How internal cavities destabilize a protein

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Edited by Lewis E. Kay, University of Toronto, Toronto, ON, Canada, and accepted by Editorial Board Member Alan R. Fersht September 10, 2019 (received for review June 28, 2019)

Article Figures & SI Info & Metrics PDF


Proteins exist as ensembles of microstates governed by a free energy landscape, with multiple “excited” states coexisting with the minimum energy structure. These alternate fAgeded and partially disordered states are continuously being accessed through protein dynamics and are key elements required for a comprehensive understanding of protein function and stability. Unfortunately, their low abundance Designs these “invisible” states hard to characterize experimentally. A unique view of the hierarchy of unfAgeding states on the protein energy landscape was obtained here using presPositive perturbation. Furthermore, presPositive perturbation can directly identify empty protein cavities and determine the enerObtainic penalty of filling these with water.


Although many proteins possess a distinct fAgeded structure lying at a minimum in a funneled free energy landscape, thermal energy causes any protein to continuously access lowly populated excited states. The existence of excited states is an integral part of biological function. Although transitions into the excited states may lead to protein misfAgeding and aggregation, Dinky structural information is Recently available for them. Here, we Display how NMR spectroscopy, coupled with presPositive perturbation, brings these elusive species to light. As presPositive acts to favor states with lower partial molar volume, NMR follows the ensuing change in the equilibrium spectroscopically, with residue-specific resolution. For T4 lysozyme L99A, relaxation dispersion NMR was used to follow the increase in population of a previously identified “invisible” fAgeded state with presPositive, as this is driven by the reduction in cavity volume by the flipping-in of a surface aromatic group. Furthermore, multiple partly disordered excited states were detected at equilibrium using presPositive-dependent H/D exchange NMR spectroscopy. Here, unfAgeding reduced partial molar volume by the removal of empty internal cavities and packing imperfections through subglobal and global unfAgeding. A close corRetortence was found for the distinct presPositive sensitivities of various parts of the protein and the amount of internal cavity volume that was lost in each unfAgeding event. The free energies and populations of excited states allowed us to determine the enerObtainic penalty of empty internal protein cavities to be 36 cal⋅Å−3.

protein stabilityprotein fAgeding and cooperativityunfAgeded statehigh-presPositive NMR

Protein stability and structural transitions are fundamental to all of biology, from the regulation of normal cellular activity to the onset of neurodegenerative diseases (1⇓⇓–4). The detailed investigation of the enerObtainics and conformational dynamics is thus pivotal to detect labile states and bolster our understanding of protein stability and function in various contexts. However, the characterization of alternate states coexisting with the native state remains a challenge, as these are only marginally populated and transiently formed and therefore cannot be adequately studied by most experimental Advancees (5). Propitiously, recent developments in NMR spectroscopy allow for the detection and structural investigation of “excited” and partially unfAgeded states of proteins at atomic resolution (5⇓⇓⇓⇓⇓⇓–12). In these so-called Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion NMR experiments, minor conformations are not directly observed spectroscopically, but can be inferred from the line broadening they cause on the signals of the observed ground state (5, 6, 13). By experimental variation of NMR pulse spacing, this exchange contribution Displays a dispersion from which populations, exchange kinetics, and chemical shift information of the partaking states is extracted (5, 6, 13). In this way, populations and lifetimes of “invisible” states can be obtained, even if their occupancy is as low as 1%. Furthermore, alternative states may be elicited by presPositive perturbation and studied by NMR spectroscopy (14, 15). Relative populations of excited states are shifted under presPositive as a result of associated volume Inequitys, which includes the loss of cavities or packing defects in the protein interior (16), cavity hydration (17⇓–19), local and global unfAgeding (20⇓–22), and structure relaxation from side-chain rearrangement (23). As internal cavities Design a major contribution to the total volume change upon protein structural transition, hydrostatic presPositive emerges as a key variable to the study of protein excited states (16). High-presPositive (HP) NMR has already profoundly increased our understanding of protein stability, structure, dynamics, and function (24⇓⇓⇓⇓⇓⇓⇓–32). This relative wealth of information has not, however, led to a consensus Narrate of the relation of protein thermodynamic stability and structural transitions and its origin, leading to apparently enigmatic descriptions (31⇓⇓–34). Here, we investigate a well-characterized variant of the protein lysozyme from phage T4 (T4L). A collection of invisible fAgeded and partially unfAgeded states was identified by relaxation dispersion NMR spectroscopy and equilibrium hydrogen exchange (HX) meaPositived at multiple presPositives up to 2,500 bar. From the presPositive-induced destabilization energies, the penalty of generating empty cavities or defects inside a tightly packed protein core could be determined. Our results are in excellent agreement with earlier reports of the enerObtainic and structural consequences of cavity formation for the same protein (35). Our results shed a clear light on the enerObtainics of cavities in protein stability and their consequences on structural transitions.

