Effects of helix and fingertip mutations on the thermostability of xyn11A investigated by molecular dynamics simulations and enzyme activity assays

Local conformational changes and global unfolding pathways of wildtype xyn11A recombinant and its mutated structures were studied through a series of atomistic molecular dynamics (MD) simulations, along with enzyme activity assays at three incubation temperatures to investigate the effects of mutations at three different sites to the thermostability. The first mutation was to replace an unstable negatively charged residue at a surface beta turn near the active site (D32G) by a hydrophobic residue. The second mutation was to create a disulphide bond (S100C/N147C) establishing a strong connection between an alpha helix and a distal beta hairpin associated with the thermally sensitive Thumb loop, and the third mutation add an extra hydrogen bond (A155S) to the same alpha helix. From the MD simulations performed, MM/PBSA energy calculations of the unfolding energy were in a good agreement with the enzyme activities measured from the experiment, as all mutated structures demonstrated the improved thermostability, especially the S100C/N147C proved to be the most stable mutant both by the simulations and the experiment. Local conformational analysis at the catalytic sites and the xylan access region also suggested that mutated xyn11A structures could accommodate xylan binding. However, the analysis of global unfolding pathways showed that structural disruptions at the beta sheet regions near the N-terminal were still imminent. These findings could provide the insight on the molecular mechanisms underlying the enhanced thermostability due to mutagenesis and changes in the protein unfolding pathways for further protein engineering of the GH11 family xylanase enzymes.     

Leap-dynamics: efficient sampling of conformational space of proteins and peptides in solution

A molecular simulation scheme, called Leap-dynamics, that provides efficient sampling of protein conformational space in solution is presented. The scheme is a combined approach using a fast sampling method, imposing conformational 'leaps' to force the system over energy barriers, and molecular dynamics (MD) for refinement. The presence of solvent is approximated by a potential of mean force depending on the solvent accessible surface area. The method has been successfully applied to N-acetyl-L-alanine-N-methylamide (alanine dipeptide), sampling experimentally observed conformations inaccessible to MD alone under the chosen conditions. The method predicts correctly the increased partial flexibility of the mutant Y35G compared to native bovine pancreatic trypsin inhibitor. In particular, the improvement over MD consists of the detection of conformational flexibility that corresponds closely to slow motions identified by nuclear magnetic resonance techniques.     

Filling small, empty protein cavities: structural and energetic consequences

Most proteins contain small cavities that can be filled by replacing cavity-lining residues by larger ones. Since shortening mutations in hydrophobic cores tend to destabilize proteins, it is expected that cavity-filling mutations may conversely increase protein stability. We have filled three small cavities in apoflavodoxin and determined by NMR and equilibrium unfolding analysis their impact in protein structure and stability. The smallest cavity (14 A3) has been filled, at two different positions, with a variety of residues and, in all cases, the mutant proteins are locally unfolded, their structure and energetics resembling those of an equilibrium intermediate of the thermal unfolding of the wild-type protein. In contrast, two slightly larger cavities of 20 A3 and 21 A3 have been filled with Val to Ile or Val to Leu mutations and the mutants preserve both the native fold and the equilibrium unfolding mechanism. From the known relationship, observed in shortening mutations, between stability changes and the differential hydrophobicity of the exchanged residues and the volume of the cavities, the filling of these apoflavodoxin cavities is expected to stabilize the protein by approximately 1.5 kcal mol(-1). However, both urea and thermal denaturation analysis reveal much more modest stabilizations, ranging from 0.0 kcal mol(-1) to 0.6 kcal mol(-1), which reflects that the accommodation of single extra methyl groups in small cavities requires some rearrangement, necessarily destabilizing, that lowers the expected theoretical stabilization. As the size of these cavities is representative of that of the typical small, empty cavities found in most proteins, it seems unlikely that filling this type of cavities will give rise to large stabilizations.     

