Native State Of Protein Research Articles

Protein folding, protein dynamics and the topology of self-motions.

It has long been recognized that segments of the protein main chain are like robotic manipulators and inverse kinematics methods from robotics have been applied to model loops to bridge gaps in protein comparative modelling. The complex internal motion of a redundant manipulator with fixed ends is called a self-motion and its character is determined by the relative position of its ends. Self-motions that are topologically equivalent (homotopic) occupy the same continous region of the configuration space. Topologically inequivalent (non-homotopic) regions are separated by co-regular surfaces and crossing a co-regular surface can result in a sudden dramatic change in the character of the self-motion. It is shown, using a five-residue type I β-turn, that these concepts apply to protein segments and that as the ends of the five-residue segment come closer together, a co-regular surface is crossed, and the structure is locked in to becoming either a type I or type I' turn. It is also shown that the type II turn is topologically equivalent to the type I' turn, not the type I turn. These results have implications for both native-state protein dynamics and protein folding.

Trimethylamine N-oxide (TMAO) doubly locks the hydrophobic core and surfaces of protein against desiccation stress.

Interactions between proteins and osmolytes are ubiquitous within cells, assisting in response to environmental stresses. However, our understanding of protein-osmolyte interactions underlying desiccation tolerance is limited. Here, we employ solid-state NMR (ssNMR) to derive information about protein conformation and site-specific interactions between the model protein, SH3, and the osmolyte trimethylamine N-oxide (TMAO). The data show that SH3-TMAO interactions maintain key structured regions during desiccation and facilitate reversion to the protein's native state once desiccation stress is even slightly relieved. We identify 10 types of residues at 28 sites involved in the SH3-TMAO interactions. These sites comprise hydrophobic, positively charged, and aromatic amino acids located in SH3's hydrophobic core and surface clusters. TMAO locks both the hydrophobic core and surface clusters through its zwitterionic and trimethyl ends. This double locking is responsible for desiccation tolerance and differs from ideas based on exclusion, vitrification, and water replacement. ssNMR is a powerful tool for deepening our understanding of extremely weak protein-osmolyte interactions and providing insight into the evolutionary mechanism of environmental tolerance.

Amino-Acid Characteristics in Protein Native State Structures.

The molecular machines of life, proteins, are made up of twenty kinds of amino acids, each with distinctive side chains. We present a geometrical analysis of the protrusion statistics of side chains in more than 4000 high-resolution protein structures. We employ a coarse-grained representation of the protein backbone viewed as a linear chain of Cα atoms and consider just the heavy atoms of the side chains. We study the large variety of behaviors of the amino acids based on both rudimentary structural chemistry as well as geometry. Our geometrical analysis uses a backbone Frenet coordinate system for the common study of all amino acids. Our analysis underscores the richness of the repertoire of amino acids that is available to nature to design protein sequences that fit within the putative native state folds.

Including the Ensemble of Unstructured Conformations in the Analysis of Protein's Native State by High-Pressure NMR Spectroscopy.

The analysis of pressure induced changes in the chemical shift of proteins allows statements on structural fluctuations proteins exhibit at ambient pressure. The inherent issue of separating general pressure effects from structural related effects on the pressure dependence of chemical shifts has so far been addressed by considering the characteristics of random coil peptides on increasing pressure. In this work, chemically and pressure denatured states of the cold shock protein B from Bacillus subtilis (BsCspB) have been assigned in 2D 1H-15N HSQC NMR spectra and their dependence on increasing hydrostatic pressure has been evaluated. The pressure denatured polypeptide chain has been used to separate general from structural related effects on 1H and 15N chemical shifts of native BsCspB and the implications on the interpretation of pressure induced changes in the chemical shift regarding the structure of BsCspB are discussed. It has been found that the ensemble of unstructured conformations of BsCspB shows different responses to increasing pressure than random coil peptides do. Thus, the approach used for considering the general effects that arise when hydrostatic pressure increases changes the structural conclusions that are drawn from high pressure NMR spectroscopic experiments that rely on the analysis of chemical shifts.

