Unfolding Pathway Research Articles

Different unfolding pathways of homologous alpha amylases from Bacillus licheniformis (BLA) and Bacillus amyloliquefaciens (BAA) in GdmCl and urea

Understanding the factors governing stability of proteins is fundamentally and industrially important topic in protein science. Bacterial alpha amylases are industrially important enzymes which are involved in the breakage of α-1, 4-glycosidic bonds in starch. Current study is focussed on elucidating the role of non-covalent interactions in the differential stability of alpha amylases from thermophilic like Bacillus licheniformis (BLA) and mesophilic Bacillus amyloliquefaciens (BAA). The conformational stability of BLA is slightly higher than BAA in GdmCl which are 2.94 and 2.53 kcal/mol respectively. BLA does not unfold even in 8.0 M urea at pH 7.0, while for BAA, the conformational stability in urea is calculated to be 2.22 kcal/mol. A structure-function relationship study of BLA reveals the non-coincidental unfolding by far UV-CD, enzyme activity and tryptophan fluorescence which indicates the presence of partially unfolded intermediates. The existence of intermediates in BLA during GdmCl induced unfolding was further confirmed by ANS fluorescence. The unfolding kinetics of both enzymes showed biphasic nature with slower unfolding of BLA compare to BAA pointing towards the higher kinetic stability of BLA than BAA. Taken together, our work demonstrates that the higher stability of BLA is mainly due to the combination of ionic and hydrophobic interactions.

Al (III) metal augment thermal aggregation and fibrillation in protein: Role of metal toxicity in neurological diseases.

Protein fibrillation is a leading cause of innumerable neurodegenerative diseases. The exact underlying mechanism associated with the formation of fibrils is yet to be known. Recently, the role of metal ions resulting into fibrillation of proteins has gained attention of the scientific community. In this piece of work, we have investigated the effect of the aluminum (Al) metal ion on the kinetics of aggregation of bovine serum albumin (BSA) protein under physiological conditions by employing several biophysical and microscopic techniques. Quenching of tryptophan fluorescence was observed along with 9 nm blue shift, demonstrating BSA becomes more hydrophobic during unfolding pathway of thermal denaturation. Moreover, ANS (8-Anilino-1-naphthalene sulfonic acid) binding shows quenching in fluorescence intensity with increasing time of incubation at 65 °C, suggesting unfolding leading to the disruption of hydrophobic patches in BSA. Besides, Thioflavin T intensity indicated a significant acceleration in BSA fibrillation at a ratio of 1:1 and 1:2 of BSA and Al (III) metal ion respectively. In addition, circular dichroism (CD) spectroscopy study revealed the transition of BSA from α-helical conformation to the β-sheet rich structure. Molecular docking analysis demonstrated significant binding affinity (−1.2 kcal/mol) of Al (III) with BSA involving Phe501, Phe506, Val575, Thr578, Gln579, Leu531 residues. Transmission electron microscopy (TEM) reaffirm augmentation of thermal-induced BSA fibril formation in the presence of Al (III) metal ions. This study highlights the metal chelating potency as the possible therapeutic target for neurological diseases.

Unfolding pathway and intermolecular interactions of the cytochrome subunit in the bacterial photosynthetic reaction center

Precise folding of photosynthetic proteins and organization of multicomponent assemblies to form functional entities are fundamental to efficient photosynthetic electron transfer. The bacteriochlorophyll b-producing purple bacterium Blastochloris viridis possesses a simplified photosynthetic apparatus. The light-harvesting (LH) antenna complex surrounds the photosynthetic reaction center (RC) to form the RC-LH1 complex. A non-membranous tetraheme cytochrome (4Hcyt) subunit is anchored at the periplasmic surface of the RC, functioning as the electron donor to transfer electrons from mobile electron carriers to the RC. Here, we use atomic force microscopy (AFM) and single-molecule force spectroscopy (SMFS) to probe the long-range organization of the photosynthetic apparatus from Blc. viridis and the unfolding pathway of the 4Hcyt subunit in its native supramolecular assembly with its functional partners. AFM images reveal that the RC-LH1 complexes are densely organized in the photosynthetic membranes, with restricted lateral protein diffusion. Unfolding of the 4Hcyt subunit represents a multi-step process and the unfolding forces of the 4Hcyt α-helices are approximately 121 picoNewtons. Pulling of 4Hcyt could also result in the unfolding of the RC L subunit that binds with the N-terminus of 4Hcyt, suggesting strong interactions between RC subunits. This study provides new insights into the protein folding and interactions of photosynthetic multicomponent complexes, which are essential for their structural and functional integrity to conduct photosynthetic electron flow.