Molecular Dynamics Simulations and Principal Component Analysis on Human Laforin Mutation W32G and W32G/K87A

Mutations in human laforin lead to an autosomal neurodegenerative disorder Lafora disease. In N-terminal carbohydrate binding domain of laforin, two mutations W32G and K87A are reported as highly disease causing laforin mutants. Experimental studies reported that mutations are responsible for the abolishment of glycogen binding which is a critical function of laforin. Our current computational study focused on the role of conformational changes in human laforin structure due to existing single mutation W32G and prepared double mutation W32G/K87A related to loss of glycogen binding. We performed 10 ns molecular dynamics (MD) simulation studies in the Gromacs package for both mutations and analyzed the trajectories. From the results, the global properties like root mean square deviation, root mean square fluctuation, radius of gyration, solvent accessible surface area and hydrogen bonds showed structural changes in atomic level observed in W32G and W32G/K87A laforin mutants. The conformational change induced by mutants influenced the loss of the overall stability of the native laforin. Moreover, the change in overall motion of protein was analyzed by principal component analysis and results showed protein clusters expanded more than native and also change in direction in case of double mutant in conformational space. Overall, our report provides theoretical information on loss of structure–function relationship due to flexible nature of laforin mutants. In conclusion, comparative MD simulation studies support the experimental data on W32G and W32G/K87A related to the lafora disease mechanism on glycogen binding.     

Temperature‐induced unfolding behavior of proteins studied by tensorial elastic network model

Motivated by single molecule experiments and recent molecular dynamics (MD) studies, we propose a simple and computationally efficient method based on a tensorial elastic network model to investigate the unfolding pathways of proteins under temperature variation. The tensorial elastic network model, which relies on the native state topology of a protein, combines the anisotropic network model, the bond bending elasticity, and the backbone twist elasticity to successfully predicts both the isotropic and anisotropic fluctuations in a manner similar to the Gaussian network model and anisotropic network model. The unfolding process is modeled by breaking the native contacts between residues one by one, and by assuming a threshold value for strain fluctuation. Using this method, we simulated the unfolding processes of four well‐characterized proteins: chymotrypsin inhibitor, barnase, ubiquitein, and adenalyate kinase. We found that this step‐wise process leads to two or more cooperative, first‐order‐like transitions between partial denaturation states. The sequence of unfolding events obtained using this method is consistent with experimental and MD studies. The results also imply that the native topology of proteins “encrypts” information regarding their unfolding process. Proteins 2016; 84:1767–1775. © 2016 Wiley Periodicals, Inc.     

pH-induced equilibrium unfolding of apomyoglobin: Substitutions at conserved Trp14 and Met131 and non-conserved Val17 positions

A number of residues in globins family are well conserved but are not directly involved in the primary oxygen-carrying function of these proteins. A possible role for these conserved, non-functional residues has been suggested in promoting a rapid and correct folding process to the native tertiary structure. To test this hypothesis, we have studied pH-induced equilibrium unfolding of mutant apomyoglobins with substitutions of the conserved residues Trp14 and Met131, which are not involved in the function of myoglobin, by various amino acids. This allowed estimating their impact on the stability of various conformational states of the proteins and selecting conditions for a folding kinetics study. The results obtained from circular dichroism, tryptophan fluorescence, and differential scanning microcalorimetry for these mutant proteins were compared with those for the wild type protein and for a mutant with the non-conserved Val17 substituted by Ala. In the native folded state, all of the mutant apoproteins have a compact globular structure, but are destabilized in comparison to the wild type protein. The pH-induced denaturation of the mutant proteins occurs through the formation of a molten globule-like intermediate similar to that of the wild type protein. Thermodynamic parameters for all of the proteins were calculated using the three state model. Stability of equilibrium intermediates at pH ~4.0 was shown to be slightly affected by the mutations. Thus, all of the above substitutions influence the stability of the native state of these proteins. The cooperativity of conformational transitions and the exposed to solvent protein surface were also changed, but not for the substitution at Val17.     

Pyrazinamide drug resistance in RpsA mutant (∆438A) of Mycobacterium tuberculosis: Dynamics of essential motions and free-energy landscape analysis

Pyrazinamide is an essential first-line antitubercular drug which plays pivotal role in tuberculosis treatment. It is a prodrug that requires amide hydrolysis by mycobacterial pyrazinamidase enzyme for conversion into pyrazinoic acid (POA). POA is known to target ribosomal protein S1 (RpsA), aspartate decarboxylase (PanD), and some other mycobacterial proteins. Spontaneous chromosomal mutations in RpsA have been reported for phenotypic resistance against pyrazinamide. We have constructed and validated 3D models of the native and Δ438A mutant form of RpsA protein. RpsA protein variants were then docked to POA and long range molecular dynamics simulations were carried out. Per residue binding free-energy calculations, free-energy landscape analysis, and essential dynamics analysis were performed to outline the mechanism underlying the high-level PZA resistance conferred by the most frequently occurring deletion mutant of RpsA. Our study revealed the conformational modulation of POA binding site due to the disruptive collective modes of motions and increased conformational flexibility in the mutant than the native form. Residue wise MMPBSA decomposition and protein-drug interaction pattern revealed the difference of energetically favorable binding site in the wild-type (WT) protein in comparison with the mutant. Analysis of size and shape of minimal energy landscape area delineated higher stability of the WT complex than the mutant form. Our study provides mechanistic insights into pyrazinamide resistance in Δ438A RpsA mutant, and the results arising out of this study will pave way for design of novel and effective inhibitors targeting the resistant strains of Mycobacterium tuberculosis.     

