Protein Main Chain 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.

Effect of Free Cysteine Residues to Serine Mutation on Cellodextrin Phosphorylase.

Cellodextrin phosphorylase (CDP) plays a key role in energy-efficient cellulose metabolism of anaerobic bacteria by catalyzing phosphorolysis of cellodextrin to produce cellobiose and glucose 1-phosphate, which can be utilized for glycolysis without consumption of additional ATP. As the enzymatic phosphorolysis reaction is reversible, CDP is also employed to produce cellulosic materials in vitro. However, the enzyme is rapidly inactivated by oxidation, which hinders in vitro utilization in aerobic environments. It has been suggested that the cysteine residues of CDP, which do not form disulfide bonds, are responsible for the loss of activity, and the aim of the present work was to test this idea. For this purpose, we replaced all 11 free cysteine residues of CDP from Acetivibrio thermocellus (formerly known as Clostridium thermocellum) with serine, which structurally resembles cysteine in our previous work. Herein, we show that the resulting CDP variant, named CDP-CS, has comparable activity to the wild-type enzyme, but shows increased stability to oxidation during long-term storage. X-Ray crystallography indicated that the mutations did not markedly alter the overall structure of the enzyme. Ensemble refinement of the crystal structures of CDP and CDP-CS indicated that the C372S and C625S mutations reduce structural fluctuations in the protein main chain, which may contribute to the increased stability of CDP-CS to oxidation.

Characterization of HIPEs stabilized by soy protein isolate with excellent rheological properties for 3D printing: Bridging the correlation among structure, function and application

Currently, the correlation between how protein structural changes affected the function of high internal phase emulsions (HIPEs) and the regulation of 3D printing performance was still unclear. Herein, this study was aimed to research the relationship between interface properties and stability of soy protein isolate (SPI) at different degrees of oxidation and prepare HIPEs with desirable rheological properties and a solid-like structure to use for 3D printing. The findings of this study suggested that moderate oxidation led to the disruption of the protein's main chain skeleton, resulting in enhancing protein flexibility and surface hydrophobicity. The HIPEs stabilized by this condition owned higher apparent viscosity, over 70% of thixotropic recovery rate, sufficient yield stress (160.8 Pa) and higher G′ and G″. This HIPEs with excellent properties could be the food-grade 3D printing inks to produce samples with high resolution. In addition, this HIPEs exhibited a slow-release effect on free fatty acids (FFA) in the gastrointestinal digestion. Finally, the findings of the correlation analysis indicated that the flexibility and surface hydrophobicity of SPI was a key factor to affect the quality of HIPEs and 3D printing. In conclusion, moderate oxidation could change the structure and interfacial characteristics of SPI, thus endowing HIPEs stabilized by modified SPI with better properties and expanding the field of SPI in the food industry. These findings not only were of great significance for guiding protein production and providing new protein-based 3D printable materials, but also this provided new possibilities for preparing fat substitutes with good appearance.

Trans-cinnamaldehyde fumigation inhibits Escherichia coli by affecting the mechanism of intracellular biological macromolecules

This study aimed to determine the antibacterial mechanism of cinnamaldehyde fumigation in Escherichia coli (E. coli). Through vapour fumigation, cinnamaldehyde was confirmed to exhibit effective antibacterial activity against E. coli. The minimum inhibitory concentration (MIC) and minimum bacterial concentration (MBC) were 0.25 μL/mL and 0.5 μL/mL, respectively. Based on transmission electron microscopy, the wrinkled bacterial cells observed after fumigation could be related to the leakage of intracellular substances. Laser tweezers Raman spectroscopy revealed changes in the main chain of proteins, the hydrogen bond system and spatial structure, and single- and double-stranded DNA breaks. In addition, breakage of the fatty acyl chain backbone was found to affect the vertical order degree of the lipid bilayer and cell membrane fluidity, thereby inhibiting the growth of E. coli. Overall, our findings indicate that cinnamaldehyde fumigation inhibits E. coli growth by inducing changes in intracellular biological macromolecules.