Analyse des Nativen Zustands von Proteinen durch Hochdruck NMR‐Spektroskopie unter Berücksichtigung des Ensembles Unstrukturierter Konformationen

AbstractDie Analyse druckabhängiger Änderungen der chemischen Verschiebung von Proteinen erlaubt es, Aussagen über strukturelle Fluktuationen zu machen, die diese bereits bei Umgebungsdruck aufweisen. Das Problem, allgemeine Effekte, die mit einer Erhöhung des hydrostatischen Drucks einhergehen, von strukturell bedingten Effekten auf die chemische Verschiebung zu trennen, wurde bisher durch das Einbeziehen der Eigenschaften unstrukturierter Modellpeptide angegangen. In dieser Arbeit wurden chemische Verschiebungen in 2D 1H‐15N‐HSQC Spektren von chemisch als auch druckdenaturierten Zuständen des Kälteschockproteins B aus Bacillus Subtilis (BsCspB) zugeordnet und ihre Abhängigkeit von Änderungen des hydrostatischen Drucks ausgewertet. Die durch hydrostatischen Druck denaturierte Polypeptidkette wurde verwendet, um allgemeine Effekte von strukturbedingten Effekten auf 1H und 15N chemische Verschiebungen zu trennen. Daraus resultierende Interpretationen der druckabhängigen Änderungen der chemischen Verschiebungen werden im strukturellen Kontext diskutiert. Es wurde festgestellt, dass das Ensemble unstrukturierter Konformationen von BsCspB anders auf eine Erhöhung des Drucks reagiert, als es unstrukturierte Modellpeptide tun. Daraus kann geschlussfolgert werden, dass der Ansatz, mit dem man allgemeine Effekte, die durch eine Erhöhung des Drucks hervorgerufen werden, abschätzt, die strukturellen Rückschlüsse ändert, die anhand einer Analyse von chemischen Verschiebungen getroffen werden.

Abstract 7092: Cancer biomarker discovery with scalable biofluid proteomics

Abstract Biofluid proteomics opens opportunities for the discovery of early detection, diagnostic, and prognostic cancer biomarkers through non-invasive or minimally invasive screening. Plasma and serum are accessible sample types, however, the high dynamic range of proteins creates analytical challenges for the sensitive detection of potential biomarkers. In this work, we compare two proteomic approaches for large-scale cancer biomarker discovery: the Olink Proximity Extension Assay and the Seer Proteograph Product Suite. Olink Explore 3072 is an immunoassay that gains specificity by using dual antibody recognition to measure approx. 3,000 proteins in each sample. Antibody pairs are tagged with oligonucleotides, which hybridize and are amplified before measurement by next generation sequencing. Seer Proteograph is an enrichment strategy that uses a proprietary mix of functionalized magnetic nanoparticles with affinity for certain amino acids and functional groups in native state proteins. The resulting peptides are measured using untargeted mass spectrometry to identify proteins at the peptide level, paving the way for the identification of proteoforms and peptide variants. Aliquots of human plasma from 78 samples representing a range of cancer indications were obtained through the Discovery Life Sciences biobank and used for analysis by both the Olink and Proteograph workflows. Olink analysis achieved Pearson correlation scores of greater than or equal to 0.8 when correlating each of seven process controls to each other for all approx. 3000 proteins. On average, greater than or equal to 75% of the proteins were detected across all panels in all sample groups except for Normal and Benign groups (64% and 74% respectively). After stringent FDR filtering to ensure only high-quality data was analyzed, the Seer Proteograph workflow quantified 4,323 proteins across all 78 samples. Pearson correlation scores of greater than or equal to 0.96 were achieved when comparing digestion controls, process controls, and sample cleanup controls. Both platforms achieved detection of proteins across the dynamic range of plasma, including low abundance proteins. The Olink Explore 3072 and Seer Proteograph platforms were found to be complementary, with approx. 15% overlap in measured proteins. Cancer biomarker discovery at the proteomic level is bolstered by recent advances in analytical capabilities, as revealed by the Olink Explore and Seer Proteograph platforms. Importantly, complementary implementation of these two workflows will enable comprehensive biomarker analysis. Citation Format: Danielle B. Gutierrez, Jessica L. Moore, Jaison Arivalagan, Tiffany Louie, Leslie Wilkinson, Ray Clemens. Cancer biomarker discovery with scalable biofluid proteomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7092.

