the protein (Figure 3), decreasing DG8 of unfolding by 2.6 kcal mol 1 at 25 8C (Table 1), which is significantly more destabilizing than a Met at the same position. To confirm that the thermally-induced transitions observed with both the CN and N3 probes correspond to the unfolding of the protein, we also characterized the unfolding of each labeled protein by circular dichroism spectroscopy (see the Supporting Information). Despite requiring very different protein concentrations, the DG8 values determined by circular dichroism and by FT IR are highly correlated (R = 0.95), suggesting that each IR transition indeed reflects the global unfolding of the protein. CN and N3 probes have been used extensively to characterize the microenvironments within proteins.[11, 19, 21–25] However, our data are consistent with the suggestion that both probes are only sensitive to hydration. Since the H bonds involved are introduced by the probe itself, this renders the data difficult to interpret in terms of the native protein. Boxer and co-workers have noted that it is possible to quantify the contribution of local electrostatics to nitrile peak shifts based on the relationship of IR frequency shifts and 13C NMR chemical shifts,[5, 14] and our data suggest that such corrections are likely to be necessary. Moreover, the data reveal that both extrinsic probes are prone to perturb the protein. In all but two cases, both the CN and the N3 probe destabilize the native state of nSH3 by 0.4 to 2.8 kcal mol 1, which is significant given that the total stability of the native protein is only 3.4 kcal mol 1. Thus, the environments and dynamics characterized by these probes may not correspond to those of the native protein. Destabilization appears not to depend on the specific amino acid modified, but rather on its position in the protein, with the destabilization being larger for more buried residues and smaller for more surface-exposed residues (Figure 3 a). The relative extents of the site-specific destabilization generally agree with those determined by mutation of other SH3 domain proteins.[49, 50] The only cases in which the protein was not destabilized were (CN)Phe186 and Aha181, which are both solvent exposed. The increased tolerance to incorporation of these probes at surface-exposed positions, along with their sensitivity to solvation, 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemphyschem.org suggests that they are likely most useful as probes of surface hydration, as suggested previously based on solvent studies.[32, 34] From this perspective, the uniquely large blueshift observed upon thermally induced folding of (CN)Phe186 is particularly interesting, as it suggests that solvation at the protein– water interface is particularly strong, which is consistent with other studies indicating that proteins are hydrated by a layer of high-density water molecules.[51, 52] This is also consistent with (CN)Phe186 being the only case where modification stabilizes the folded state of the protein. While CN and N3 probes have each been used to characterize protein folding,[3, 23, 24, 53] our data suggest that their use may be problematic due to site-specific destabilization. For example, (CN)Phe is much more destabilizing if used to replace Phe153 than if used to replace Tyr186. Correspondingly, the use of either CN or N3 probes to study the folding of nSH3 would have erroneously suggested a multistep folding process involving the population of intermediates, as opposed to the simple two-state process revealed by the use of the non-perturbative C D probes (Figure 3). The results of this systematic analysis using nSH3 adds to a growing literature[3, 13, 19, 24, 35] that suggests that these probes are destabilizing, and combined these results suggest that the destabilization may be general. Thus, in contrast to C D labels, which are strictly non-perturbative, the results of studies based on the use of CN and N3 probes must be interpreted with caution to ensure that they are biologically relevant and not an artifact of the chromophore or its perturbation of the protein. Keywords: labeling · IR spectroscopy · perturbation · protein environments · protein folding
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Received: January 8, 2014 Published online on February 12, 2014
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IR Probes of Protein Microenvironments: Utility and Potential for Perturbation Ramkrishna Adhikary, Jçrg Zimmermann, Philip E. Dawson, and Floyd E. Romesberg*[a] A variety of IR-active moieties with absorptions that are distinct from those of proteins have been developed as probes of local protein environments, including carbon-deuterium bonds (C D), cyano groups (CN), and azides (N3); however, no systematic analysis of their utility in a protein has been published. Previously, we characterized the N-terminal Src homology 3 domain of the murine adapter protein Crk-II (nSH3) with C D bonds site-selectively incorporated throughout, and showed that it is relatively rigid and electrostatically heterogeneous and that it thermally unfolds under equilibrium conditions via a simple two-state mechanism. We now report the synthesis and characterization of eight variants of nSH3 with CN and/or N3 probes at five of the same positions. In agreement with previous studies, the position-dependent spectra suggest that both probes are predominantly sensitive to hydration, and not to their local electrostatic environments. Importantly, both probes also tend to significantly perturb the protein if they are not incorporated at surface-exposed positions. Thus, unlike C D labels, which are both sensitive to their environment and non-perturbative, CN and N3 probes should be used with caution.