Protein mechanics probed using simple molecular models

BackgroundSingle-molecule experimental techniques such as optical tweezers or atomic force microscopy are a direct probe of the mechanical unfolding/folding of individual proteins. They are also a means to investigate free energy landscapes. Protein force spectroscopy alone provides limited information; theoretical models relate measurements to thermodynamic and kinetic properties of the protein, but do not reveal atomic level information. By building a molecular model of the protein and probing its properties through numerical simulation, one can gauge the response to an external force for individual interatomic interactions and determine structures along the unfolding pathway. In combination, single-molecule force probes and molecular simulations contribute to uncover the rich behavior of proteins when subjected to mechanical force. Scope of reviewWe focus on how simplified protein models have been instrumental in showing how general properties of the free energy landscape of a protein relate to its response to mechanical perturbations. We discuss the role of simple protein models to explore the complexity of free energy landscapes and highlight important conceptual issues that more chemically accurate models with all-atom representations of proteins and solvent cannot easily address. Major conclusionsNative-centric, coarse-grained models, despite simplifications in chemical detail compared to all-atom models, can reproduce and interpret experimental results. They also highlight instances where the theoretical framework used to interpret single-molecule data is too simple. However, these simple models are not able to reproduce experimental findings where non-native contacts are involved. General significanceMechanical forces are ubiquitous in the cell and it is increasingly clear that the way a protein responds to mechanical perturbation is important.

The Interplay between Molten Globules and Heme Disassociation Defines Human Hemoglobin Disassembly

Hemoglobin functions as a tetrameric oxygen transport protein, with each subunit containing a heme cofactor. Its denaturation, either in vivo or in vitro, involves autoxidation to methemoglobin, followed by cofactor loss and globin unfolding. We have proposed a global disassembly scheme for human methemoglobin, linking hemin (ferric protoporphyrin IX) disassociation and apoprotein unfolding pathways. The model is based on the evaluation of circular dichroism and visible absorbance measurements of guanidine-hydrochloride-induced disassembly of methemoglobin and previous measurements of apohemoglobin unfolding. The populations of holointermediates and equilibrium disassembly parameters were estimated quantitatively for adult and fetal hemoglobins. The key stages are characterized by hexacoordinated hemichrome intermediates, which are important for preventing hemin disassociation from partially unfolded, molten globular species during early disassembly and late-stage assembly events. Both unfolding experiments and independent small angle x-ray scattering measurements demonstrate that heme disassociation leads to the loss of tetrameric structural integrity. Our model predicts that after autoxidation, dimeric and monomeric hemichrome intermediates occur along the disassembly pathway inside red cells, where the hemoglobin concentration is very high. This prediction suggests why misassembled hemoglobins often get trapped as hemichromes that accumulate into insoluble Heinz bodies in the red cells of patients with unstable hemoglobinopathies. These Heinz bodies become deposited on the cell membranes and can lead to hemolysis. Alternatively, when acellular hemoglobin is diluted into blood plasma after red cell lysis, the disassembly pathway appears to be dominated by early hemin disassociation events, which leads to the generation of higher fractions of unfolded apo subunits and free hemin, which are known to damage the integrity of blood vessel walls. Thus, our model provides explanations of the pathophysiology of hemoglobinopathies and other disease states associated with unstable globins and red cell lysis and also insights into the factors governing hemoglobin assembly during erythropoiesis.