An evolved pyrrolysyl-tRNA synthetase with polysubstrate specificity expands the toolbox for engineering enzymes with incorporation of noncanonical amino acids

Aminoacyl-tRNA synthetase (aaRS) is a core component for genetic code expansion (GCE), a powerful technique that enables the incorporation of noncanonical amino acids (ncAAs) into a protein. The aaRS with polyspecificity can be exploited in incorporating additional ncAAs into a protein without the evolution of new, orthogonal aaRS/tRNA pair, which hence provides a useful tool for probing the enzyme mechanism or expanding protein function. A variant (N346A/C348A) of pyrrolysyl-tRNA synthetase from Methanosarcina mazei (MmPylRS) exhibited a wide substrate scope of accepting over 40 phenylalanine derivatives. However, for most of the substrates, the incorporation efficiency was low. Here, a MbPylRS (N311A/C313A) variant was constructed that showed higher ncAA incorporation efficiency than its homologous MmPylRS (N346A/C348A). Next, N-terminal of MbPylRS (N311A/C313A) was engineered by a greedy combination of single variants identified previously, resulting in an IPE (N311A/C313A/V31I/T56P/A100E) variant with significantly improved activity against various ncAAs. Activity of IPE was then tested toward 43 novel ncAAs, and 16 of them were identified to be accepted by the variant. The variant hence could incorporate nearly 60 ncAAs in total into proteins. With the utility of this variant, eight various ncAAs were then incorporated into a lanthanide-dependent alcohol dehydrogenase PedH. Incorporation of phenyllactic acid improved the catalytic efficiency of PedH toward methanol by 1.8-fold, indicating the role of modifying protein main chain in enzyme engineering. Incorporation of O-tert-Butyl-L-tyrosine modified the enantioselectivity of PedH by influencing the interactions between substrate and protein. Enzymatic characterization and molecular dynamics simulations revealed the mechanism of ncAAs affecting PedH catalysis. This study provides a PylRS variant with high activity and substrate promiscuity, which increases the utility of GCE in enzyme mechanism illustration and engineering.Graphical

DeepMainmast: integrated protocol of protein structure modeling for cryo-EM with deep learning and structure prediction.

Three-dimensional structure modeling from maps is an indispensable step for studying proteins and their complexes with cryogenic electron microscopy. Although the resolution of determined cryogenic electron microscopy maps has generally improved, there are still many cases where tracing protein main chains is difficult, even in maps determined at a near-atomic resolution. Here we developed a protein structure modeling method, DeepMainmast, which employs deep learning to capture the local map features of amino acids and atoms to assist main-chain tracing. Moreover, we integrated AlphaFold2 with the de novo density tracing protocol to combine their complementary strengths and achieved even higher accuracy than each method alone. Additionally, the protocol is able to accurately assign the chain identity to the structure models of homo-multimers, which is not a trivial task for existing methods.

Free-Energy Landscape Analysis of Protein-Ligand Binding: The Case of Human Glutathione Transferase A1

Glutathione transferases (GSTs) are a superfamily of enzymes which have in common the ability to catalyze the nucleophilic addition of the thiol group of reduced glutathione (GSH) onto electrophilic and hydrophobic substrates. This conjugation reaction, which occurs spontaneously but is dramatically accelerated by the enzyme, protects cells against damages caused by harmful molecules. With some exceptions, GSTs are catalytically active as homodimers, with monomers generally constituted of 200 to 250 residues organized into two subdomains. The first is the N-terminal subdomain, which contains an active site named G site, where GSH is hosted in catalytic conformation and which is generally highly conserved among GSTs. The second subdomain, hydrophobic, which binds the substrate counterpart (H site), can vary from one GST to another, resulting in structures able to recognize different substrates. In the present work, we performed all-atom molecular dynamics simulations in explicit solvent of human GSTA1 in its APO form, bound to GSH ligand and bound to GS-conjugated ligand. From MD, two probes were analyzed to (i) decipher the local conformational changes induced by the presence of the ligand and (ii) map the communication pathways involved in the ligand-binding process. These two local probes are, first, coarse-grained angles (θ,γ), representing the local conformation of the protein main chain and, second, dihedral angles χ representing the local conformation of the amino-acid side chains. From the local probes time series, effective free-energy landscapes along the amino-acid sequence were analyzed and compared between the three different forms of GSTA1. This methodology allowed us to extract a network of 33 key residues, some of them being located in the experimentally well-known binding sites G and H of GSTA1 and others being located as far as 30Å from the original binding sites. Finally, the collective motions associated with the network of key residues were established, showing a strong dynamical coupling between residues Gly14-Arg15 and Gln54-Val55, both in the same binding site (intrasite) but also between binding sites of each monomer (intersites).