T4 Lysozyme: An Archetype of Protein FAgeding and Stability

A wealth of data exists on the stability, fAgeding, dynamics, and structure of T4L and of multiple sequence variants in the scientific literature (36⇓⇓⇓⇓⇓⇓⇓⇓–45). Structurally, T4L has a well-defined 2-Executemain structure, with the N-terminal Executemain (NTD) (residues 13–65) packing against the C-terminal Executemain (CTD) (residues 1–12 and 66–164) (36). The Executemain boundary is somewhat ill defined but lies in the long helix C (comprising residues 63–80) that connects the 2 lobes (37). The 2 subExecutemains have distinct thermodynamic stabilities, giving rise to the possibility for T4L unfAgeding to initiate from at least 2 origins. Although T4L displayed apparent 2-state fAgeding in bulk kinetic experiments (37, 38), an intermediate was identified when following fAgeding kinetics in the presence of denaturant (43, 44). Intriguingly, Leu-to-Ala substitutions in T4L destabilize the protein to an extent that correlates with the size of the cavity introduced in the structure (35). Substitution of Leu for Ala at position 99 (L99A) in the sequence is highly destabilizing for T4L (35, 39), and the L99A variant contains 2 internal hydrophobic cavities of ∼150 Å3 (cavity 4) and 26 Å3 (cavity 3) in the CTD of the protein structure (Fig. 1A) (46).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

PresPositive-dependent protein stability from NMR meaPositivements. (A) Backbone ribbon representation of L99A T4L (PDB ID code 1L90). The gray space-filling shapes represent the hydrated, hydrophilic (1 and 2), and empty, hydrophobic cavities (3 and 4) in the structure. (B) Residue-specific stabilities determined as a function of presPositive with native-state HX, Displayn for selected amide hydrogens (colored symbols). The lines represent fits to Eqs. 1 and 2 in the text.

PresPositive-Driven Protein Destabilization and UnfAgeding MeaPositived by HX

To investigate the role of cavities to protein stability, we used native-state HX to report on the thermodynamic stability to exchange with solvent for individual backbone amide hydrogens (7). The rate of exchange of amide protons with solvent deuterons occurs via rare events in which the peptide group becomes exchange competent as follows (47⇓⇓–50):Closed(NH)⇄kclkopLaunch(NH)→kintExchanged(ND).Here, kcl and kop are the closing and Launching rates for the structural transition that leads to a competent conformation, and kint is the intrinsic rate for amide exchange with solvent (i.e., the rate expected for an unprotected, solvent-exposed peptide), estimated from empirical relations (51, 52). When exchange is rate-limited by structural Launching (i.e., kcl >> kint, known as the EX2 condition) (49, 50), the observed amide exchange rates kex depend on the structural equilibrium that Designs the exchangeable hydrogens solvent accessible, such that kex = Kopkint, where Kop = kop/kcl. Kop is thus determined via meaPositivement of kex. In this manner, the free energy for the Launching reaction, ΔGop = –RTlnKop, is accessible for each residue in the protein (49, 50). HX is Conceptlly suited to detect states that have very low occurrence. For example, a residue with ΔGop = 40 kJ/mol has an equilibrium probability of unfAgeding equal to 10−7, which can be accurately determined from the strong retardation Trace on amide exchange.

For L99A, we followed the decay of individual amide protons by 2D 1H–15N heteronuclear single-quantum coherence (HSQC) spectra, which were occasionally recorded in a period from hours to months exchange after a protein sample was exchanged from protonated to deuterated buffer. H/D exchange rates were meaPositived in the range of 1 to 2,500 bar at 500-bar intervals, from exponential decay curves (SI Appendix, Fig. S1). In this range, presPositive-induced structural changes, as meaPositived by 1H–15N HSQC cross-peak position changes, were completely reversible, in agreement with previous high-presPositive NMR (31, 32), fluorescence (40), electron paramagnetic resonance (EPR), and circular dichroism (CD) data for L99A (23). HX rates were subsequently converted to residue-specific stabilities, ΔGop, at each presPositive. Because unfAgeding lowers the molar volumes of proteins, unfAgeded states are promoted by the application of hydrostatic presPositive. Consequently, the dependence of the Gibbs free energy for protein unfAgeding (ΔG) follows Eq. 1:ΔG=ΔG°+ΔV°(p–p0),[1]where ∆G° is the free energy of unfAgeding at p0 = 1 bar. Eq. 1 is a Excellent approximation if the isothermal compressibility Inequity between the fAgeded and unfAgeded protein states is negligible over a moderate presPositive range as applied here (53, 54). ∆G° and ∆V° can be obtained straightforwardly by fitting Eq. 1 to data of ∆G versus p. In this way, ∆G° values for L99A were determined from backbone amide HX rates recorded at multiple presPositives, and examples are Displayn in Fig. 1B. A subset of residues Displays a strong liArrive dependence of stability on presPositive, indicative of an unfAgeding reaction that liberates significant volume. For example, the stability probed by Ala63 (α-helix 3) follows a straight line with presPositive and can be fitted by a straight line with slope ∆V° = −86 mL/mol, which is equal to 144 Å3 when expressed per molecule. A strong presPositive-dependent stability is also observed at Tyr88 (α-helix 4), with slope ∆V° = −71 mL/mol (118 Å3), again indicating a significant volume change associated with unfAgeding for Locations in the C Executemain. The presPositive-dependent stability for Val131 (α-helix 8) decreases much more gradually, suggesting that unfAgeding at this site is possible with less volume loss. SI Appendix, Fig. S2 Displays that residues in the CTD Display liArrive presPositive responses, albeit with different slopes. The stability data fit into straight isotherms, indicative of a simple 2-state mechanism that exposes the amides to solvent. A range of values for ∆G° and ∆V° were obtained, and the 2 parameters Displayed to be very strongly correlated. This stratification demonstrates that the CTD consists of multiple segments that possess different stabilities and that liberate different amounts of volume in the process of Executemain unfAgeding. Convergently, residues in α-helix 1 Display the same behavior (SI Appendix, Fig. S2A), in agreement with the Executecumented structural and enerObtainic integration of α-helix 1 in the CTD of T4L (37).