The Effect of Chain Connectivity on the Thermodynamic, Kinetic and Mechanical Properties of Azurin

Azurin, a blue-copper protein, is responsible for electron transfer in Pseudomonas aeruginosa. It has widely been studied for its stability, electronic and electron transport properties, with potential applications in biomaterials-based devices. The amino acid chain connectivity is of fundamental importance in modulating the protein stability and folding. Hence, here we studied the thermodynamic, kinetic and mechanical properties of a circular permutant of apo-azurin (Azu-CPN42), with its N-terminus at Asn42, and compared them with those of the wild-type apo-azurin (WT-Azu). While the spectroscopic and copper-binding properties of Azu-CPN42 are unaltered, we observe changes in protein stability. Equilibrium unfolding studies using GdHCl suggest a drop of ∼0.6M in Cm and ∼13 kJ/mol in free energy of unfolding for Azu-CPN42 compared to that of WT-Azu. Kinetic studies reveal increase in the spontaneous unfolding rate by two-orders of magnitude, while the spontaneous folding rate being similar. Mechanical properties probed using single-molecule atomic force microscope suggest parallel unfolding pathways via 2- and 3-states, similar to that of WT-Azu reported previously. Interestingly, the intermediate stability is lower than that of the native state of Azu-CPN42 by ∼10pN, in accord with that of WT-Azu. As anticipated, the mechanical unfolding pathways of Azu-CPN42 and WT-Azu are different, as the two proteins are pulled along different N-C termini. This resulted in different mechanical intermediates, with a broad distribution of unfolding contour lengths (∼3-25 nm) for Azu-CPN42 while those of WT-Azu are centered at 7.5 ± 1.5 nm. This huge diversity may arise due to mechanical unraveling from both C- and N- termini as opposed to only C-terminal unraveling observed in WT-Azu. Our study reveals a decreased thermodynamic and kinetic stability and altered mechanical unfolding pathway of azurin upon changing its chain connectivity through circular permutation.

Reconstruction of ARNT PAS-B Unfolding Paths by Steered MD and Artificial Neural Network Reveals New Putative Binding Conformations

Several proteins have been identified that can switch between different folds, expanding their functional role through previously unknown binding interactions. In this context, experimental evidence suggests that the PAS-B domain of the Aryl Hydrocarbon Receptor Nuclear Translocator protein (ARNT) has a fragile β-sheet structure that can undergo a +3 register shift upon a single mutation, changing its dimerization surface. Both unfolding and interconversion mechanisms require the same uncharacterized partially-unfolded state. Single-molecule Atomic Force Microscopy measurements (AFM) confirmed that the ARNT PAS-B domain is mechanically labile and revealed two distinct unfolding pathways, one following a single-stage mechanism, and the other showing an intermediate state. An atomistic characterisation of unfolding paths and relevant intermediate states has been carried out to identify new ARNT conformations possibly involved in partner recruitment. The unfolding pathways for ARNT PAS-B were simulated by steered Molecular Dynamics. Multiple replicas were recorded to explore different paths at low pulling speed. The overall model of unfolding was then reconstructed using a self-organizing map (SOM), a type of artificial neural network with an explicit visual representation. The map was trained on inter-residue distances of native contacts. The SOM model clearly depicts two alternative pathways in a human-understandable two-dimensional map. Retracing of single replicas on the map highlights a two-stage mechanism with initial unfolding of the C-terminal strand in 1/3 of the simulations, in agreement with AFM findings. Shortest path analysis on the map correctly detects replicas with minimal steering work. The partially-folded states extracted from the map could have a role in selectivity for partner recruitment during ARNT transcriptional activity.

