Protein Interior Research Articles

Waterless structures in the Protein Data Bank.

The absence of solvent molecules in high-resolution protein crystal structure models deposited in the Protein Data Bank (PDB) contradicts the fact that, for proteins crystallized from aqueous media, water molecules are always expected to bind to the protein surface, as well as to some sites in the protein interior. An analysis of the contents of the PDB indicated that the expected ratio of the number of water molecules to the number of amino-acid residues exceeds 1.5 inatomic resolution structures, decreasing to 0.25 at around 2.5 Å resolution. Nevertheless, almost 800 protein crystal structures determined at a resolution of 2.5 Å or higher are found in the current release of the PDB without any water molecules, whereas some other depositions have unusually low or high occupancies of modeled solvent. Detailed analysis of these depositions revealed that the lack of solvent molecules might be an indication of problems with either the diffraction data, the refinement protocol, the deposition process or a combination of these factors. It is postulated that problems with solvent structure should be flagged by the PDB and addressed by the depositors.

Thermal Energy Transport through Nonbonded Native Contacts in Protein.

Within the protein interior, where we observe various types of interactions, nonuniform flow of thermal energy occurs along the polypeptide chain and through nonbonded native contacts, leading to inhomogeneous transport efficiencies from one site to another. The folded native protein serves not merely as thermal transfer medium but, more importantly, as sophisticated molecular nanomachines in cells. Therefore, we are particularly interested in what sort of "communication" is mediated through native contacts in the folded proteins and how such features are quantitatively depicted in terms of local transport coefficients of heat currents. To address the issue, we introduced a concept of inter-residue thermal conductivity and characterized the nonuniform thermal transport properties of a small globular protein, HP36, using equilibrium molecular dynamics simulation and the Green-Kubo formula. We observed that the thermal transport of the protein was dominated by that along the polypeptide chain, while the local thermal conductivity of nonbonded native contacts decreased in the order of H-bonding > π-stacking > electrostatic > hydrophobic contacts. Furthermore, we applied machine learning techniques to analyze the molecular mechanism of protein thermal transport. As a result, the contact distance, variance in contact distance, and H-bonding occurrence probability during MD simulations are found to be the top three important determinants for predicting local thermal transport coefficients.

Wet Conformation of Prion-Like Domain and Intimate Correlation of Hydration and Conformational Fluctuations.

Proteins with prion-like domains (PLDs) are involved in neurodegeneration-associated aggregation and are prevalent in liquid-like membrane-less organelles. These PLDs contain amyloidogenic stretches but can maintain dynamic disordered conformations, even in the condensed phase. However, the molecular mechanism underlying such intricate conformational properties of PLDs remains elusive. Here we employed molecular dynamics simulations to investigate the conformational properties of a prototypical PLD system (i.e., FUS PLD). According to our simulation results, PLD adopts a wet collapsed conformation, wherein most residues maintain sufficient hydration with the abundance of internal water. These internal water molecules can rapidly exchange between the protein interior and the bulk, enabling intensive coupling of the entire protein with its hydration environment. The dynamic exchange of water molecules is intimately correlated to the overall conformational fluctuations of PLD. Furthermore, the abundance of dynamic internal water suppresses the formation of aggregation-prone ordered structures. These results collectively elucidate the crucial role of internal water in sustaining the dynamic disordered conformation of the PLD and inhibiting its aggregation propensity.

Discovery of a Hidden Pocket beneath the NES Groove by Novel Noncovalent CRM1 Inhibitors.

Protein localization is frequently manipulated to favor tumor initiation and progression. In cancer cells, the nuclear export factor CRM1 is often overexpressed and aberrantly localizes many tumor suppressors via protein-protein interactions. Although targeting protein-protein interactions is usually challenging, covalent inhibitors, including the FDA-approved drug KPT-330 (selinexor), were successfully developed. The development of noncovalent CRM1 inhibitors remains scarce. Here, by shifting the side chain of two methionine residues and virtually screening against a large compound library, we successfully identified a series of noncovalent CRM1 inhibitors with a stable scaffold. Crystal structures of inhibitor-protein complexes revealed that one of the compounds, B28, utilized a deeply hidden protein interior cavity for binding. SAR analysis guided the development of several B28 derivatives with enhanced inhibition on nuclear export and growth of multiple cancer cell lines. This work may benefit the development of new CRM1-targeted therapies.