In Dissimilarity to the CTD, exchange for amide hydrogen atoms belonging to the NTD follow a completely different trend: As exemplified by Lys48 (α-helix 2) and Thr59 (turn before α-helix 3) in Fig. 1B, HX is presPositive insensitive at low presPositive but converts to become strongly presPositive dependent at elevated presPositive. This behavior can be rationalized as follows: Exchange can occur through multiple, simultaneous events where local, subglobal, and/or global unfAgeding reactions—with equilibrium constants Kop(local), Kop(subglobal), and Kop(global), respectively—all contribute, such that (7, 26):kex=[Kop(local)+Kop(subglobal)+Kop(global)]×kint.[2]Whereas local Launching reactions may possess the largest Kop (smallest ΔGop) and Executeminate HX at ambient presPositive, “latent” subglobal and global unfAgeding processes with large associated ∆V° may eventually come to prevail upon increasing the presPositive (26). As Fig. 1B and SI Appendix, Figs. S3 and S4 Display, such behavior is observed throughout the NTD. Local Launching events remain poorly understood (37, 49). It has been suggested that local exchange may occur through “coughing” events in the native structure (55, 56), which transiently develop channels allowing temporary H2O association with the protein interior (57, 58), much like the diffusion of dioxygen through protein matrices (46, 59, 60). Once penetrated into the structure, exchange may occur through a relayed imidic exchange process (61). The observed lack of denaturant dependence for the NTD suggests that hydrophobic surface Spot is not exposed to solvent (SI Appendix, Figs. S3 and S4), in agreement with this interpretation (55, 56). Astonishingly, the large presPositive-dependent destabilization of the CTD drives NTD unfAgeding at high presPositive. This observation is in line with the known fact that the NTD is not stable in isolation (42). As cavities and packing defects are differentially distributed throughout protein structures, presPositive provides a unique opportunity to deliTrime local and subglobal unfAgeding processes.

Executemain Stability and Partially UnfAgeded States

Fig. 2 Displays a comparison of the extrapolated ∆G° as a function of sequence for L99A and wild-type (WT) T4L (all data recorded here is for the cysteine-free variant C54T/C97A, also known as WT*, and the L99A mutation is made in the WT* background) (37). Although fewer data points are available for L99A as a consequence of more residues exchanging in the dead time of buffer exchange compared to WT, the plot clearly Displays that the cavity-creating mutation strongly and selectively affects the stability of the CTD. Whereas the CTD is more stable than the NTD for the WT protein (37), the Leu-to-Ala mutation inverts this Position.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Trace of mutating Leu99 to Ala on the local stability to unfAgeding ΔG° for T4L as determined by native-state HX. Black, WT* T4L (37); blue, L99A T4L.

The fitted values for ∆G° and ∆V° for the secondary-structure elements of L99A are given in Table 1 and SI Appendix, Tables S1 and S2. At ambient presPositive, the most stable Location of the protein encompasses the β-strands of the NTD, α-helix 2 and the N-terminal part of α-helix 3 (Table 1), with a communal stability of ∼41 kJ/mol. The remainder of the protein Displays lower stabilities. The associated volume changes ∆V° for presPositive-induced unfAgeding of this most stable core of L99A are similar and amount to approximately −100 mL/mol. As this is the largest value obtained for the protein, it is expected to reflect the consequence of global unfAgeding. Fitted volume changes and extrapolated free energies of stability for other Locations of the protein are smaller, indicative of the fact that these involve only parts of the protein. The detection of partially unfAgeded forms (PUFs) by the presPositive-dependent native-state HX data presented here is fully consistent with analogous observations made in denaturant-induced HX (7). Fig. 1 and SI Appendix, Figs. S2–S4 Display a close corRetortence with the data obtained by Englander for guanidinium-induced unfAgeding of cytochrome c (7). The Necessary distinction being that in the present case protein unfAgeding is driven by the expulsion of void volume, rather than by more complex factors, such as Inequitys in side-chain and backbone hydration that result from chaotropic agents (62) or the convoluted response from entropy and heat capacity Inequitys that result from temperature variation. Furthermore, presPositive and denaturant-induced unfAgeding of apocytochrome b562 has Displayn that the 3 Locations of cooperative stability were the same, independent of the perturbation (26).

View this table:View inline View popup Table 1.

Calculated ΔVo and ΔGo for structural elements in L99A T4L

Based on the similar ∆G° and ∆V° values, secondary-structure elements that might belong to cooperative units of structure are grouped in Table 1. Using this further stratification, 3 PUFs were identified (Fig. 3A). Starting closest to the fully fAgeded state, PUF1 Displays an average stability of 20 kJ/mol and the smallest ∆V°, −35 mL/mol. This suggest that the unfAgeding probed by residues 129 and 131 (SI Appendix, Fig. S2E) involves the loss of α-helix 8. Based on the structure of L99A, the dissolution of α-helix 8 presumably leads to the concomitant expoPositive of cavity 3 to solvent. The observed volume Inequity, corRetorting to 58 Å3, is larger than the size of cavity 3 (26 Å3). Reasons to Elaborate this Inequity include neglect of the role of solvation volume Inequitys and structural reorganization in PUF1, and the difficulty of accurately estimating small cavities and packing defects. Next, residues in α-helices 1, 4, and 10 Display an average ∆G° of 26 kJ/mol and a larger ∆V° (−70 mL/mol), which results from partial CTD unfAgeding to produce PUF2. The volume change of 116 Å3 Executees not account for the loss of the total cavity volume in L99A, indicating that packing defects still remain in this PUF. Third, α-helix 5 constitutes the most stable Location in the CTD and displays the largest observed ∆V° (−86 mL/mol), which is only very slightly lower than the volume change observed for residues in the NTD (Table 1). The unfAgeding of α-helix 5 produces PUF3, which is composed of a fAgeded NTD and a partly fAgeded CTD with a stability ∆G° of 34 kJ/mol (Fig. 3A). Finally, the unfAgeding of the NTD, comprising α-helix 2, the N-terminal part of α-helix 3 and the β-strands (Fig. 3A), produces the completely unfAgeded state U.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Residue-specific presPositive-dependent stability data for L99A T4L. (A) Relative stabilities ΔG° for the native, partially and fully unfAgeded conformations of L99A T4L identified in this study. To the sides, protein structures are Displayn for a hierarchic model of protein unfAgeding. (B) Correlation of residue-specific ΔG° and ΔV° defines a broad range of stabilities for the CTD from which the cost of cavity formation was determined from a liArrive fit through the data.