A Tale of Two Chains: Geometries of a Chain Model and Protein Native State Structures.

Linear chain molecules play a central role in polymer physics with innumerable industrial applications. They are also ubiquitous constituents of living cells. Here, we highlight the similarities and differences between two distinct ways of viewing a linear chain. We do this, on the one hand, through the lens of simulations for a standard polymer chain of tethered spheres at low and high temperatures and, on the other hand, through published experimental data on an important class of biopolymers, proteins. We present detailed analyses of their local and non-local structures as well as the maps of their closest contacts. We seek to reconcile the startlingly different behaviors of the two types of chains based on symmetry considerations.

Effect of Protein Surface Hydrophobicity and Surface Amines on Soy Adhesive Strength.

Soy is considered one of the most promising natural materials for manufacturing wood adhesives due to its low cost, high protein content, and ready availability. However, more cost-effective ways of improving its wet shear strength are needed to achieve wider market acceptance. Protein adhesive wet strength depends on the use of (typically expensive) crosslinking additives as well as the processing/denaturation of the protein. It has been commonly stated in the literature that protein denaturation leads to higher bond strength by activating the surface and exposing the reactive groups. Therefore, we investigated how differences in surface reactive groups (surface hydrophobicity and reactive amine groups) brought on with different denaturation treatments relate to bonding performance. Fourteen soy protein isolates (SPIs) with different denaturation histories were investigated. Characterization of the SPIs included surface hydrophobicity, surface amine content, extent of protein hydrolysis, and bond strength (wet and dry, with and without polyamidoamine epichlorohydrin (PAE) crosslinking agent) by ASTM D7998. The molecular weight patterns showed that proteins denatured by extensive hydrolysis had very low bond strengths. Adding the crosslinker, PAE, improved all the shear strength values. We found that the number of water-accessible reactive amine groups on protein surfaces had no impact on the adhesive strength, even with the amine-reactive crosslinker, PAE. Conversely, increased surface hydrophobicity was beneficial to adhesive strength in all cases, though this correlation was only statistically significant for wet strength without PAE. While, in general, denatured proteins are typically thought to form better bonds than native state proteins, this work suggests that it matters how proteins are denatured, and what surfaces become exposed. Denaturation by hydrolysis did not improve bond strength, and extensive hydrolysis seemed highly detrimental. Moreover, exposing hydrophobic surface groups was beneficial, but exposing covalent bond-forming reactive amine groups was not.

Characterization of Spatial Differences in Two Misfolded Proteins During Aggresome Formation.

Cells have evolved an elaborate network of folding and degradation pathways to maintain the native state of proteins. If these pathways are disrupted (e.g., mutation) or their capacity is exceeded then protein aggregates form in cells. Cells sequester these aggregated proteins into aggresome or aggresome-like bodies as a protective mechanism. In this study, we co-expressed two model misfolded proteins in HEK293 cells and measured aggresome formation upon proteasomal inhibition. We observed spatial differences in early time points of aggresome formation upon co-expression of the misfolded proteins compared to individual expression in cells.

Disallowed spots in protein structures

Ramachandran (ϕ, ψ) steric map was introduced in 1963 to describe available conformation space for protein structures. Subsequently, residues were observed in high-energy disallowed regions of the map. To unequivocally identify the locations of disallowed conformations of residues, we got 36 noise-free protein structures (resolution ≤1 Å, Rwork/Rfree ≤ 0.10). These stringent criteria were applied to rule out data or model errors or any crystallographic disorders. No disallowed conformation was found in the dataset. Further, we also examined disallowed conformations in a larger dataset (resolution ≤1.5 Å, devoid of any model errors, or disorders). The observed locations of disallowed residues are referred as disallowed spots. These spots include short loops of 3–5 residues, and locations where residues participate in disulfide bonding or intramolecular interactions or inter-molecular interactions with neighboring water, metals or ligands. Conformational sampling revealed that short loops in between secondary structures hardly have any opportunity to relieve from conformational strain. Residues involved in interactions, which provide energetic compensation for high-energy conformational states, were relieved from strain once the causative interaction was removed. The present study aims to identify disallowed spots in the native state of proteins, wherein residues are forced to be trapped in high-energy disallowed conformations. Moreover, it was also observed that pre-Pro, Ser, Asp, trans-Pro, Val, Asn & Gly have higher tendency to occur in disallowed conformation, which could be attributed to factors such as conformational restrictions, residue propensity of secondary structures and compensating sidechain and mainchain interactions, stabilizing turn-mimics.