Induced Circular Dichroism Analysis of Thermally Induced Conformational Changes on Protein Binding Sites Under a Crowding Environment.

Protein-ligand interactions in crowded cellular environments play a crucial role in biological functions. The crowded environment can perturb the overall protein structure and local conformation, thereby influencing the binding pathway of protein-ligand reactions within the cellular milieu. Therefore, a detailed understanding of the local conformation is crucial for elucidating the intricacies of protein-ligand interactions in crowded cellular environments. In this study, we investigated the feasibility of induced circular dichroism (ICD) using 8-anilinonaphthalene-1-sulfonic acid (ANS) for local conformational analysis at the binding site in a crowding environment. Bovine serum albumin (BSA) concentration-dependent measurements were performed to assess the feasibility of ANS-ICD for analyzing protein interior binding sites. The results showed distinct changes in the ANS-ICD spectra of BSA solutions, indicating their potential for analyzing the internal conformation of proteins. Moreover, temperature-dependent measurements were performed in dilute and crowding environments, revealing distinct denaturation pathways of BSA binding sites. Principal component analysis of ANS-ICD spectral changes revealed lower temperature pre-denaturation in the crowded solution than that in the diluted solution, suggesting destabilization of binding sites owing to self-crowding repulsive interactions. The established ANS-ICD method can provide valuable conformational insights into protein-ligand interactions in crowded cellular environments.

Understanding Cysteine Reactivity in Protein Environments with Electric Fields.

The role cysteine residues play in proteins is mediated by their protonation state, whereby the thiolate form of the side chain is highly reactive while the thiol form is more inert. However, the pKa of cysteine residues is hard to predict as it can differ widely from its reference value in solution, an effect that is accentuated by local effects in the heterogeneous protein environment. Here, we present a new approach to the prediction of cysteine reactivity based on electric field calculations at the thiol/thiolate group. We validated our approach by predicting the protonation state of cysteine residues in different protein environments (in the active site, at the protein surface, and buried within the protein interior), including Cys-25 in papaya protease omega, which was proven problematic for the more traditional constant pH molecular dynamics (MD) technique. We predict pKa shifts consistent with experimental observations, and the decomposition of the electric fields into contributions from molecular fragments provides a direct handle to rationalize local pH and pKa effects in proteins without introducing parameters other than those of the force field used for MD simulations.

Intra-protein interacting collective modes in the terahertz frequency region.

Understanding how proteins work requires a thorough understanding of their internal dynamics. Proteins support a wide range of motions, from the femtoseconds to seconds time scale, relevant to crucial biological functions. In this context, the term "protein collective dynamics" refers to the complex patterns of coordinated motions of numerous atoms throughout the protein in the sub-picosecond time scale (terahertz frequency region). It is hypothesized that these dynamics have a substantial impact on the regulation of functional dynamical mechanisms, including ligand binding and allosteric signalling, charge transport direction, and the regulation of thermodynamic and thermal transport properties. Using the theoretical framework of hydrodynamics, the collective dynamics of proteins had previously been described in a manner akin to that of simple liquids, i.e. in terms of a single acoustic-like excitation, related to intra-protein vibrational motions. Here, we employ an interacting-mode model to analyse the results from molecular dynamics simulations and we unveil that the vibrational landscape of proteins is populated by multiple acoustic-like and low-frequency optic-like modes, with mixed symmetry and interfering with each other. We propose an interpretation at the molecular level of the observed scenario that we relate to the side-chains and the hydrogen-bonded networks dynamics. The present insights provide a perspective for understanding the molecular mechanisms underlying the energy redistribution processes in the interior of proteins.