Unfolding of hen egg lysozyme by molecular dynamics simulations at 300K: insight into the role of the interdomain interface

We present the results of two 1.2 ns molecular dynamics (MD) unfolding simulations on hen egg lysozyme in water at 300K, performed using a new procedure called PEDC (Path Exploration With Distance Constraints). This procedure allows exploration of low energy structures as a function of increasing RMSD from the native structure, and offers especially the possibility of extensive exploration of the conformational space during the initial unfolding stages. The two independent MD simulations gave similar chronology of unfolding events: disruption of the active site, kinking of helix C, partial unfolding of the three-stranded beta-sheet to a two-stranded sheet (during which the helices A, B, and D remain to a great extent native), and finally unfolding of the beta-domain and partial unfolding of the alpha-domain in which hydrophobic clusters persist. We show particularly that the loss of hydrophobic contacts between the beta-sheet turn residues Leu55 and Ile56 and the hydrobic patch of the alpha-domain destabilizes the beta-domain and leads to its unfolding, suggesting that the correct embedding of these residues in the alpha-beta interface may constitute the rate limiting step in folding. These results are in accord with experimental observations on the folding/unfolding behavior of hen egg lysozyme at room temperature. They would also explain the loss of stability and the tendency to aggregation observed for the mutant Leu55Thr, and the slow refolding kinetics observed in the analogous amyloidogenic variant of human lysozyme.     

Multi‐scale characterization of the energy landscape of proteins with application to the C3D/Efb‐C complex

We present a novel multi‐level methodology to explore and characterize the low energy landscape and the thermodynamics of proteins. Traditional conformational search methods typically explore only a small portion of the conformational space of proteins and are hard to apply to large proteins due to the large amount of calculations required. In our multi‐scale approach, we first provide an initial characterization of the equilibrium state ensemble of a protein using an efficient computational conformational sampling method. We then enrich the obtained ensemble by performing short Molecular Dynamics (MD) simulations on selected conformations from the ensembles as starting points. To facilitate the analysis of the results, we project the resulting conformations on a low‐dimensional landscape to efficiently focus on important interactions and examine low energy regions. This methodology provides a more extensive sampling of the low energy landscape than an MD simulation starting from a single crystal structure as it explores multiple trajectories of the protein. This enables us to obtain a broader view of the dynamics of proteins and it can help in understanding complex binding, improving docking results and more. In this work, we apply the methodology to provide an extensive characterization of the bound complexes of the C3d fragment of human Complement component C3 and one of its powerful bacterial inhibitors, the inhibitory domain of Staphylococcus aureus extra‐cellular fibrinogen‐binding domain (Efb‐C) and two of its mutants. We characterize several important interactions along the binding interface and define low free energy regions in the three complexes. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Ligand-linked stability of mutants of the C-domain of calmodulin

There is a necessary energetic linkage between ligand binding and stability in biological molecules. The critical glutamate in Site 4 was mutated to create two mutants of the C-domain of calmodulin yielding E140D and E140Q. These proteins were stably folded in the absence of calcium, but had dramatically impaired binding of calcium. We determined the stability of the mutant proteins in the absence and presence of calcium using urea-induced unfolding monitored by circular dichroism (CD) spectroscopy. These calcium-dependent unfolding curves were fit to models that allowed for linkage of stability to binding of a single calcium ion to the native and unfolded states. Simultaneous analysis of the unfolding profiles for each mutant yielded estimates for calcium-binding constants that were consistent with results from direct titrations monitored by fluorescence. Binding to the unfolded state was not an important energetic contributor to the ligand-linked stability of these mutants.     