Role of Zero-Order Loop in Protein Unfolding Case Study with Apoazurin

Pseudomonas aeruginosa apoazurin (apo, without the copper cofactor) is a two-state folder that has a single disulfide bond between residues 3 and 26. This bond covalently connects the N-termini of beta-strands 1 and 3; thereby it creates a zero-order loop. The loop restricts the conformational space for the apoazurin polypeptide. In order to understand the role played by the zero-order loop, we used molecular dynamics (MD) simulations to compare two variants of apoazurin; one variant called “loop” which contained the disulfide and another called “open” in which the disulfide bond between residues 3 and 26 was removed. MD simulations were performed to probe the unfolding pathway and stability of the two apoazurin variants at different urea concentrations and temperatures. Our results show that the folded structure apoazurin is somewhat more stable due to the presence of the disulfide bond. However, the disulfide bond plays a prominent role in the apoazurin unfolding mechanism: we find that it changes both the folding-transition state and the unfolded-state ensemble of conformations.

Mg2+ Impacts the Twister Ribozyme through Push-Pull Stabilization of Nonsequential Phosphate Pairs

RNA molecules perform a variety of biological functions for which the correct three-dimensional structure is essential, including as ribozymes where they catalyze chemical reactions. Metal ions, especially Mg2+, neutralize these negatively charged nucleic acids and specifically stabilize RNA tertiary structures as well as impact the folding landscape of RNAs as they assume their tertiary structures. Specific binding sites of Mg2+ in folded conformations of RNA have been studied extensively; however, the full range of interactions of the ion with compact intermediates and unfolded states of RNA is challenging to investigate, and the atomic details of the mechanism by which the ion facilitates tertiary structure formation is not fully known. Here, umbrella sampling combined with oscillating chemical potential Grand Canonical Monte Carlo/molecular dynamics simulations are used to capture the energetics and atomic-level details of Mg2+-RNA interactions that occur along an unfolding pathway of the Twister ribozyme. The free energy profiles reveal stabilization of partially unfolded states by Mg2+, as observed in unfolding experiments, with this stabilization being due to increased sampling of simultaneous interactions of Mg2+ with two or more nonsequential phosphate groups. Notably, these results indicate a push-pull mechanism in which the Mg2+-RNA interactions actually lead to destabilization of specific nonsequential phosphate-phosphate interactions (i.e., pushed apart), whereas other interactions are stabilized (i.e., pulled together), a balance that stabilizes unfolded states and facilitates the folding of Twister, including the formation of hydrogen bonds associated with the tertiary structure. This study establishes a better understanding of how Mg2+-ion interactions contribute to RNA structural properties and stability.

Investigation of guanidinium chloride-induced unfolding pathway of sphingosine kinase 1

Sphingosine kinase 1 (SphK1) is a lipid kinase which plays vital role in the regulation of varieties of biological processes including, cell growth, apoptosis and mitogenesis. In the present study, we investigated the guanidinium chloride (GdmCl)-induced denaturation of SphK1 at pH 8.0 and 25 °C using two different spectroscopic probes, i.e., mean residue ellipticity at 222 nm ([θ]222) and fluorescence emission maxima (λmax). A significant overlap between the transition curves obtained from both the spectral properties indicate that GdmCl-induced unfolding of SphK1 follows two-state process i.e., Native (N) ⇌ Denatured (D) state. Interestingly, a visible protein aggregation was observed at low concentrations of GdmCl ([GdmCl] ≤ 1.5 M). The analysis of transition curves was done to estimate the thermodynamic parameters associated with the stability of SphK1. To complement our experimental findings, 100 ns molecular dynamics (MD) simulations were performed. Spectroscopic studies together with MD simulations provided mechanistic insights of unfolding pathway of SphK1 along with its stability parameters.

Mechanical Unfolding and Refolding of Single Membrane Proteins by Atomic Force Microscopy.

Atomic force microscopy (AFM)-based single-molecule force spectroscopy allows direct physical manipulation of single membrane proteins under near-physiological conditions. It can be applied to study mechanical properties and molecular interactions as well as unfolding and folding pathways of membrane proteins. Here, we describe the basic procedure to study membrane proteins by single-molecule force spectroscopy and discuss general requirements of the experimental setup as well as common pitfalls typically encountered when working with membrane proteins in AFM.