Native and nonnative reactions in ethanolamine ammonia-lyase are actuated by different dynamics

We address the contribution of select classes of solvent-coupled configurational fluctuations to the complex choreography involved in configurational and chemical steps in an enzyme by comparing native and nonnative reactions conducted at different protein internal sites. The low temperature, first-order kinetics of covalent bond rearrangement of the cryotrapped substrate radical in coenzyme B12-dependent ethanolamine ammonia-lyase (EAL) from Salmonella enterica display a kink, or increase in slope, of the Arrhenius plot with decreasing temperature. The event is associated with quenching of a select class of reaction-actuating collective fluctuations in the protein hydration layer. For comparison, a nonnative, radical reaction of the protein interior cysteine sulfhydryl group with hydrogen peroxide (H2O2) is introduced by cryotrapping EAL in an aqueous H2O2 eutectic system. The low-temperature aqueous H2O2 protein hydration and mesodomain solvent phases surrounding cryotrapped EAL are characterized by using TEMPOL spin probe electron paramagnetic resonance spectroscopy, including a freezing transition of the eutectic phase that orders the protein hydration layer. Kinetics of the cysteine-H2O2 reaction in the EAL protein interior are monitored by DEPMPO spin trapping of hydroxyl radical product. In contrast to the native reaction, the linear Arrhenius relation for the nonnative cysteine-H2O2 reaction is maintained through the solvent-protein ordering transition. The nonnative reaction is coupled to the generic local, incremental fluctuations that are intrinsic to globular proteins. The comparative approach supports the proposal that select coupled solvent-protein configurational fluctuations actuate the native reaction, and suggests that select dynamical coupling contributes to the degree of catalysis in enzymes.

Extracellular protein homeostasis: The dawning of a new age for human disease therapies?

Extracellular protein homeostasis: The dawning of a new age for human disease therapies?

Multiplexed selectivity screening of anti-GPCR antibodies.

G protein-coupled receptors (GPCRs) control critical cellular signaling pathways. Therapeutic agents including anti-GPCR antibodies (Abs) are being developed to modulate GPCR function. However, validating the selectivity of anti-GPCR Abs is challenging because of sequence similarities among individual receptors within GPCR subfamilies. To address this challenge, we developed a multiplexed immunoassay to test >400 anti-GPCR Abs from the Human Protein Atlas targeting a customized library of 215 expressed and solubilized GPCRs representing all GPCR subfamilies. We found that ~61% of Abs tested were selective for their intended target, ~11% bound off-target, and ~28% did not bind to any GPCR. Antigens of on-target Abs were, on average, significantly longer, more disordered, and less likely to be buried in the interior of the GPCR protein than the other Abs. These results provide important insights into the immunogenicity of GPCR epitopes and form a basis for designing therapeutic Abs and for detecting pathological auto-Abs against GPCRs.

Insights into Nitrosoalkane Binding to Myoglobin Provided by Crystallography of Wild-Type and Distal Pocket Mutant Derivatives.

Nitrosoalkanes (R-N═O; R = alkyl) are biological intermediates that form from the oxidative metabolism of various amine (RNH2) drugs or from the reduction of nitroorganics (RNO2). RNO compounds bind to and inhibit various heme proteins. However, structural information on the resulting Fe-RNO moieties remains limited. We report the preparation of ferrous wild-type and H64A sw MbII-RNO derivatives (λmax 424 nm; R = Me, Et, Pr, iPr) from the reactions of MbIII-H2O with dithionite and nitroalkanes. The apparent extent of formation of the wt Mb derivatives followed the order MeNO > EtNO > PrNO > iPrNO, whereas the order was the opposite for the H64A derivatives. Ferricyanide oxidation of the MbII-RNO derivatives resulted in the formation of the ferric MbIII-H2O precursors with loss of the RNO ligands. X-ray crystal structures of the wt MbII-RNO derivatives at 1.76-2.0 Å resoln. revealed N-binding of RNO to Fe and the presence of H-bonding interactions between the nitroso O-atoms and distal pocket His64. The nitroso O-atoms pointed in the general direction of the protein exterior, and the hydrophobic R groups pointed toward the protein interior. X-ray crystal structures for the H64A mutant derivatives were determined at 1.74-1.80 Å resoln. An analysis of the distal pocket amino acid surface landscape provided an explanation for the differences in ligand orientations adopted by the EtNO and PrNO ligands in their wt and H64A structures. Our results provide a good baseline for the structural analysis of RNO binding to heme proteins possessing small distal pockets.