Scrambled isomers as key intermediates in the oxidative folding of ligand binding module 5 of the low density lipoprotein receptor

The ligand binding module five (LA5) of the low density lipoprotein receptor is a small, single-domain protein of 40 residues and three disulfide bonds with a calcium binding motif that is essential for its structure and function. Several mutations in LA5 have been reported to cause familial hypercholesterolemia by impairing a proper folding of the module. The current study reports the oxidative folding and reductive unfolding pathways of wild type and mutant LA5 modules through kinetic and structural analysis of the trapped intermediates. Wild type LA5 folding involves an initial phase of nonspecific packing where the sequential oxidation of its cysteines gives rise to complex equilibrated populations of intermediates. In the presence of calcium, the attainment of a coordination-competent conformation becomes the rate-limiting step of folding while binding of the ion funnels both thermodynamically and kinetically the folding reaction toward the native state. In the absence of calcium, a scrambled isomer (termed Xa) constitutes the global free energy minimum of the folding process. Xa and the native form are stable, inter-convertible species whose relative populations at equilibrium appear displaced in disease-linked mutants toward the scrambled form. Because stable scrambled isomers such as Xa avoid the exposition of reactive cysteines in misfolded modules, they might constitute a strategy to prevent wrong interactions with other domains during folding of the receptor. Comparison of the folding pathways of wild type and mutant LA5 provides the molecular basis to understand how LA modules fold into a functional conformation or upon mutation misfold and lead to disease.     

Diabetes mellitus caused by mutations in human insulin: analysis of impaired receptor binding of insulins Wakayama,Los Angeles and Chicago using pharmacoinformatics

Several naturally occuring mutations in the human insulin gene are associated with diabetes mellitus. The three known mutant molecules, Wakayama, Los Angeles and Chicago were evaluated using molecular docking and molecular dynamics (MD) to analyse mechanisms of deprived binding affinity for insulin receptor (IR). Insulin Wakayama, is a variant in which valine at position A3 is substituted by leucine, while in insulin Los Angeles and Chicago, phenylalanine at positions B24 and B25 is replaced by serine and leucine, respectively. These mutations show radical changes in binding affinity for IR. The ZDOCK server was used for molecular docking, while AMBER 14 was used for the MD study. The published crystal structure of IR bound to natural insulin was also used for MD. The binding interactions and MD trajectories clearly explained the critical factors for deprived binding to the IR. The surface area around position A3 was increased when valine was substituted by leucine, while at positions B24 and B25 aromatic amino acid phenylalanine replaced by non-aromatic serine and leucine might be responsible for fewer binding interactions at the binding site of IR that leads to instability of the complex. In the MD simulation, the normal mode analysis, rmsd trajectories and prediction of fluctuation indicated instability of complexes with mutant insulin in order of insulin native insulin < insulin Chicago < insulin Los Angeles < insulin Wakayama molecules which corresponds to the biological evidence of the differing affinities of the mutant insulins for the IR.     

Identification of Potential Small Molecule Allosteric Modulator Sites on IL-1R1 Ectodomain Using Accelerated Conformational Sampling Method

The interleukin-1 receptor (IL-1R) is the founding member of the interleukin 1 receptor family which activates innate immune response by its binding to cytokines. Reports showed dysregulation of cytokine production leads to aberrant immune cells activation which contributes to auto-inflammatory disorders and diseases. Current therapeutic strategies focus on utilizing antibodies or chimeric cytokine biologics. The large protein-protein interaction interface between cytokine receptor and cytokine poses a challenge in identifying binding sites for small molecule inhibitor development. Based on the significant conformational change of IL-1R type 1 (IL-1R1) ectodomain upon binding to different ligands observed in crystal structures, we hypothesized that transient small molecule binding sites may exist when IL-1R1 undergoes conformational transition and thus suitable for inhibitor development. Here, we employed accelerated molecular dynamics (MD) simulation to efficiently sample conformational space of IL-1R1 ectodomain. Representative IL-1R1 ectodomain conformations determined from the hierarchy cluster analysis were analyzed by the SiteMap program which leads to identify small molecule binding sites at the protein-protein interaction interface and allosteric modulator locations. The cosolvent mapping analysis using phenol as the probe molecule further confirms the allosteric modulator site as a binding hotspot. Eight highest ranked fragment molecules identified from in silico screening at the modulator site were evaluated by MD simulations. Four of them restricted the IL-1R1 dynamical motion to inactive conformational space. The strategy from this study, subject to in vitro experimental validation, can be useful to identify small molecule compounds targeting the allosteric modulator sites of IL-1R and prevent IL-1R from binding to cytokine by trapping IL-1R in inactive conformations.     