Constant velocity pulling and unfolding of thyroid hormone receptor by steered molecular dynamics

Unfolding pathways of T3 liganded thyroid hormone receptor (THRT3) can be studied by using the protocols of steered molecular dynamics (SMD). Theory of constant velocity pulling has been implemented to the structure of THRT3 in a neutral water-ion solution equilibrated up to 20 ns. The globular form of THRT3 is completely unfolded extending N-C termini from 38 Å to 876 Å at a constant speed of 0.1 Å/ps by means of 8.5 ns long SMD simulations. The peak force measured in the intermediate conformations is related to a burst of backbone H-bonds among a-helices and b-hairpins. With decrease in H-bonds, electrostatic energy increases by losing gradually the secondary structure and separating a and b-strands in solution. The force at the end (t > 8.5 ns) increases steeply with the large increase in bond-angle and bond-length potentials when the system becomes completely unfolded. The hydrophobic ligand binding domain (LBD) of THR-b with load bearing H-bonds protects T3 from water attack. Even after complete unfolding of THR-b LBD, the position of T3 is not deviated more than 2.5 Å and a large number of water molecules remain in the surrounding of this domain area. This is a strong evidence for the mechanochemical stability of a receptor protein’s LBD towards hormone activated gene expressions followed by ligand binding and dissociation.
 BIBECHANA 17 (2020) 50-57

Shared unfolding pathways of unrelated immunoglobulin-like β-sandwich proteins.

The Dynameomics project contains native state and unfolding simulations of 807 protein domains, where each domain is representative of a different metafold; these metafolds encompass ~97% of protein fold space. There is a long-standing question in structural biology as to whether proteins in the same fold family share the same folding/unfolding characteristics. Using molecular dynamics simulations from the Dynameomics project, we conducted a detailed study of protein unfolding/folding pathways for 5 protein domains from the immunoglobulin (Ig)-like β-sandwich metafold (the highest ranked metafold in our database). The domains have sequence similarities ranging from 4 to 15% and are all from different SCOP superfamilies, yet they share the same overall Ig-like topology. Despite having very different amino acid sequences, the dominant unfolding pathway is very similar for the 5 proteins, and the secondary structures that are peripheral to the aligned, shared core domain add variability to the unfolding pathway. Aligned residues in the core domain display consensus structure in the transition state primarily through conservation of hydrophobic positions. Commonalities in the obligate folding nucleus indicate that insights into the major events in the folding/unfolding of other domains from this metafold may be obtainable from unfolding simulations of a few representative proteins.

Delineating the conformational dynamics of intermediate structures on the unfolding pathway of β-lactoglobulin in aqueous urea and dimethyl sulfoxide

The funnel shaped energy landscape model of the protein folding suggests that progression of folding proceeds through multiple pathways, having the multiple intermediates which leads to multidimensional free-energy surface. Herein, we applied all-atom MD simulation to conduct a comparative study on the structure of β-lactoglobulin (β-LgA) in aqueous mixture of 8 M urea and 8 M dimethyl sulfoxide (DMSO), at different temperatures. The cumulative results of multiple simulations suggest a common unfolding pathway of β-LgA, occurred through the stable and meta-stable intermediates (I), in both urea and DMSO. However, the free-energy landscape (FEL) analyses show that the structural transitions of I-states are energetically different. In urea, FEL shows distinct ensemble of intermediates, I1 and I2, separated by the energy barrier of ∼3.0 kcal mol−1. Similarly, we find the population of two distinct I1 and I2 states in DMSO, however, the I1 appeared transiently around ∼30–35 ns and is short-lived. But, the I2 ensemble is observed structurally compact and long-lived (∼50–150 ns) as compared to unfolding in urea. Furthermore, the I1 and I2 are separated through a high energy barrier of ∼6.0 kcal mol−1. Thus, our results provide the structural insights of intermediates which essentially bear the signature of a different unfolding pathway of β-LgA in urea and DMSO.Abbreviationsβ-LgAβ-lactoglobulinDMSOdimethyl sulfoxideFELfree-energy landscapeGdmClguanidinium chlorideIintermediate stateMGmolten globule statePMEparticle mesh EwaldQfraction of native contactsRMSDroot mean square deviationRMSFroot mean square fluctuationRgradius of gyrationSASAsolvent Accessible Surface AreascSASAthe side chain SASATrptryptophanCommunicated by Ramaswamy H. Sarma