Surface-Enhanced Raman Spectroscopy (SERS) for characterization SARS-CoV-2

We used SERS with silver nanoparticles (AgNPs) as the active substrate to develop a, simple, quick, and accurate method for the detection and characterization SARS-CoV-2 without the need for RNA isolation and purification. Inactivated SARS-CoV-2 was used. The SERS signals were more than 105times enhanced than the normal Raman (NR) spectra. The SERS spectra of SARS-CoV-2 fingerprint revealed pronounced intensity signals of nucleic acids; aromatic amino acid side chains: 1007 cm−1 (Phe marker), 1095 cm−1 (CN and PO2− markers), 1580 cm−1 (Tyr, Trp markers). Vibrations of the protein main chain: 1144 cm−1 (CN and NH2 markers), 1221 cm−1 (CN and NH markers), 1270 cm−1 (NH2 marker), 1453 cm−1 (CHCH2 marker). All of these biomolecules could be adsorbed on the AgNPs surface's dense hot patches. The intensity of the SERS band varied with the concentration of SARS-CoV-2, with a virus detection limit of less than 103 vp/mL and RSDs of 20 %.

Protein Structural Modeling for Electron Microscopy Maps Using VESPER and MAINMAST.

An increasing number of protein structures are determined by cryo‐electron microscopy (cryo‐EM) and stored in the Electron Microscopy Data Bank (EMDB). To interpret determined cryo‐EM maps, several methods have been developed that model the tertiary structure of biomolecules, particularly proteins. Here we show how to use two such methods, VESPER and MAINMAST, which were developed in our group. VESPER is a method mainly for two purposes: fitting protein structure models into an EM map and aligning two EM maps locally or globally to capture their similarity. VESPER represents each EM map as a set of vectors pointing toward denser points. By considering matching the directions of vectors, in general, VESPER aligns maps better than conventional methods that only consider local densities of maps. MAINMAST is a de novo protein modeling tool designed for EM maps with resolution of 3–5 Å or better. MAINMAST builds a protein main chain directly from a density map by tracing dense points in an EM map and connecting them using a tree‐graph structure. This article describes how to use these two tools using three illustrative modeling examples. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Protein structure model fitting using VESPER Alternate Protocol: Atomic model fitting using VESPER web server Basic Protocol 2: Protein de novo modeling using MAINMAST

Analysis of Serum from Acute Leukemia Patients Using Surface-Enhanced Raman Spectroscopy (SERS)

In this paper, we show surface-enhanced Raman spectroscopy (SERS) of serum from acute leukemia and analyze the SERS through the multivariate statistical method of principal component analysis (PCA). First, the changes in the structure of proteins, lipids, carbohydrates, and other substances in the serum of patients with acute leukemia are analyzed from the molecular perspective. The results showed that the ordered structure of the protein main chain and the side chain conformation was weakened, the main chain structure was almost broken, and the protein became loose and disordered. In addition, the content of esters, amino acids, glycoproteins, and carbohydrate-related substances decreases, indicating a change in the environment. Finally, the PCA method was used to analyze the SERS signal of the serum of healthy people and patients with acute leukemia to accurately distinguish the two serum samples. The above conclusions can provide a favorable experimental basis for diagnosing leukemia and the study of the biochemical mechanism involved.

Hydrogen Bond and Geometry Effects of Thioamide Backbone Modifications.

Thioamide substitution of backbone peptide bonds can probe interactions along the main chain of proteins. Despite theoretical predictions of the enhanced hydrogen bonding propensities of thioamides, previous studies often do not consider the geometric constraints imposed by folded peptide secondary structure. This work addresses drawbacks in previous studies that ignored the geometry dependence and local dielectric properties of thioamide hydrogen bonding and identifies cases where thioamides may be either stronger or weaker hydrogen-bonding partners than amides.

2-Mercaptomethyl Thiazolidines (MMTZs) Inhibit All Metallo-β-Lactamase Classes by Maintaining a Conserved Binding Mode.