Insulin assembly damps conformational fluctuations: Raman analysis of amide I linewidths in native states and fibrils

The crystal structure of insulin has been investigated in a variety of dimeric and hexameric assemblies. Interest in dynamics has been stimulated by conformational variability among crystal forms and evidence suggesting that the functional monomer undergoes a conformational change on receptor binding. Here, we employ Raman spectroscopy and Raman microscopy to investigate well-defined oligomeric species: monomeric and dimeric analogs in solution, native T(6) and R(6) hexamers in solution and corresponding polycrystalline samples. Remarkably, linewidths of Raman bands associated with the polypeptide backbone (amide I) exhibit progressive narrowing with successive self-assembly. Whereas dimerization damps fluctuations at an intermolecular beta-sheet, deconvolution of the amide I band indicates that formation of hexamers stabilizes both helical and non-helical elements. Although the structure of a monomer in solution resembles a crystallographic protomer, its encagement in a native assembly damps main-chain fluctuations. Further narrowing of a beta-sheet-specific amide I band is observed on reorganization of insulin in a cross-beta fibril. Enhanced flexibility of the native insulin monomer is in accord with molecular dynamics simulations. Such conformational fluctuations may initiate formation of an amyloidogenic nucleus and enable induced fit on receptor binding.     

Identification of two new LDL-receptor mutations causing homozygous familial hypercholesterolemia in a South African of Indian origin

South Africans of Indian origin have a high frequency of Familial Hypercholesterolemia (FH). Fibroblasts from a South African Indian FH homozygote, D, expressed about 30% of the normal number of LDL receptors. These receptors showed defective LDL binding. Sequence and haplotype analysis revealed that D had two different mutant LDL receptor alleles: FH Durban-1 is a point mutation [asp₆₉(GAT) to tyr(TAT)] in ligand-binding repeat 2 and FH Durban-2 is a point mutation [glu₁₁₉GAG) to lys(AAG)] in ligand-binding repeat three of the LDL receptor. Single-strand conformational polymorphism analysis, which was used in the initial detection of these mutations, was also employed for subsequent population screening assays. These mutations were not detected in amy of the South African Indian FH of hypercholesterolemic patients that were screened.     

Phage P22 tailspike protein: removal of head-binding domain unmasks effects of folding mutations on native-state thermal stability.

A shortened, recombinant protein comprising residues 109-666 of the tailspike endorhamnosidase of Salmonella phage P22 was purified from Escherichia coli and crystallized. Like the full-length tailspike, the protein lacking the amino-terminal head-binding domain is an SDS-resistant, thermostable trimer. Its fluorescence and circular dichroism spectra indicate native structure. Oligosaccharide binding and endoglycosidase activities of both proteins are identical. A number of tailspike folding mutants have been obtained previously in a genetic approach to protein folding. Two temperature-sensitive-folding (tsf) mutations and the four known global second-site suppressor (su) mutations were introduced into the shortened protein and found to reduce or increase folding yields at high temperature. The mutational effects on folding yields and subunit folding kinetics parallel those observed with the full-length protein. They mirror the in vivo phenotypes and are consistent with the substitutions altering the stability of thermolabile folding intermediates. Because full-length and shortened tailspikes aggregate upon thermal denaturation, and their denaturant-induced unfolding displays hysteresis, kinetics of thermal unfolding were measured to assess the stability of the native proteins. Unfolding of the shortened wild-type protein in the presence of 2% SDS at 71 degrees C occurs at a rate of 9.2 x 10(-4) s(-1). It reflects the second kinetic phase of unfolding of the full-length protein. All six mutations were found to affect the thermal stability of the native protein. Both tsf mutations accelerate thermal unfolding about 10-fold. Two of the su mutations retard thermal unfolding up to 5-fold, while the remaining two mutations accelerate unfolding up to 5-fold. The mutational effects can be rationalized on the background of the recently determined crystal structure of the protein.     