Microwave radiation induces modifications in the protein fractions of tef flours and modulates their derived techno-functional properties

The impact of microwave (MW) treatments on the structure, solubility, and techno-functional properties of the proteins in starchy matrices is still poorly understood. This study aimed to investigate the effects of MW intensity by applying 1, 2, and 6 min of radiation on two tef flour varieties moistened at 15 % and 25 %. The fractionation method recovered ∼83 % of the total protein content in untreated flours. The interaction between treatment time and moisture content (MC) significantly influenced the extraction of protein fractions. Samples treated at 25 %MC showed significant reductions in albumins (up to −74 %), globulins (up to −79 %), and prolamins (up to −32 %). The SDS-extractable proteins of both tef flours presented similar molecular weights (12–100 kDa). SDS-PAGE analysis revealed decreased band intensity in MW-treated samples compared to untreated flours, and confocal analysis showed changes in the native state of proteins in treated samples. Shorter treatments at low MC significantly improved the emulsifying stability of tef flours, particularly in brown tef flour, with an enhancement of up to 203 %. The hydration properties significantly increased in flours treated at 25 %MC for 6 min. Pearson correlation analysis demonstrated the influence of treatment time and MC on protein recovery and functional properties of tef flours.

Accurate prediction by AlphaFold2 for ligand binding in a reductive dehalogenase and implications for PFAS (per- and polyfluoroalkyl substance) biodegradation

Despite the success of AlphaFold2 (AF2), it is unclear how AF2 models accommodate for ligand binding. Here, we start with a protein sequence from Acidimicrobiaceae TMED77 (T7RdhA) with potential for catalyzing the degradation of per- and polyfluoroalkyl substances (PFASs). AF2 models and experiments identified T7RdhA as a corrinoid iron-sulfur protein (CoFeSP) which uses a norpseudo-cobalamin (BVQ) cofactor and two Fe4S4 iron-sulfur clusters for catalysis. Docking and molecular dynamics simulations suggest that T7RdhA uses perfluorooctanoic acetate (PFOA) as a substrate, supporting the reported defluorination activity of its homolog, A6RdhA. We showed that AF2 provides processual (dynamic) predictions for the binding pockets of ligands (cofactors and/or substrates). Because the pLDDT scores provided by AF2 reflect the protein native states in complex with ligands as the evolutionary constraints, the Evoformer network of AF2 predicts protein structures and residue flexibility in complex with the ligands, i.e., in their native states. Therefore, an apo-protein predicted by AF2 is actually a holo-protein awaiting ligands.

Separating the thermodynamic impact of non-specific interfacial interactions from confinement effects on protein stability.

The intracellular milieu is incredibly complex, containing myriad degrees of spatial restriction and highly variable interfacial chemistry. It is well established that this environmental complexity impacts diffusive properties, stability, and functional rates of proteins. The relative thermodynamic contributions of confinement versus non-specific interfacial interactions (quinary interactions), however, remain poorly understood. We are using reverse micelles (RMs) as a tool to differentiate between these effects. RMs are spontaneously organizing nanopools of water stabilized by surfactants in bulk non-polar solvent. By using optimized surfactant mixtures, the native states of proteins can be maintained in the RM water core. We have executed folding studies on a wide variety of proteins using varied surfactant mixtures with uniform degrees of confinement. We investigate both equilibrium folding thermodynamics and folding kinetics using various spectroscopic approaches. Our findings begin to unveil the complex relationship between a protein's thermodynamic stability and its local solvating environment, helping to dissect the differential impacts of confinement and quinary interactions.

Mechanism of protein stabilization in crowded solution and preservation in desiccated sugar glass.