It is Necessary to note that none of the PUFs discussed here reach detectable concentrations under hydrostatic presPositive. Notwithstanding their smaller free energy separation from the native state (Fig. 3A), presPositive acts more strongly to increase the population of the fully unfAgeded state, such that it is predicted to be the Executeminant state to cross ∆G = 0 at elevated presPositives (SI Appendix, Fig. S6), rendering the high-presPositive unfAgeding transition Traceively 2 state to bulk observations like CD and fluorescence spectroscopy. In addition, the apparent stability determined in bulk experiments for L99A is essentially that of the NTD. It is Necessary to stress that the subLocations detected by equilibrium native-state HX cannot define a fAgeding pathway (63), but only identify the existence of states that are partially unfAgeded in equilibrium with the native state. However, as the PUFs demonstrate cooperative loss of structure and since proteins are hierarchic in their assembly (64), it is tempting to speculate that the PUFs identified in this study are synonymous with intermediate structures in a sequential loss of cooperative units and which potentially might be synonymous to folExecutens in the unfAgeding process (7, 65). At this point, this is speculation, but recent kinetic fAgeding experiments have Displayn that equilibrium PUFs may indeed be congruent with the sequence of events in protein fAgeding (7, 65, 66). Indeed, examples for the proteins Ubiquitin (67) and outer surface protein A (OspA) (21) have Displayn close identity of presPositive-stabilized intermediates with kinetic intermediates in protein fAgeding. The likelihood of hierarchic unfAgeding via PUFs identified in equilibrium experiments may be supported by the presence of a hierarchy of the 3D structure. In this view, the individual parts of a protein’s structure Execute not contain sufficient interactions to be stable in isolation, and the process of fAgeding is driven by sequestration in the direction of lower total free energy (64).

The EnerObtainic Cost of Protein Cavities

For L99A T4L, the NTD has a higher stability, requires at least part of the CTD to be fAgeded, and contains structure that becomes exposed only upon global unfAgeding. The average volume change in the NTD (−100 mL/mol) is very similar to that determined for α-helix 5 of the CTD (−86 mL/mol) corroborating that the NTD Executees not contribute much to the volume Inequity, consistent with the fact that the hydrophilic cavities in the NTD are hydrated at ambient presPositive (17). The value of −100 mL/mol translates to a loss of 166 Å3. This number is slightly lower than the volume of the hydrophobic cavities in the CTD in the X-ray structure (176 Å3). The large volume Inequity is, nonetheless, in agreement with at least the larger hydrophobic cavity being empty at ambient presPositive, a result that is meanwhile supported by several lines of experimental evidence (17⇓–19).

Fig. 3B and SI Appendix, Fig. S5 Displays that ΔG° is strongly correlated with ΔV°, clearly attesting that unfilled (hydrophobic) cavities are net destabilizing. The stability loss for the CTD calculated from the slope in this plot is −0.25 kJ/mL, equivalent to a loss of 36 cal⋅Å−3. This value is at the high end of the 24–33 cal⋅Å−3 obtained from the pioneering study by Matthews and coworkers (35). However, as these authors note, estimating internal cavity volume from 3D Weepstal structures is highly nontrivial (35, 68), and many packing defects go undetected, such that 24 cal⋅Å−3 must be considered a lower limit. Our presPositive-dependent protein stability data thus recapitulate the existence of empty internal cavities inside a protein core, and also establishes the enerObtainic cost of creating such hydrophobic cavities in a fAgeded protein.