Binding affinity and conformational change predictions for a series of inhibitors with RuBisCO in a carbon dioxide gas and water environment by multiple computational methods

Ribulose 1, 5-bisphosphate carboxylase-oxygenase (RuBisCO), a Calvin cycle enzyme, is responsible for absorbing the majority of atmospheric CO2 into photosynthetic organisms (McKinlay and Harwood, 2010) [1]. RuBisCO has been the subject of a great deal of research over the years, but its progress is still constrained by the low activity of the RuBisCO enzyme (Timm et al., 2016) [2]. It has not yet been documented how tightly bound inhibitors induce decreased efficiency and how this is related to CO2. In our study, we investigated and validated the binding of five natural inhibitors and substrates to RuBisCO using a variety of analytical techniques such as accelerated molecular dynamics simulation (Perez et al., 2009) [3], binding free energy calculation (Sakae et al., 2020) [4], and protein interior analysis such as a dynamical cross-correlation matrix, a free energy landscape, a dictionary of secondary structure of proteins, and principal component analysis (Marelius et al., 1998) [5]. The results provided evidence in support of the tight binding and activity control of RuBisCO, in fact, The level of inhibitor binding ability calculated by us well echoes the level of inhibition (Frank et al., 1998; Zhu and Jensen, 1991; Keys et al., 1995; Pearce, 2006) [6–9] rate under physiological conditions, which reflects the accuracy of the calculation model. According to the results of the dynamic simulation, significant dynamic changes that occurred before and after the tight binding and were influenced by different aspects of the major and secondary structures were discovered. The analysis indicates that the close binding of the oxidation-produced inhibitor to RuBisCO was controlled by the interaction of key amino acids, the degree of bending of the inhibitor, and the critical LOOP area. Further, the CO2 simulation conditions (Tcherkez, 2016) [10] described the CO2 concentrating mechanism's carbon concentration mechanism and offered fresh insights into the RuBisCO enzyme (Andrews and Whitney, 2005) [11] (CCM). Analysis was done on CO2’s crucial function in the binding of RuBisCO.

Different pKa Shifts of Internal GLU8 in Human β-Endorphin Amyloid Revealing a Coupling of Internal Ionization and Stepwise Fibril Disassembly.

As a functional amyloid, human β-endorphin amyloid fibril features a β-solenoid conformation and store peptide hormones within acidic secretory granules, which would be released into the blood through fibril disassembly when the cellular milieu pH increases from acidic to neutral level on exocytosis. To gain detailed atomic mechanism of β-endorphin amyloid fibrils' pH-responsive disassembly, we conduct constant pH molecular dynamics simulations to investigate the structural and dynamical properties of β-endorphin amyloid fibrils in experiencing the environmental pH changes. Our results demonstrate a clear pKa shift of the internal ionizable residue of GLU8, and this shift becomes even more pronounced when it is buried more deeply in the amyloid fibrils. The unusual pKa of GLU8 reveals that its protonation state changes from the protonated state in the acidic secretory granule to the deprotonated state in the neutral pH conditions in the blood, where the deprotonation of GLU8 leads to unfavorable interactions within the hydrophobic core of the amyloid and subsequent fibril disassembly. The different pKa shifts of GLU8 relative to its positions in the amyloid fibril indicate that the β-endorphin amyloid fibril disassembly is a stepwise process, accounting for the experimental observation that the disassembly always initiates from the outermost layer. This study reveals the critical role of the protonation state of GLU8 in amyloid fibrils' pH-responsive disassembly, and provides clear insights for understanding the structural transitions of amyloids in hormone secretion. This study also provides theoretical basis for designing pH-sensitive biological tools for specific use with precise positioning of ionizable residues into the hydrophobic interior of proteins.

ProtRAP: Predicting Lipid Accessibility Together with Solvent Accessibility of Proteins in One Run

Solvent accessibility has been extensively used to characterize and predict the chemical properties of the surface residues of soluble proteins. However, there is not yet a widely accepted quantity of the same dimension for the study of lipid-accessible residues of membrane proteins. In this study, we propose that lipid accessibility, defined in a similar way to solvent accessibility, can be used to characterize the lipid-accessible residues of membrane proteins. Moreover, we developed a deep learning-based method, ProtRAP (Protein Relative Accessibility Predictor), to predict the relative lipid accessibility and relative solvent accessibility of residues from a given protein sequence, which can infer which residues are likely accessible to lipids, accessible to solvent, or buried in the protein interior in one run.