Unfolding crystallins: the destabilizing role of a beta-hairpin cysteine in betaB2-crystallin by simulation and experiment

The thermodynamic and kinetic stabilities of the eye lens family of betagamma-crystallins are important factors in the etiology of senile cataract. They control the chance of proteins unfolding, which can lead to aggregation and loss of transparency. betaB2-Crystallin orthologs are of low stability and comprise two typical betagamma-crystallin domains, although, uniquely, the N-terminal domain has a cysteine in one of the conserved folded beta-hairpins. Using high-temperature (500 K) molecular dynamics simulations with explicit solvent on the N-terminal domain of rodent betaB2-crystallin, we have identified in silico local flexibility in this folded beta-hairpin. We have shown in vitro using two-domain human betaB2-crystallin that replacement of this cysteine with a more usual aromatic residue (phenylalanine) results in a gain in conformational stability and a reduction in the rate of unfolding. We have used principal components analysis to visualize and cluster the coordinates from eight separate simulated unfolding trajectories of both the wild-type and the C50F mutant N-terminal domains. These data, representing fluctuations around the native well, show that although the mutant and wild-type appear to behave similarly over the early time period, the wild type appears to explore a different region of conformational space. It is proposed that the advantage of having this low-stability cysteine may be correlated with a subunit-exchange mechanism that allows betaB2-crystallin to interact with a range of other beta-crystallin subunits.     

人亲环素18抑制肌酸激酶的去折叠

新生肽链折叠过程中容易出现错误折叠与聚沉,从而导致折叠病等病理现象. 分子伴侣具有辅助其他蛋白质正确折叠,保护蛋白质分子结构的功能.本文选用人肌肌酸激酶为靶蛋白,研究了肽基脯氨酰顺反异构酶人亲环素18（human cyclophilin 18,hCyp18）对人肌肌酸激酶去折叠的作用,发现hCyp18能够抑制人肌肌酸激酶在热变性与化学变性过程中的失活与构象变化,并抑制人肌肌酸激酶在化学变性过程中的聚沉,因此推断hCyp18具有针对人肌肌酸激酶的分子伴侣功能.本文同时研究了hCyp18与人肌肌酸激酶的结合作用,对hCyp18的作用机制进行了初步探讨.     

Effects of Pro1266Leu mutation on structure and function of glycoprotein Ib binding domain of von Willebrand factor

Glycoprotein Ibα (GpIbα) binding ability of A1 domain of von Willebrand factor (vWF) facilitates platelet adhesion that plays a crucial role in maintaining hemostasis and thrombosis at the site of vascular damage. There are both “loss as well as gain of function” mutations observed in this domain. Naturally occurring “gain of function” mutations leave self-activating impacts on the A1 domain which turns the normal binding to characteristic constitutive binding with GPIbα. These “gain of function” mutations are associated with the von Willebrand disease type 2B. In recent years, studies focused on understanding the mechanism and conformational patterns attached to these phenomena have been conducted, but the conformational pathways leading to such binding patterns are poorly understood as of now. To obtain a microscopic picture of such events for the better understanding of pathways, we used molecular dynamics (MD) simulations along with principal component analysis and normal mode analysis to study the effects of Pro1266Leu (Pro503Leu in structural context) mutation on the structure and function of A1 domain of vWF. MD simulations have provided atomic-level details of intermolecular motions as a function of time to understand the dynamic behavior of A1 domain of vWF. Comparative analysis of the trajectories obtained from MD simulations of both the wild type and Pro503Leu mutant suggesting appreciable conformational changes in the structure of mutant which might provide a basis for assuming the “gain of function” effects of these mutations on the A1 domain of vWF, resulting in the constitutive binding with GpIbα.     

Unfolding of Green Fluorescent Protein mut2 in wet nanoporous silica gels

Many of the effects exerted on protein structure, stability, and dynamics by molecular crowding and confinement in the cellular environment can be mimicked by encapsulation in polymeric matrices. We have compared the stability and unfolding kinetics of a highly fluorescent mutant of Green Fluorescent Protein, GFPmut2, in solution and in wet, nanoporous silica gels. In the absence of denaturant, encapsulation does not induce any observable change in the circular dichroism and fluorescence emission spectra of GFPmut2. In solution, the unfolding induced by guanidinium chloride is well described by a thermodynamic and kinetic two-state process. In the gel, biphasic unfolding kinetics reveal that at least two alternative conformations of the native protein are significantly populated. The relative rates for the unfolding of each conformer differ by almost two orders of magnitude. The slower rate, once extrapolated to native solvent conditions, superimposes to that of the single unfolding phase observed in solution. Differences in the dependence on denaturant concentration are consistent with restrictions opposed by the gel to possibly expanded transition states and to the conformational entropy of the denatured ensemble. The observed behavior highlights the significance of investigating protein function and stability in different environments to uncover structural and dynamic properties that can escape detection in dilute solution, but might be relevant for proteins in vivo.