The highly crowded cellular milieu includes countless different macromolecules and osmolytes aimed at regulating osmotic stress while protecting proteins and other macromolecules. Of special interest is the formation of cellular glassy matrices composed of sugars, which strongly correlate with the ability of some yeast, nematodes, and tardigrades to survive desiccation and extreme temperature changes. Remarkably, these carbohydrates stabilize proteins in crowded solutions and preserve them in the dry glassy state. Yet, although considerable efforts have been directed to discern how crowding stabilizes proteins, the molecular-level stabilization mechanism has remained elusive. We have followed the effect of various osmolytes on the native state stability of model miniproteins in crowded solutions using circular dichroism (CD) spectroscopy and our newly developed mean-field theory for crowding. We show that protein native state stability and folding thermodynamic signature can be explained using three distinct types of interactions: excluded volume, solvent-osmolyte non-ideal mixing, and osmolyte-protein soft-interactions. Furthermore, using our combination of synchrotron radiation CD spectroscopy together with sampling enhanced molecular dynamics (MD) simulations we have been able to shed light on the stability and new emerging nanostructures of proteins when they are embedded in sugar glass.

Distribution and solvent exposure of Hsp70 chaperone binding sites across the Escherichia coli proteome.

Many proteins must interact with molecular chaperones to achieve their native state in the cell. Yet, how chaperone binding-site characteristics affect the folding process is poorly understood. The ubiquitous Hsp70 chaperone system prevents client-protein aggregation by holding unfolded conformations and by unfolding misfolded states. Hsp70 binding sites of client proteins comprise a nonpolar core surrounded by positively charged residues. However, a detailed analysis of Hsp70 binding sites on a proteome-wide scale is still lacking. Further, it is not known whether proteins undergo some degree of folding while chaperone bound. Here, we begin to address the above questions by identifying Hsp70 binding sites in 2258 Escherichia coli (E. coli) proteins. We find that most proteins bear at least one Hsp70 binding site and that the number of Hsp70 binding sites is directly proportional to protein size. Aggregation propensity upon release from the ribosome correlates with number of Hsp70 binding sites only in the case of large proteins. Interestingly, Hsp70 binding sites are more solvent-exposed than other nonpolar sites, in protein native states. Our findings show that the majority of E. coli proteins are systematically enabled to interact with Hsp70 even if this interaction only takes place during a fraction of the protein lifetime. In addition, our data suggest that some conformational sampling may take place within Hsp70-bound states, due to the solvent exposure of some chaperone binding sites in native proteins. In all, we propose that Hsp70-chaperone-binding traits have evolved to favor Hsp70-assisted protein folding devoid of aggregation.

CoDNaS-Q: a database of conformational diversity of the native state of proteins with quaternary structure

A collection of conformers that exist in a dynamical equilibrium defines the native state of a protein. The structural differences between them describe their conformational diversity, a defining characteristic of the protein with an essential role in multiple cellular processes. Since most proteins carry out their functions by assembling into complexes, we have developed CoDNaS-Q, the first online resource to explore conformational diversity in homooligomeric proteins. It features a curated collection of redundant protein structures with known quaternary structure. CoDNaS-Q integrates relevant annotations that allow researchers to identify and explore the extent and possible reasons of conformational diversity in homooligomeric protein complexes. CoDNaS-Q is freely accessible at http://ufq.unq.edu.ar/codnasq/ or https://codnas-q.bioinformatica.org/home. The data can be retrieved from the website. The source code of the database can be downloaded from https://github.com/SfrRonaldo/codnas-q.

Characterization of structural and functional properties of soybean 11S globulin during the renaturation after the guanidine hydrochloride denaturation