Detection of simple proteins by direct surface-enhanced Raman scattering based on the Hofmeister ion-specific effect

Surface-enhanced Raman scattering (SERS) spectroscopy has unique advantages in detecting biomolecules, but label-free determination of proteins with low scattering cross-sections remains challenging. In this study, such proteins’ SERS signals have been optimized using the Hofmeister effect between protein molecules and CsI solution at physiological concentrations (A 100 mmol/L Cesium iodide, CsI). Cs+ as chaotro cation ion has a complex interaction mechanism with protein, can not only deprive hydrated water molecules on the surface of protein but also penetrate into the hydrophobic interior of protein. In addition to the above advantages, I- in excess CsI solution with appropriate concentration can removes the interference of citric acid-based impurities on the surface of silver nanoparticles, and Cs+ in excess CsI solution attracts the aggregation of negatively charged silver nanoparticles and cause local electromagnetic field enhancement to achieve high sensitivity in protein detection. This has been combined with principal component analysis to perform a comprehensive analysis of several proteins. Molecular dynamics simulations have been performed to study the mechanism of interaction between CsI and proteins. In addition, the vibrational peak of water has been used as an internal standard to quantify the protein content, and a good linear relationship between peak intensity and concentration was obtained.

Programming xenon diffusion in maltose-binding protein

Protein interiors contain void space that can bind small gas molecules. Determination of gas pathways and kinetics in proteins has been an intriguing and challenging task. Here, we combined computational methods and the hyperpolarized xenon-129 chemical exchange saturation transfer (hyper-CEST) NMR technique to investigate xenon (Xe) exchange kinetics in maltose-binding protein (MBP). A salt bridge ∼9 Å from the Xe-binding site formed upon maltose binding and slowed the Xe exchange rate, leading to a hyper-CEST 129Xe signal from maltose-bound MBP. Xe dissociation occurred faster than dissociation of the salt bridge, as shown by 13C NMR spectroscopy and variable-B1 hyper-CEST experiments. “Xe flooding” molecular dynamics simulations identified a surface hydrophobic site, V23, that has good Xe binding affinity. Mutations at this site confirmed its role as a secondary exchange pathway in modulating Xe diffusion. This shows the possibility for site-specifically controlling xenon protein-solvent exchange. Analysis of the available MBP structures suggests a biological role of MBP’s large hydrophobic cavity to accommodate structural changes associated with ligand binding and protein-protein interactions.

Macromolecular Crowding Significantly Affects the Conformational Features and Carbohydrate Binding Properties of CIA17, a PP2-Type Lectin from Coccinia indica.

The effect of macromolecular crowding on the conformational features and carbohydrate binding properties of CIA17, a PP2-type lectin, was investigated employing polymeric dextrans D6, D40, and D70 (Mr ∼ 6, 40, and 70 kDa, respectively) as crowders. While the secondary structure of CIA17 was significantly affected by D6, with a considerable decrease in the number of β-sheets and β-turns with a corresponding increase in the number of unordered structures, relatively smaller changes were induced by D40 and D70. However, differential scanning calorimetry (DSC) studies revealed that the thermal stability of the protein remains unchanged in the presence of crowders. While the larger dextrans, D70 and D40, induced modest quenching (∼10%) of the protein fluorescence by a static pathway, the smaller D6 induced a higher degree of quenching (37%), which involved both static and collisional quenching processes. The results of fluorescence correlation spectroscopy measurements together with DSC studies suggested that CIA17 forms larger oligomers in the presence of D40 and D70 but D6 prevents the formation of higher-order oligomers. The association constant for the CIA17-chitooligosaccharide interaction increased by ∼30% and 160% in the presence of D40 and D70, respectively, but decreased by ∼30% in the presence of D6. The higher binding affinity can be attributed to the excluded volume effect, i.e., an increased effective concentration of the protein in the presence of D40 and D70, whereas D6, being smaller, possibly penetrates into the protein interior, disrupting the water structure around the protein and also inducing conformational changes, resulting in weaker binding. These observations demonstrate that molecular crowding significantly affects the carbohydrate binding characteristics of lectins, which can modulate their physiological function.

Characterizing Fast Conformational Exchange of Aromatic Rings Using Residual Dipolar Couplings: Distinguishing Jumplike Flips from Other Exchange Mechanisms.