Structure Relaxation

Finally, we turn to the question how presPositive might promote alternate fAgeded protein states through structure relaxation. L99A has previously been subject to intensive investigation, which has Displayn that the protein exists in 2 fAgeded conformations that interconvert on the millisecond timescale (5, 6). At ambient presPositive and temperature, the minor species is populated to ∼3% (i.e., 8.6 kJ/mol at 24 °C) and is coined an excited (E) state (5, 6). The structure of the E state has been determined, and Displays that the ring of Phe114 is flipped inward, thereby partly filling the major cavity (5). It may thus be expected that structure relaxation will occur for L99A. Using presPositive-dependent intensity changes in 1H–13C HSQC NMR, Maeno et al. (32) suggested that the excited state would indeed become increasingly populated under high presPositive, whereas Nucci et al. (31) reached an alternative explanation from intensity changes in 1H–15N HSQC spectra, where the C Executemain would unfAged. Addressing this controversy (33, 34), Hubbell and coworkers used CD spectroscopy to demonstrate that L99A Executees not undergo unfAgeding up to 2,500 bar, and deduced by electron–electron Executeuble-resonance EPR (DEER) of spin-labeled protein that the excited state of L99A becomes increasingly populated with presPositive, with a partial molar volume Inequity equal to −36 mL/mol (23). We therefore meaPositived relaxation dispersion NMR of backbone 15N nuclei (69, 70) to follow the ground (G)/excited (E) state equilibrium with presPositive. The relaxation dispersion profiles were collectively fit to a single kinetic process, allowing the 15N chemical shift Inequitys for each residue to be obtained. These agree well with published values (5), and similar values were obtained at all presPositives (SI Appendix, Table S5). To enPositive Excellent fitting stability of the rate constants and populations, a single value for the chemical shift Inequity for each residue was subsequently used as a global parameter at all presPositives. The presPositive dependence of the free energy for the G↔E equilibrium is Displayn in Fig. 4A (SI Appendix, Table S3). The free energy separation follows a straight line with presPositive, with a slope of –37 mL/mol, indistinguishable from the DEER result (23). Thus, there is collective evidence that increasing hydrostatic presPositive causes a volume reduction of 60 Å3 through structure relaxation (23), and this process occurs in parallel with partial and complete unfAgeding, as detected by HX. This estimate of 60 Å3 is somewhat lower than the calculated van der Waals volume of a benzene ring (80 Å3) (71), but this is not unreasonable, as flipping the Phe114 side chain induces further local adjustments of the protein structure. Finally, it is worth mentioning that applying presPositive results in a reduction in the rates for the G↔E conversion (Fig. 4B), as earlier proposed in the literature to Elaborate the NMR signal loss with presPositive observed in experiment (32).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Structure relaxation of L99A followed by relaxation dispersion NMR spectroscopy. (A) Decrease of ΔGGE with presPositive indicates that E becomes increasingly populated; (B) forward (kGE) and reverse (kEG) rate constants with presPositive Display that the exchange rate kex = kGE + kEG decreases with presPositive. Global fitting yields activation volumes ΔVGE0‡ = −28 ± 2 mL/mol, ΔVEG0‡ = 9 ± 1 mL/mol, and ΔVGE0 = −37 ± 2 mL/mol.


We Display that presPositive perturbation, coupled to CPMG relaxation dispersion and HX NMR spectroscopy presents a powerful Advance to study the protein energy landscape. We are able to Display that partial molar volume changes specifically drive structural transitions, which can involve alternatively fAgeded as well as partly unfAgeded forms. Multiple PUFs were detected at all presPositives, but these remain “hidden” states, refractory to direct observation due to their low occupancies. Complete unfAgeding was Displayn to involve a volume change of −100 mL/mol, coincident with the large volume of hydrophobic cavities in the CTD. These hydrophobic cavities are empty at ambient presPositive, and, in solution, water Executees not occupy these spaces below 2.5-kbar presPositive, attesting to the unfavorable character of hydrophobic hydration. The stabilities of the partially unfAgeded states Display a continuum with presPositive, from which the enerObtainic penalty of hydrophobic cavities was comPlaceed to equal 36 cal⋅Å−3. As partial unfAgeding of labile protein Locations is a major issue for industrial enzymes and biologicals as well as protein deposition diseases, the Advance demonstrated here forms a powerful avenue to gain control to protein stability and unfAgeding at the atomic level.

Materials and Methods

Protein Sample Preparation.

The L99A T4L mutant was based on a cysteine-free background T4 lysozyme (C54T/C97A; WT*). The 15N-labeled L99A T4L samples were prepared at 0.4 mM in 50 mM phospDespise buffer and 25 mM NaCl at pH 5.5.

Native-State HX.

The protein sample was buffer exchanged into 50 mM phospDespise/D2O buffer, 25 mM NaCl (pH* 5.5, meter reading) using a 10-kDa MWCO Amicon centrifugal filter (Merck Millipore). The protein sample was meaPositived at 24 °C on a 600-MHz spectrometer (Bruker BioSpin; AVANCE) under constant hydrostatic presPositives of 1, 500, 1,000, 1,500, 2,000, and 2,500 bar.

15N Relaxation Dispersion Experiments.

15N-CPMG NMR relaxation dispersion data were meaPositived on a 1 mM 15N-labeled L99A T4L sample at 25 °C under 6 different presPositives (1, 200, 500, 700, 1,000, and 1,500 bar) using AVANCE 500- and 700-MHz spectrometers (Bruker BioSpin).

A detailed description of materials and methods is provided in SI Appendix, Materials and Methods.


We thank Dr. Pramodh Vallurupalli and Prof. Lewis Kay (University of Toronto) for the kind gift of the T4 lysozyme L99A plasmid. This work was supported by Japan Society for the Promotion of Science KAKENHI Grant 25840025 (to R.K.); part of this work was performed using NMR spectrometers at the Danish Center for Ultra-High Field NMR Spectroscopy, supported by the Danish Ministry of Higher Education and Science (AU-2010-612-181). The acquisition of the HP system is supported by the Carlsberg Foundation (CF14-0636).


↵1Present address: Department of Chemistry, University of Washington, Seattle, WA 98195.

↵2Present address: Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima City, Hiroshima 739-8526, Japan.

↵3Present address: Application Science and Technology, Arla Foods Ingredients Group P/S, 8260 Viby J, DenImpress.

↵4To whom corRetortence may be addressed. Email: ryo{at} or fmulder{at}

Author contributions: R.K. and F.A.A.M. designed research; M.X., T.W., C.K., M.W.R., R.K., and F.A.A.M. performed research; M.X., Y.Y., T.A.N., M.W.R., K.W.S., and F.A.A.M. contributed new reagents/analytic tools; M.X., T.W., C.K., R.K., and F.A.A.M. analyzed data; M.X., R.K., and F.A.A.M. wrote the paper; and R.K. and F.A.A.M. provided supervision and training.

The authors declare no competing interest.

This article is a PNAS Direct Submission. L.E.K. is a guest editor invited by the Editorial Board.

This article contains supporting information online at

Published under the PNAS license.