Vibrational spectroscopy has become increasingly popular for the characterization of protein microenvironments. This is due to the development of IR probes that have environmentally sensitive absorptions around 2200 cm 1, which allows for their straightforward observation and characterization in the otherwise prohibitively congested protein IR spectrum. The first such probes to be incorporated into a protein were carbondeuterium (C D) bonds, which were used to replace non-exchangeable C H bonds.[1] Cyano (CN) groups had previously been proposed as probes of electrostatics,[2] and CN-derivatized amino acids have since been incorporated into model peptides[3–10] and proteins.[11–19] More recently, azide (N3) groups have also been examined as probes and have been incorporated into peptides[20] and proteins.[21–28] The CN and N3 probes have the advantage of being strong chromophores; however, unlike C D labels, CN and N3 substituents are extrinsic probes that may introduce interactions that are not present in the [a] Dr. R. Adhikary, Dr. J. Zimmermann, Prof. P. E. Dawson, Prof. F. E. Romesberg Department of Chemistry The Scripps Research Institute 10550 North Torrey Pines Road La Jolla, CA 92037 (USA) E-mail: [email protected] Supporting Information for this article is available on the WWW under http://dx.doi.org/10.1002/cphc.201400017.
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native protein, and sensitivity to these artificial interactions may desensitize them to their native protein environment. Indeed, previous studies have indicated that both probes are predominantly sensitive to hydrogen bonding,[5, 29–34] interactions introduced by the probes themselves. Moreover, as extrinsic probes they might perturb the protein, a possibility exacerbated by the size of the N3 group and by the H-bonding propensity of both CN and N3 groups. For example, Londergan and co-workers demonstrated that cyanylation of cysteine destabilized a model helix, and more so if in the middle than near either terminus.[13] Raleigh and co-workers showed that replacing Met1 or Ile4 of the N-terminal domain of the L9 ribosomal protein with azidohomoalanine destabilized the protein to thermal unfolding by 0.8 and 1.9 kcal mol 1, respectively,[24] while replacing Phe5 with p-cyanophenylalanine, resulted in a slight stabilization to chaotropic denaturation.[35] In addition, Fayer and co-workers demonstrated that the incorporation of two p-cyanophenylalanine probes destabilizes the 35-residue villin headpiece subdomain.[3] Finally, we previously demonstrated that the incorporation of p-cyanophenylalanine at either Tyr67 or Phe82 of cytochrome c destabilizes the protein to denaturation by guanidine hydrochloride.[19] However, no systematic comparison of these probes in a protein has been reported. Src homology 3 (SH3) domains mediate molecular recognition in many different proteins, fold into a conserved structure (Figure 1 a), and have emerged as a paradigm for biological
Figure 1. a) Structure of nSH3 (PDB 1CKA) showing labeled residues. b) Structure of probe-bearing amino acids used in this study.
molecular recognition[36] and protein folding.[37–39] Our recent characterization of the N-terminal SH3 domain of the murine adapter protein Crk-II (nSH3) with C D bonds incorporated at eleven different positions revealed that the protein folds into a relatively rigid structure with different sites experiencing unique electrostatic microenvironments and that thermal deChemPhysChem 2014, 15, 849 – 853
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Table 1. Thermodynamic fit parameters.