Atomic Force Microscopy as a Powerful Multifunctional Tool for Probing the Behaviors of Single Proteins.

Proteins play a crucial role in regulating life activities and therefore investigating the behaviors of proteins is of critical significance for understanding the underlying mechanisms guiding the physiological and pathological processes of living organisms. Traditional molecular biochemical methods for protein assays are based on ensemble measurements which reflect the averaged behaviors of the whole molecular populations, veiling the rare activities of small molecular subpopulations. Achievements obtained in the past decades have proved that atomic force microscopy (AFM) is a powerful multifunctional tool for characterizing the behaviors of single proteins at work in aqueous conditions with unprecedented spatiotemporal resolution, which provides novel insights into nanoscopic molecular mechanisms and contributes much to the communities of protein biology. In this article, we review the recent advances in AFM-based single-protein analysis from several facets (including morphological imaging, single-molecule force recognition, mechanical unfolding pathways, and high-speed AFM molecular detection), and provide perspectives for future progression.

Unfolding pathway and its identifiability in heterogeneous chains of bistable units

We investigate the behavior of a chain of bistable units with an heterogeneous distribution of energy jumps between the folded and unfolded states. For homogeneous chains, loaded by soft or hard devices, all units at each switching occurrence have the same probability to unfold and it is therefore impossible to identify an unfolding pathway. Conversely, the heterogeneity represents a quenched disorder from the statistical mechanics point of view, and is able to break the symmetry eventually generating an unfolding pathway. We prove that the most probable pathway is realized by arranging the energy jumps in ascending order. Hence, the mechanics of this system is able to implement a statistical sorting procedure. We quantitatively evaluate the identifiability of the obtained unfolding pathway in terms of the variance of the heterogeneous energy jumps and the temperature. This concept is applied to both deterministic and random distributions of energy jumps within the chain.

Reversible folding energetics of Yersinia Ail barrel reveals a hyperfluorescent intermediate

Deducing the molecular details of membrane protein folding has lately become an important area of research in biology. Using Ail, an outer membrane protein (OMP) from Yersina pestis as our model, we explore details of β-barrel folding, stability, and unfolding. Ail displays a simple transmembrane β-barrel topology. Here, we find that Ail follows a simple two-state mechanism in its folding and unfolding thermodynamics. Interestingly, Ail displays multi-step folding kinetics. The early kinetic intermediates in the folding pathway populate near the unfolded state (βT ≈ 0.20), and do not display detectable changes in the local environment of the two interface indoles. Interestingly, tryptophans regulate the late events of barrel rearrangement, and Ail thermodynamic stability. We show that W149 → Y/F/A substitution destabilizes Ail by ~0.13–1.7 kcal mol−1, but retains path–independent thermodynamic equilibrium of Ail. In surprising contrast, substituting W42 and retaining W149 shifts the thermodynamic equilibrium to an apparent kinetic retardation of only the unfolding process, which gives rise to an associated increase in scaffold stability by ~0.3–1.1 kcal mol−1. This is accompanied by the formation of an unusual hyperfluorescent state in the unfolding pathway that is more structured, and represents a conformationally dynamic unfolding intermediate with the interface W149 now lipid solvated. The defined role of each tryptophan and poorer folding efficiency of Trp mutants together presents compelling evidence for the importance of interface aromatics in the unique (un)folding pathway of Ail, and offers interesting insight on alternative pathways in generalized OMP assembly and unfolding mechanisms.