Aromatic ring flips are a hallmark of protein dynamics. They are experimentally studied by NMR spectroscopy, where recent advances have led to improved characterization across a wide range of time scales. Results on different proteins have been interpreted as continuous diffusive ring rotations or jumplike flips, leading to diverging views of the protein interior as being fluidlike or solidlike, respectively. It is challenging to distinguish between these mechanisms and other types of conformational exchange because chemical-shift-mediated line broadening provides only conclusive evidence for ring flips only if the system can be moved from the slow- to intermediate/fast-exchange regime. Moreover, whenever the chemical shift difference between the two symmetry-related sites is close to zero, it is not generally possible to determine the exchange time scale. Here we resolve these issues by measuring residual dipolar coupling (RDC)-mediated exchange contributions using NMR relaxation dispersion experiments on proteins dissolved in dilute liquid crystalline media. Excellent agreement is found between the experimental difference in RDC between the two symmetry-related sites and the value calculated from high-resolution X-ray structures, demonstrating that dynamics measured for F52 in the B1 domain of protein G reports on distinct, jumplike flips rather than other types of conformational exchange.

Pressure denaturation of β-lactoglobulin: Volume changes for genetic A and B variants

Using densitometry of the partial molar volume in dilute aqueous solution at 25 °C, the genetic variant A of bovine β-lactoglobulin was found to be larger than the genetic variant B by 101.0 mL mol−1. Moderate high hydrostratic pressure increased the partial molar volume of the A-variant and B-variant up to 1.5% and 2.0%, respectively, with 30 min holding time. The observed changes in volume were not related to thiol/disulphide exchange and polymerisation reactions, but were a consequence of an increasing water binding at the protein surface in balance with an increasing water binding of the exposed protein interior. The effect was larger for the A-variant in agreement with its lower denaturation temperature and less robustness against enzymatic hydrolysis. Pressure denaturation of the β-lactoglobulin variants occurs, despite pressure-induced protein volume increase, resulting from an increase in water binding with an overall decrease in reaction volume for the denaturation process.

Modulation of GPCR Rhodopsin Function by Membrane Lipids and Water

G protein‐coupled receptors (GPCRs) are responsible for the transduction of signals across lipid membranes and are the largest family of targets (~35%) for currently approved drugs. They exist as dynamic conformational ensembles with multiple inactive and active conformational substates described by an energy landscape model. Consequently, understanding the modulation of conformational dynamics of GPCRs by soft matter such as membrane lipids and cellular water in the receptor’s environment is critical to understanding GPCR efficacy and selectivity. Here we show how membrane lipids and cellular water play crucial roles in shaping the energy landscape of GPCR activation, thus acting as allosteric modulators. We investigated ways in which the receptor hydration level and lipid bilayer composition influence the metarhodopsin equilibrium of the archetypical GPCR rhodopsin in native and POPC recombinant membranes after light activation. Using different polyethylene glycol (PEG) solutions, the osmotic pressure on rhodopsin was varied, while shifting of the metarhodopsin equilibrium was probed using UV‐Visible spectroscopy. Our results show a flood of ~80‐100 water molecules into the rhodopsin interior during photoactivation, forming a solvent‐swollen Meta‐II active state. Dehydrating conditions favor Meta‐I through the efflux of water from the protein interior yet simultaneously increase bilayer thickness, which should favor Meta‐II. Because the osmotic effect on the protein is more significant than the effect of the lipid bilayer, the overall equilibrium is generally shifted to Meta‐I. However, small osmolytes favored the Meta‐II state because they can penetrate the protein core, decreasing the osmotic effect on the protein. Furthermore, the metarhodopsin equilibrium was shifted towards the Meta‐I state in POPC recombinant membranes compared to the native membrane environment. Analysis of transducin C‐terminal peptide‐binding isotherms revealed that the G‐prottein binding affinity is significantly decreased when the lipid environment is changed from the native lipids to POPC lipids. The POPC lipid membrane has zero spontaneous curvature that shifts the equilibrium towards the more compact, inactive Meta‐I state. By contrast, the native lipid membrane environment possesses a negative spontaneous curvature that favors the more expanded state of Meta‐II. Our results delineate the crucial role of soft matter in regulating the metarhodopsin equilibrium in a membrane environment.