C D labeled[a] (CN)Phe143 (CN)Phe153 (CN)Phe186 (N3)Phe143 (N3)Phe153 (N3)Phe186 Aha159 Aha181 (d3)Met159
Tm [8C]
DH8 [kcal mol 1]
DS8 [cal mol 1 K 1]
DG8(25 8C) [kcal mol 1]
55.6 0.1 46.9 0.5 29.2 0.3 55.3 0.4 52.6 1.3 34.6 1.5 52.8 0.8 34.2 0.5 52.8 0.7 47.0 0.7
37.2 0.5 30.0 1.0 45.5 6.8 42.6 4.4 34.1 3.0 36.1 2.3 35.7 1.1 26.8 1.0 36.9 3.7 35.0 0.2
113 2 94 4 150 24 130 14 105 12 117 12 110 5 87 5 113 13 109 2
3.4 0.1 2.1 0.1 0.6 0.1 3.9 0.4 2.9 0.3 1.1 0.1 3.0 0.1 0.8 0.1 3.2 0.4 2.4 0.1
[a] Common transition observed with C D bonds incorporated at nine different sites within nSH3.[40]
naturation induces a global two-state unfolding transition (Tm = 55.6 0.1 8C and DG8 = 3.4 0.1 kcal mol 1 at 25 8C, Table 1).[40] To explore the relative utility of CN and N3 probes, we used tert-butyloxycarbonyl (Boc)-solid phase peptide synthesis to synthesize eight nSH3 variants with CN or N3 probes site selectively incorporated at five of the positions characterized previously with C D bonds (Figure 1 a and Supporting Information). IR spectra of 10 mm samples of each folded protein were acquired at either 6 or 24 8C (depending on the stability of the protein), and for comparison, spectra of thermally unfolded protein were acquired at 93 8C. To facilitate comparison of the folded and unfolded states, spectra were fit with a minimum number of pseudo-Voigt functions. (CN)Phe and (N3)Phe (Figure 1 b) have been used extensively as probe-bearing mimics of Phe or Tyr.[3–8, 11, 17, 19, 25–27] We first synthesized and characterized nSH3 with these modified amino acids replacing Phe143, Phe153, or Tyr186 [denoted as (CN)Phe143, (CN)Phe153, (CN)Phe186; and (N3)Phe143, (N3)Phe153, (N3)Phe186, respectively; Figure 2]. At 93 8C, each CN-labeled protein showed a similar spectrum consisting of one absorption band that was well fit with a single pseudoVoigt function, and that was virtually identical to that of the Boc-protected (CN)Phe (see the Supporting Information), suggesting, as expected, that each residue is solvent exposed in the unfolded protein. The same conclusion was drawn based
on the absorptions of C D bonds incorporated at the same positions.[40] The low-temperature spectra of the CN-labeled proteins also showed a single absorption band that was well fit with a single pseudo-Voigt function. Previously, the requirement of multiple functions to fit the spectra of (CN)Phe incorporated into a model peptide was taken as evidence of conformational heterogeneity,[8] and thus the quality of the fits using a single pseudo-Voigt function suggests that the CN probes experience a single, well-defined environment in the folded state. Similarly, no evidence for conformational heterogeneity was observed with C D bonds incorporated at the same positions.[40] As with the C D probes, and in contrast to the high-temperature spectra, the low-temperature spectra varied with the probe’s position within the protein. Relative to 93 8C, the frequency of the (CN)Phe186 absorption at 24 8C is blue-shifted by ~ 4 cm 1. The absorption frequencies of (CN)Phe143 and (CN)Phe153 at 6 8C are also blue-shifted relative to their high-temperature spectra, but less so (1.6 and 3.1 cm 1, respectively). Blueshifts with decreasing temperature were also observed with Boc-protected (CN)Phe in the same solvent (see the Supporting Information), and previously by others with nitriles in aqueous solution. These blueshifts are thought to result from increased H bonding with water at low temperatures, which strengthens the C N bond by reducing its antibonding character.[29, 30] Moreover, solvent studies have convincingly demonstrated that the absorption frequency is predominantly sensitive to H-bond formation, and that H bonding induces a blueshift.[31–33, 41] In addition, the data are inconsistent with electrostatics as the origin of the position-dependent spectra, because the more buried positions of (CN)Phe143 and (CN)Phe153 would be expected to provide a more hydrophobic environment, and thus based on a literature precedent induce a greater blueshift than the surface-exposed and more polar environment of (CN)Phe186.[5, 41, 42] Thus, we assign the site-specific blueshifts to increased nitrile hydration. Correspondingly, the larger blueshift observed for (CN)Phe186 suggests that it is the most hydrated in the folded state, which is consistent with its surfaceexposed position in the nSH3 structure (Figure 1). The smaller blueshifts observed for (CN)Phe143 and (CN)Phe153 suggest that in the folded state they are less hydrated, consistent with their more buried positions.
Figure 2. Low-temperature (upper panels) and high-temperature (lower panels) spectra of (CN)Phe- and (N3)Phe-labeled nSH3. Data is shown as circles, best fit as bold lines, and individual pseudo-Voigt profiles as thin lines, respectively.