3D structure stability of the HIV-1 TAR RNA in ion solutions: A coarse-grained model study.

As an extremely common structural motif, RNA hairpins with bulge loops [e.g., the human immunodeficiency virus type 1 (HIV-1) transactivation response (TAR) RNA] can play essential roles in normal cellular processes by binding to proteins and small ligands, which could be very dependent on their three-dimensional (3D) structures and stability. Although the structures and conformational dynamics of the HIV-1 TAR RNA have been extensively studied, there are few investigations on the thermodynamic stability of the TAR RNA, especially in ion solutions, and the existing studies also have some divergence on the unfolding process of the RNA. Here, we employed our previously developed coarse-grained model with implicit salt to predict the 3D structure, stability, and unfolding pathway for the HIV-1 TAR RNA over a wide range of ion concentrations. As compared with the extensive experimental/theoretical results, the present model can give reliable predictions on the 3D structure stability of the TAR RNA from the sequence. Based on the predictions, our further comprehensive analyses on the stability of the TAR RNA as well as its variants revealed that the unfolding pathway of an RNA hairpin with a bulge loop is mainly determined by the relative stability between different states (folded state, intermediate state, and unfolded state) and the strength of the coaxial stacking between two stems in folded structures, both of which can be apparently modulated by the ion concentrations as well as the sequences.

Relative Solvent Exposure of the Alpha-Helix and Beta-Sheet in Water Determines the Initial Stages of Urea and Guanidinium Chloride-Induced Denaturation of Alpha/Beta Proteins.

Among several denaturants, urea and guanidinium chloride (GdmCl) are the two strong and extensively used denaturants in unfolding experiments. However, the sequences of events in terms of secondary structure melting of several proteins in these two solvents have generated diverse results. A clear molecular understanding has still not been drawn to address the differential secondary structure specificity between urea and GdmCl. Here, we present a way to predict the possible unfolding route of α/β proteins, in terms of secondary structure melting, based on the observation of relative solvent exposure of the alpha-helix and beta-sheet of protein in water. We find that while beta-sheet always melts first in urea, a diverse preference is evident for GdmCl. Larger solvent exposure of the backbone of the beta-sheet helps the beta-sheet to make favorable hydrogen bonds with urea which in turn causes the initial melting of the beta-sheet of alpha/beta proteins in urea. On the other hand, because GdmCl has hydrophobic weakening ability through preferential interaction, the region which has comparatively more hydrophobic solvent exposure melts first in GdmCl. Urea- and GdmCl-induced initial unfolding pathways of the alpha/beta protein is thus determined by the relative solvent exposure of the alpha-helix and beta-sheet of protein in water. Therefore, detailed knowledge of relative solvent exposures in water can provide a hint of the possible unfolding pathway provided the mode of action of the solvent is known.

Solid-state NMR spectroscopy based atomistic view of a membrane protein unfolding pathway

Membrane protein folding, structure, and function strongly depend on a cell membrane environment, yet detailed characterization of folding within a lipid bilayer is challenging. Studies of reversible unfolding yield valuable information on the energetics of folding and on the hierarchy of interactions contributing to protein stability. Here, we devise a methodology that combines hydrogen-deuterium (H/D) exchange and solid-state NMR (SSNMR) to follow membrane protein unfolding in lipid membranes at atomic resolution through detecting changes in the protein water-accessible surface, and concurrently monitoring the reversibility of unfolding. We obtain atomistic description of the reversible part of a thermally induced unfolding pathway of a seven-helical photoreceptor. The pathway is visualized through SSNMR-detected snapshots of H/D exchange patterns as a function of temperature, revealing the unfolding intermediate and its stabilizing factors. Our approach is transferable to other membrane proteins, and opens additional ways to characterize their unfolding and stabilizing interactions with atomic resolution.