In this study, we explored how 11S globulin undergoes the renaturation with the decrease of guanidine hydrochloride (GuHCl) concentration. To analyze the changes in the structural and functional properties of 11S globulin, circular dichroism spectroscopy (CD), intrinsic fluorescence, particle size, foaming capacity (FC), foam stability (FS), emulsifying activity index (EAI) and emulsion stability index (ESI) at different GuHCl concentrations (8.0 mol/L, 4.0 mol/L, 2.0 mol/L, 0.8 mol/L, and 0.4 mol/L) were performed. 11S globulin was completely denatured after being treated with 8.0 mol/L of GuHCl. The CD spectra and particle size analyses showed that the α-helix and β-sheet contents and particle size of the renaturation process for 11S globulin were gradually close to those of native state protein and the protein structure became more orderly. The α-helix content increased from 0.2 ± 0.1% to 7.8 ± 0.3% and the hydrophobic groups inside the 11S globulin were exposed, which increased the surface hydrophobicity of the 11S globulin and changed its conformation. Regarding the functional properties, when the GuHCl concentration was diluted to 2.0 mol/L, some functional properties were improved compared with those of native state protein. The FC and FS of 11S globulin were increased by 5 and 2.2 times respectively and the EAI and ESI were increased by 1.25 times respectively. This study will provide guidance for the control of renaturation degree during 11S globulin production process to improve functional properties.

Effects of High-Pressure Treatments (Ultra-High Hydrostatic Pressure and High-Pressure Homogenization) on Bighead Carp (Aristichthys nobilis) Myofibrillar Protein Native State and Its Hydrolysate

The impacts of four treatments, ultra-high hydrostatic pressure (UHP), high-pressure homogenization (HPH), combined UHP + HPH (U-H), and HPH + UHP (H-U), on bighead carp (Aristichthys nobilis) myofibrillar protein (BMP) structure, functional hydrolysis property to pepsin, and antioxidant activity of hydrolysates were investigated. All treatments led to increase in low-molecular-weight BMP, the BMP stability, and hydrophobic group exposure, but decrease in total sulfhydryl content and BMP particle size, thus resulting in increased hydrolysis and antioxidant capacity of enzymatic hydrolysates. U-H treated BMP exhibited the highest surface hydrophobicity (924.5 ± 1.0), zeta potential absolute value (18.88 ± 0.11 mV), hydrolysis degree (54.5 ± 1.6%), and antioxidant activity, but the lowest α-helix (21.84 ± 2.61%), intrinsic fluorescence spectrum intensity, total sulfhydryl (7.24 ± 0.07 μmol/g), and mean particle size (182.57 ± 2.23 nm). Therefore, U-H might be a promising pretreatment to prepare bioactive peptide.

Crowding-induced protein destabilization in the absence of soft attractions

It is generally assumed that volume exclusion by macromolecular crowders universally stabilizes the native states of proteins and destabilization suggests soft attractions between crowders and protein. Here we show that proteins can be destabilized even by crowders that are purely repulsive. With a coarse-grained sequence-based model, we study the folding thermodynamics of two sequences with different native folds, a helical hairpin and a β-barrel, in a range of crowder volume fractions, φc. We find that the native state, N, remains structurally unchanged under crowded conditions, while the size of the unfolded state, U, decreases monotonically with φc. Hence, for all φc>0, U is entropically disfavored relative to N. This entropy-centric view holds for the helical hairpin protein, which is stabilized under all crowded conditions as quantified by changes in either the folding midpoint temperature, Tm, or the free energy of folding. We find, however, that the β-barrel protein is destabilized under low-T, low-φc conditions. This destabilization can be understood from two characteristics of its folding: 1) a relatively compact U at T<Tm, such that U is only weakly disfavored entropically by the crowders; and 2) a transient, compact, and relatively low-energy nonnative state that has a maximum population of only a few percent at φc=0, but increasing monotonically with φc. Overall, protein destabilization driven by hard-core effects appears possible when a compaction of U leads to even a modest population of compact nonnative states that are energetically competitive with N.

A Small Molecule Stabilizes the Disordered Native State of the Alzheimer's Aβ Peptide.

The stabilization of native states of proteins is a powerful drug discovery strategy. It is still unclear, however, whether this approach can be applied to intrinsically disordered proteins. Here, we report a small molecule that stabilizes the native state of the Aβ42 peptide, an intrinsically disordered protein fragment associated with Alzheimer’s disease. We show that this stabilization takes place by a disordered binding mechanism, in which both the small molecule and the Aβ42 peptide remain disordered. This disordered binding mechanism involves enthalpically favorable local π-stacking interactions coupled with entropically advantageous global effects. These results indicate that small molecules can stabilize disordered proteins in their native states through transient non-specific interactions that provide enthalpic gain while simultaneously increasing the conformational entropy of the proteins.