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CHEMPHYSCHEM COMMUNICATIONS At 93 8C, each N3-labeled protein shows similar asymmetric absorption bands, which require a major high-frequency and minor low-frequency pseudo-Voigt function for fitting, and which are similar to that of (N3)Phe under identical conditions, but red-shifted by 3.0 to 4.9 cm 1 (Figure 2 and Supporting Information). Based on solvent studies such redshifts are expected to result from reduced H bonding,[33, 34] suggesting that the N3 probes are not fully solvent exposed in the unfolded state. As the C D and CN absorptions at the same positions in the unfolded protein are virtually identical to those of the free amino acid, the protected environment must be unique to the azide probes. The N3 absorption band of (N3)Phe shows a 1.5 cm 1 blueshift upon cooling from 93 to 6 8C, but remains well fit by two pseudo-Voigt functions (see the Supporting Information). While the low-temperature spectra of the N3-labeled proteins are similar to the high-temperature spectra (Figure 2), the minor low-frequency absorption of (N3)Phe153 is more intense. Comparison to (N3)Phe at the same temperature reveals that the major bands are red-shifted by 4.7, 7.2, and 9.1 cm 1, respectively, for (N3)Phe186, (N3)Phe153, and (N3)Phe143 (the minor bands are also red-shifted, see the Supporting Information). Again, such redshifts are suggestive of reduced H bonding, and thus the data suggest that the azide moiety of (N3)Phe143 undergoes the most folding-dependent desolvation, followed by (N3)Phe153, and then (N3)Phe186. These conclusions are also consistent with the C D absorption data[40] and with the structure of nSH3. A more detailed interpretation of the N3 absorptions is complicated by a dependence on the structural details of the H bonds[22] and by the possible presence of Fermi resonances.[34, 43, 44] To characterize the effects of the (CN)Phe and (N3)Phe probes on protein stability and to explore their use as probes of protein folding, we collected spectra at 5 8C intervals between the low-temperature (folded) and high-temperature (unfolded) states (see the Supporting Information). The spectra and thermal denaturation curves of each site probed after heating were indistinguishable from those acquired without heating, suggesting that the unfolding is reversible (see the Supporting Information). All spectra at intermediate temperatures were well fit by a superposition of the low- and hightemperature spectra, and the resulting fractional concentrations were fit to two-state transitions (Figure 3 a) to determine the residue-specific values of Tm, DH8, DS8, and DG8 at 25 8C (Table 1). In most cases, a moderate to significant destabilization was observed upon the introduction of either a CN or N3 probe. The destabilization is generally smaller for the probes at positions predicted to be solvent exposed, and larger at positions predicted to be buried within the core of the protein. For example, no destabilization was observed with (CN)Phe186, but (CN)Phe153 and (N3)Phe153 were destabilized by 2.8 and 2.3 kcal mol 1, respectively, at 25 8C. Interestingly, although an unfolding transition was not observed when Tyr186 was labeled with C D bonds, due to the similar solvent-exposed environment of the side chain in the folded and unfolded states,[40] the inclusion of a CN or N3 probe at this position introduces interactions that are sensitive to the unfolding transition. 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 3. a) Overlay of the unfolding titration curves for labeled proteins (see Table 1 for Tm values). Each data point was determined in triplicate (standard deviation is shown) and fit to residue-specific, two-state transitions. b) For comparison, analogous data for C D probes at nine different positions within nSH3 are shown, revealing a common unfolding transition.[40]
Azidohomoalanine (Aha, Figure 1 b) has been used as an probe-bearing mimic of Met.[21–24] nSH3 contains a single Met residue, Met181, which like Tyr186 is solvent exposed in the crystal structure.[45] The high-temperature spectrum of the protein with Aha replacing Met181 (Aha181) is well fit with a single pseudo-Voigt function, while the 24 8C spectra required three pseudo-Voigt functions for fitting (Figure 4). The
Figure 4. Low-temperature (upper panels) and high-temperature (lower panels) spectra of Aha159, Aha181, and (d3)Met159. Data is shown as circles, best fit as bold lines, and individual pseudo-Voigt profiles as thin lines, respectively.