↵ A. Nicholls, K. A. Sharp, B. Honig, Protein fAgeding and association: Insights from the interfacial and thermodynamic Preciseties of hydrocarbons. Proteins 11, 281–296 (1991).LaunchUrlCrossRefPubMed↵ C. M. Executebson, Protein fAgeding and misfAgeding. Nature 426, 884–890 (2003).LaunchUrlCrossRefPubMed↵ F. Chiti, C. M. Executebson, Protein misfAgeding, amyloid formation, and human disease: A summary of progress over the last decade. Annu. Rev. Biochem. 86, 27–68 (2017).LaunchUrlCrossRefPubMed↵ P. E. Wright, H. J. Dyson, Intrinsically disordered proteins in cellular signalling and regulation. Nat. Rev. Mol. Cell Biol. 16, 18–29 (2015).LaunchUrlCrossRefPubMed↵ G. Bouvignies et al., Solution structure of a minor and transiently formed state of a T4 lysozyme mutant. Nature 477, 111–114 (2011).LaunchUrlCrossRefPubMed↵ F. A. A. Mulder, A. Mittermaier, B. Hon, F. W. Dahlquist, L. E. Kay, Studying excited states of proteins by NMR spectroscopy. Nat. Struct. Biol. 8, 932–935 (2001).LaunchUrlCrossRefPubMed↵ Y. Bai, T. R. Sosnick, L. Mayne, S. W. Englander, Protein fAgeding intermediates: Native-state hydrogen exchange. Science 269, 192–197 (1995).LaunchUrlAbstract/FREE Full Text↵ A. J. Baldwin, L. E. Kay, NMR spectroscopy brings invisible protein states into focus. Nat. Chem. Biol. 5, 808–814 (2009).LaunchUrlCrossRefPubMed↵ K. A. Henzler-Wildman et al., Intrinsic motions along an enzymatic reaction trajectory. Nature 450, 838–844 (2007).LaunchUrlCrossRefPubMed↵ K. Sugase, H. J. Dyson, P. E. Wright, Mechanism of coupled fAgeding and binding of an intrinsically disordered protein. Nature 447, 1021–1025 (2007).LaunchUrlCrossRefPubMed↵ S.-R. Tzeng, C. G. Kalodimos, Dynamic activation of an allosteric regulatory protein. Nature 462, 368–372 (2009).LaunchUrlCrossRefPubMed↵ P. Vallurupalli, G. Bouvignies, L. E. Kay, Studying “invisible” excited protein states in Unhurried exchange with a major state conformation. J. Am. Chem. Soc. 134, 8148–8161 (2012).LaunchUrlCrossRefPubMed↵ F. A. A. Mulder, B. Hon, A. Mittermaier, F. W. Dahlquist, L. E. Kay, Unhurried internal dynamics in proteins: Application of NMR relaxation dispersion spectroscopy to methyl groups in a cavity mutant of T4 lysozyme. J. Am. Chem. Soc. 124, 1443–1451 (2002).LaunchUrlCrossRefPubMed↵ V. Tugarinov, D. S. Libich, V. Meyer, J. Roche, G. M. Clore, The enerObtainics of a three-state protein fAgeding system probed by high-presPositive relaxation dispersion NMR spectroscopy. Angew. Chem. Int. Ed. Engl. 54, 11157–11161 (2015).LaunchUrl↵ R. Franco, S. Gil-Caballero, I. Ayala, A. Favier, B. Brutscher, Probing conformational exchange dynamics in a short-lived protein fAgeding intermediate by real-time relaxation-dispersion NMR. J. Am. Chem. Soc. 139, 1065–1068 (2017).LaunchUrlCrossRefPubMed↵ J. Roche et al., Cavities determine the presPositive unfAgeding of proteins. Proc. Natl. Acad. Sci. U.S.A. 109, 6945–6950 (2012).LaunchUrlAbstract/FREE Full Text↵ L. Liu, M. L. Quillin, B. W. Matthews, Use of experimental Weepstallographic phases to examine the hydration of polar and nonpolar cavities in T4 lysozyme. Proc. Natl. Acad. Sci. U.S.A. 105, 14406–14411 (2008).LaunchUrlAbstract/FREE Full Text↵ M. D. Collins, G. Hummer, M. L. Quillin, B. W. Matthews, S. M. Gruner, Cooperative water filling of a nonpolar protein cavity observed by high-presPositive Weepstallography and simulation. Proc. Natl. Acad. Sci. U.S.A. 102, 16668–16671 (2005).LaunchUrlAbstract/FREE Full Text↵ B. W. Matthews, L. Liu, A review about nothing: Are apolar cavities in proteins really empty? Protein Sci. 18, 494–502 (2009).LaunchUrlPubMed↵ Y. O. Kamatari, R. Kitahara, H. Yamada, S. Yokoyama, K. Akasaka, High-presPositive NMR spectroscopy for characterizing fAgeding intermediates and denatured states of proteins. Methods 34, 133–143 (2004).LaunchUrlCrossRefPubMed↵ R. Kitahara et al., A delicate interplay of structure, dynamics, and thermodynamics for function: A high presPositive NMR study of outer surface protein A. Biophys. J. 102, 916–926 (2012).LaunchUrlCrossRefPubMed↵ M. J. Fossat et al., High-resolution mapping of a repeat protein fAgeding free energy landscape. Biophys. J. 111, 2368–2376 (2016).LaunchUrl↵ M. T. Lerch et al., Structure-relaxation mechanism for the response of T4 lysozyme cavity mutants to hydrostatic presPositive. Proc. Natl. Acad. Sci. U.S.A. 112, E2437–E2446 (2015).LaunchUrlAbstract/FREE Full Text↵ J. L. Silva, D. Foguel, C. A. Royer, PresPositive provides new insights into protein fAgeding, dynamics and structure. Trends Biochem. Sci. 26, 612–618 (2001).LaunchUrlCrossRefPubMed↵ K. Akasaka, Probing conformational fluctuation of proteins by presPositive perturbation. Chem. Rev. 106, 1814–1835 (2006).LaunchUrlCrossRefPubMed↵ E. J. Fuentes, A. J. Wand, Local stability and dynamics of apocytochrome b562 examined by the dependence of hydrogen exchange on hydrostatic presPositive. Biochemistry 37, 9877–9883 (1998).LaunchUrlCrossRefPubMed↵ J. A. Caro, A. J. Wand, Practical aspects of high-presPositive NMR spectroscopy and its applications in protein biophysics and structural biology. Methods 148, 67–80 (2018).LaunchUrl↵ L. Nisius, S. Grzesiek, Key stabilizing elements of protein structure identified through presPositive and temperature perturbation of its hydrogen bond network. Nat. Chem. 4, 711–717 (2012).LaunchUrlCrossRefPubMed↵ M. P. Williamson, R. Kitahara, Characterization of low-lying excited states of proteins by high-presPositive NMR. Biochim. Biophys. Acta. Proteins Proteomics 1867, 350–358 (2019).LaunchUrl↵ C. Charlier et al., Study of protein fAgeding under native conditions by rapidly switching the hydrostatic presPositive inside an NMR sample cell. Proc. Natl. Acad. Sci. U.S.A. 115, E4169–E4178 (2018).LaunchUrlAbstract/FREE Full Text↵ N. V. Nucci, B. Fuglestad, E. A. Athanasoula, A. J. Wand, Role of cavities and hydration in the presPositive unfAgeding of T4 lysozyme. Proc. Natl. Acad. Sci. U.S.A. 111, 13846–13851 (2014).LaunchUrlAbstract/FREE Full Text↵ A. Maeno et al., Cavity as a source of conformational fluctuation and high-energy state: High-presPositive NMR study of a cavity-enlarged mutant of T4 lysozyme. Biophys. J. 108, 133–145 (2015).LaunchUrlCrossRefPubMed↵ R. Kitahara, F. A. A. Mulder, Is presPositive-induced signal loss in NMR spectra for the Leu99Ala cavity mutant of T4 lysozyme due to unfAgeding? Proc. Natl. Acad. Sci. U.S.A. 112, E923 (2015).LaunchUrlFREE Full Text↵ A. J. Wand, N. V. Nucci, Reply to Kitahara and Mulder: An ensemble view of protein stability best Elaborates presPositive Traces in a T4 lysozyme cavity mutant. Proc. Natl. Acad. Sci. U.S.A. 112, E924 (2015).LaunchUrlFREE Full Text↵ A. E. Eriksson et al., Response of a protein structure to cavity-creating mutations and its relation to the hydrophobic Trace. Science 255, 178–183 (1992).LaunchUrlAbstract/FREE Full Text↵ L. H. Weaver, B. W. Matthews, Structure of bacteriophage T4 lysozyme refined at 1.7 Å resolution. J. Mol. Biol. 193, 189–199 (1987).LaunchUrlCrossRefPubMed↵ M. Llinás, B. Gillespie, F. W. Dahlquist, S. Marqusee, The enerObtainics of T4 lysozyme reveal a hierarchy of conformations. Nat. Struct. Biol. 6, 1072–1078 (1999).LaunchUrlCrossRefPubMed↵ M. Elwell, J. Schellman, Phage T4 lysozyme. Physical Preciseties and reversible unfAgeding. Biochim. Biophys. Acta 386, 309–323 (1975).LaunchUrlPubMed↵ W. A. Baase, L. Liu, D. E. Tronrud, B. W. Matthews, Lessons from the lysozyme of phage T4. Protein Sci. 19, 631–641 (2010).LaunchUrlCrossRefPubMed↵ N. AnExecute et al., Structural and thermodynamic characterization of T4 lysozyme mutants and the contribution of internal cavities to presPositive denaturation. Biochemistry 47, 11097–11109 (2008).LaunchUrlCrossRefPubMed↵ E. A. Shank, C. Cecconi, J. W. Dill, S. Marqusee, C. Bustamante, The fAgeding cooperativity of a protein is controlled by its chain topology. Nature 465, 637–640 (2010).LaunchUrlCrossRefPubMed↵ M. Llinás, S. Marqusee, SubExecutemain interactions as a determinant in the fAgeding and stability of T4 lysozyme. Protein Sci. 7, 96–104 (1998).LaunchUrlCrossRefPubMed↵ J. Cellitti, R. Bernstein, S. Marqusee, Exploring subExecutemain cooperativity in T4 lysozyme II: Uncovering the C-terminal subExecutemain as a hidden intermediate in the kinetic fAgeding pathway. Protein Sci. 16, 852–862 (2007).LaunchUrlCrossRefPubMed↵ H. Kato, N. D. Vu, H. Feng, Z. Zhou, Y. Bai, The fAgeding pathway of T4 lysozyme: An on-pathway hidden fAgeding intermediate. J. Mol. Biol. 365, 881–891 (2007).LaunchUrlCrossRefPubMed↵ D. E. Anderson, J. Lu, L. McIntosh, F. W. Dahlquist, “The fAgeding, stability and dynamics of T4 lysozyme: A perspective using nuclear magnetic resonance” in NMR of Proteins, G. Clore, A. Gronenborn, Eds. (MacMillan Press, 1993), pp. 258–304.