frequency of the most intense low-temperature absorption was similar to that of the high-temperature absorption, and both were ~ 2.7 cm 1 red-shifted relative to the free amino acid. This suggests that the azide probe is not fully solvent exposed and that it is similarly hydrated in both the folded or unfolded state. Substitution at this solvent-exposed position does not significantly affect the stability of the folded protein (Table 1 and Figure 3). Due to their similar hydrophobicity, Leu and Met are often considered interchangeable.[46–48] Therefore, we synthesized the variant with Leu159 replaced with Aha (Aha159) to explore the use of Aha to probe the nSH3 hydrophobic core. We also synthesized and characterized the variant with (methyl-d3) methionine at the same position [(d3)Met159]. The CD3 symmetric absorption band of (d3)Met159 was well fit by a single pseudoVoigt function at both 93 and 24 8C (Figure 4). In the folded ChemPhysChem 2014, 15, 849 – 853
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CHEMPHYSCHEM COMMUNICATIONS state, the absorption band was narrower and 10.6 cm 1 redshifted relative to the unfolded state. (d3)Met159 showed a DG8 of unfolding of 2.4 0.1 kcal mol 1 at 25 8C, indicating that the substitution of Met for Leu destabilizes the protein by 1.0 kcal mol 1 (Table 1), despite their similar volume and hydrophobicity. The high-temperature spectrum of Aha159 shows a single absorption band that is well fit by a single pseudo-Voigt function and is virtually identical to that observed with Aha181. However, at 6 8C, Aha159 exhibits two clearly resolved absorption bands (Figure 4). These bands each required a pseudoVoigt function for fitting, and they are each red-shifted relative to the corresponding absorptions of the free amino acid (by 5 and 30 cm 1 for the major and the minor band, respectively), suggestive of a strongly hydrophobic environment that effectively precludes hydration, which is consistent with its position in the hydrophobic core of the protein (Figure 1 a). The N3 probe at this buried position significantly destabilized the protein (Figure 3), decreasing DG8 of unfolding by 2.6 kcal mol 1 at 25 8C (Table 1), which is significantly more destabilizing than a Met at the same position. To confirm that the thermally-induced transitions observed with both the CN and N3 probes correspond to the unfolding of the protein, we also characterized the unfolding of each labeled protein by circular dichroism spectroscopy (see the Supporting Information). Despite requiring very different protein concentrations, the DG8 values determined by circular dichroism and by FT IR are highly correlated (R = 0.95), suggesting that each IR transition indeed reflects the global unfolding of the protein. CN and N3 probes have been used extensively to characterize the microenvironments within proteins.[11, 19, 21–25] However, our data are consistent with the suggestion that both probes are only sensitive to hydration. Since the H bonds involved are introduced by the probe itself, this renders the data difficult to interpret in terms of the native protein. Boxer and co-workers have noted that it is possible to quantify the contribution of local electrostatics to nitrile peak shifts based on the relationship of IR frequency shifts and 13C NMR chemical shifts,[5, 14] and our data suggest that such corrections are likely to be necessary. Moreover, the data reveal that both extrinsic probes are prone to perturb the protein. In all but two cases, both the CN and the N3 probe destabilize the native state of nSH3 by 0.4 to 2.8 kcal mol 1, which is significant given that the total stability of the native protein is only 3.4 kcal mol 1. Thus, the environments and dynamics characterized by these probes may not correspond to those of the native protein. Destabilization appears not to depend on the specific amino acid modified, but rather on its position in the protein, with the destabilization being larger for more buried residues and smaller for more surface-exposed residues (Figure 3 a). The relative extents of the site-specific destabilization generally agree with those determined by mutation of other SH3 domain proteins.[49, 50] The only cases in which the protein was not destabilized were (CN)Phe186 and Aha181, which are both solvent exposed. The increased tolerance to incorporation of these probes at surface-exposed positions, along with their sensitivity to solvation, 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemphyschem.org suggests that they are likely most useful as probes of surface hydration, as suggested previously based on solvent studies.[32, 34] From this perspective, the uniquely large blueshift observed upon thermally induced folding of (CN)Phe186 is particularly interesting, as it suggests that solvation at the protein– water interface is particularly strong, which is consistent with other studies indicating that proteins are hydrated by a layer of high-density water molecules.[51, 52] This is also consistent with (CN)Phe186 being the only case where modification stabilizes the folded state of the protein. While CN and N3 probes have each been used to characterize protein folding,[3, 23, 24, 53] our data suggest that their use may be problematic due to site-specific destabilization. For example, (CN)Phe is much more destabilizing if used to replace Phe153 than if used to replace Tyr186. Correspondingly, the use of either CN or N3 probes to study the folding of nSH3 would have erroneously suggested a multistep folding process involving the population of intermediates, as opposed to the simple two-state process revealed by the use of the non-perturbative C D probes (Figure 3). The results of this systematic analysis using nSH3 adds to a growing literature[3, 13, 19, 24, 35] that suggests that these probes are destabilizing, and combined these results suggest that the destabilization may be general. Thus, in contrast to C D labels, which are strictly non-perturbative, the results of studies based on the use of CN and N3 probes must be interpreted with caution to ensure that they are biologically relevant and not an artifact of the chromophore or its perturbation of the protein. Keywords: labeling · IR spectroscopy · perturbation · protein environments · protein folding
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Received: January 8, 2014 Published online on February 12, 2014
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