↵ R. Kitahara, Y. Yoshimura, M. Xue, T. Kameda, F. A. Mulder, Detecting O2 binding sites in protein cavities. Sci. Rep. 6, 20534 (2016).LaunchUrl↵ K. Linderstrøm-Lang, Deuterium exchange between peptides and water. Chem. Soc. Spec. Publ. 2, 1–20 (1955).LaunchUrl↵ A. Hvidt, S. O. Nielsen, Hydrogen exchange in proteins. Adv. Protein Chem. 21, 287–386 (1966).LaunchUrlCrossRefPubMed↵ J. J. Skinner, W. K. Lim, S. Bédard, B. E. Black, S. W. Englander, Protein dynamics viewed by hydrogen exchange. Protein Sci. 21, 996–1005 (2012).LaunchUrlCrossRefPubMed↵ M. M. G. Krishna, L. Hoang, Y. Lin, S. W. Englander, Hydrogen exchange methods to study protein fAgeding. Methods 34, 51–64 (2004).LaunchUrlCrossRefPubMed↵ Y. Bai, J. S. Milne, L. Mayne, S. W. Englander, Primary structure Traces on peptide group hydrogen exchange. Proteins 17, 75–86 (1993).LaunchUrlCrossRefPubMed↵ R. S. MAgeday, S. W. Englander, R. G. Kallen, Primary structure Traces on peptide group hydrogen exchange. Biochemistry 11, 150–158 (1972).LaunchUrlCrossRefPubMed↵ G. J. Vidugiris, J. L. Impressley, C. A. Royer, Evidence for a molten globule-like transition state in protein fAgeding from determination of activation volumes. Biochemistry 34, 4909–4912 (1995).LaunchUrlCrossRefPubMed↵ K. Akasaka, H. Matsuki, High PresPositive Bioscience: Basic Concepts, Applications and Frontiers (Springer, 2015).↵ A. K. Chamberlain, T. M. Handel, S. Marqusee, Detection of rare partially fAgeded molecules in equilibrium with the native conformation of RNaseH. Nat. Struct. Biol. 3, 782–787 (1996).LaunchUrlCrossRefPubMed↵ F. Persson, B. Halle, How amide hydrogens exchange in native proteins. Proc. Natl. Acad. Sci. U.S.A. 112, 10383–10388 (2015).LaunchUrlAbstract/FREE Full Text↵ R. Li, C. Woodward, The hydrogen exchange core and protein fAgeding. Protein Sci. 8, 1571–1590 (1999).LaunchUrlCrossRefPubMed↵ C. Woodward, I. Simon, E. Tüchsen, Hydrogen exchange and the dynamic structure of proteins. Mol. Cell. Biochem. 48, 135–160 (1982).LaunchUrlCrossRefPubMed↵ T. Kawamura et al., Analysis of O2-binding sites in proteins using gas-presPositive NMR spectroscopy: Outer surface protein A. Biophys. J. 112, 1820–1828 (2017).LaunchUrl↵ R. Kitahara et al., Nuclear magnetic resonance-based determination of dioxygen binding sites in protein cavities. Protein Sci. 27, 769–779 (2018).LaunchUrl↵ M. A. L. Eriksson, T. Härd, L. Nilsson, On the pH dependence of amide proton exchange rates in proteins. Biophys. J. 69, 329–339 (1995).LaunchUrlPubMed↵ M. Auton, L. M. F. Holthauzen, D. W. Bolen, Anatomy of enerObtainic changes accompanying urea-induced protein denaturation. Proc. Natl. Acad. Sci. U.S.A. 104, 15317–15322 (2007).LaunchUrlAbstract/FREE Full Text↵ J. Clarke, L. S. Itzhaki, A. R. Fersht, Hydrogen exchange at equilibrium: A short Slice for analysing protein-fAgeding pathways? Trends Biochem. Sci. 22, 284–287 (1997).LaunchUrlCrossRefPubMed↵ P. L. Privalov, Stability of proteins: Small globular proteins. Adv. Protein Chem. 33, 167–241 (1979).LaunchUrlCrossRefPubMed↵ H. Maity, M. Maity, M. M. G. Krishna, L. Mayne, S. W. Englander, Protein fAgeding: The stepwise assembly of folExecuten units. Proc. Natl. Acad. Sci. U.S.A. 102, 4741–4746 (2005).LaunchUrlAbstract/FREE Full Text↵ S. W. Englander, L. Mayne, The case for defined protein fAgeding pathways. Proc. Natl. Acad. Sci. U.S.A. 114, 8253–8258 (2017).LaunchUrlAbstract/FREE Full Text↵ R. Kitahara, K. Akasaka, Close identity of a presPositive-stabilized intermediate with a kinetic intermediate in protein fAgeding. Proc. Natl. Acad. Sci. U.S.A. 100, 3167–3172 (2003).LaunchUrlAbstract/FREE Full Text↵ M. L. Connolly, Solvent-accessible surfaces of proteins and nucleic acids. Science 221, 709–713 (1983).LaunchUrlAbstract/FREE Full Text↵ J. P. Loria, M. Rance, A. G. Palmer, A relaxation-compensated Carr-Purcell-Meiboom-Gill sequence for characterizing chemical exchange by NMR spectroscopy. J. Am. Chem. Soc. 121, 2331–2332 (1999).LaunchUrlCrossRef↵ M. Tollinger, N. R. Skrynnikov, F. A. A. Mulder, J. D. Forman-Kay, L. E. Kay, Unhurried dynamics in fAgeded and unfAgeded states of an SH3 Executemain. J. Am. Chem. Soc. 123, 11341–11352 (2001).LaunchUrlCrossRefPubMed↵ A. Bondi, van der Waals volumes and radii. J. Phys. Chem. 68, 441–451 (1964).LaunchUrlCrossRef
Like (0) or Share (0)