Metallo-β-lactamase (MBL) production in Gram-negative bacteria is an important contributor to β-lactam antibiotic resistance. Combining β-lactams with β-lactamase inhibitors (BLIs) is a validated route to overcoming resistance, but MBL inhibitors are not available in the clinic. On the basis of zinc utilization and sequence, MBLs are divided into three subclasses, B1, B2, and B3, whose differing active-site architectures hinder development of BLIs capable of "cross-class" MBL inhibition. We previously described 2-mercaptomethyl thiazolidines (MMTZs) as B1 MBL inhibitors (e.g., NDM-1) and here show that inhibition extends to the clinically relevant B2 (Sfh-I) and B3 (L1) enzymes. MMTZs inhibit purified MBLs in vitro (e.g., Sfh-I, Ki 0.16 μM) and potentiate β-lactam activity against producer strains. X-ray crystallography reveals that inhibition involves direct interaction of the MMTZ thiol with the mono- or dizinc centers of Sfh-I/L1, respectively. This is further enhanced by sulfur-π interactions with a conserved active site tryptophan. Computational studies reveal that the stereochemistry at chiral centers is critical, showing less potent MMTZ stereoisomers (up to 800-fold) as unable to replicate sulfur-π interactions in Sfh-I, largely through steric constraints in a compact active site. Furthermore, in silico replacement of the thiazolidine sulfur with oxygen (forming an oxazolidine) resulted in less favorable aromatic interactions with B2 MBLs, though the effect is less than that previously observed for the subclass B1 enzyme NDM-1. In the B3 enzyme L1, these effects are offset by additional MMTZ interactions with the protein main chain. MMTZs can therefore inhibit all MBL classes by maintaining conserved binding modes through different routes.

Two Methods, One Goal: Structural Differences between Cocrystallization and Crystal Soaking to Discover Ligand Binding Poses.

In lead optimization, protein crystallography is an indispensable tool to analyze drug binding. Binding modes and non‐covalent interaction inventories are essential to design follow‐up synthesis candidates. Two protocols are commonly applied to produce protein–ligand complexes: cocrystallization and soaking. Because of its time and cost effectiveness, soaking is the more popular method. Taking eight ligand hinge binders of protein kinase A, we demonstrate that cocrystallization is superior. Particularly for flexible proteins, such as kinases, and larger ligands cocrystallization captures more reliable the correct binding pose and induced protein adaptations. The geometrical discrepancies between soaking and cocrystallization appear smaller for fragment‐sized ligands. For larger flexible ligands that trigger conformational changes of the protein, soaking can be misleading and underestimates the number of possible polar interactions due to inadequate, highly impaired positions of protein amino‐acid side and main chain atoms. Thus, if applicable cocrystallization should be the gold standard to study protein–ligand complexes.

Tunneling matrix element and tunneling pathways of protein electron transfer calculated with a fragment molecular orbital method

Practical ways to calculate the tunneling matrix elements and analyze the tunneling pathways for protein electron-transfer (ET) reactions with a fragment molecular orbital (FMO) method are presented. The straightforward use of minimal basis sets only for the atoms involved in the covalent bond detachment in FMO can properly describe the ETs through the protein main-chains with the cost-effective two-body corrections (FMO2) without losing the quality of double-zeta basis sets. The current FMO codes have been interfaced with density functional theory, polarizable continuum model, and model core potentials, with which the FMO-based protein ET calculations can consider the effects of electron correlation, solvation, and transition-metal redox centers. The reasonable performance of the FMO-based ET calculations is demonstrated for three different sets of protein-ET model molecules: (1) hole transfer between two tryptophans covalently bridged by a polyalanine linker in the ideal α-helix and β-strand conformations, (2) ET between two plastoquinones covalently bridged by a polyalanine linker in the ideal α-helix and β-strand conformations, and (3) hole transfer between ruthenium (Ru) and copper (Cu) complexes covalently bridged by a stretch of a polyglycine linker as a model for Ru-modified derivatives of azurin.

Curvature and Torsion of Protein Main Chain as Local Order Parameters of Protein Unfolding.

Thermal protein unfolding resembles a global (two-state) phase transition. At the local scale, protein unfolding is, however, heterogeneous and probe dependent. Here, we consider local order parameters defined by the local curvature and torsion of the protein main chain. Because chemical shifts (CS's) measured by NMR spectroscopy are extremely sensitive to the local atomic environment, CS has served as a local probe of thermal unfolding of proteins by varying the position of the atomic isotope along the amino acid sequence. The variation of the CS of each Cα atom along the sequence as a function of the temperature defines a local heat-induced denaturation curve. We demonstrate that these local heat-induced denaturation curves mirror the local protein nativeness defined by the free energy landscape of the local curvature and torsion of the protein main chain described by the Cα-Cα virtual bonds. Comparison between molecular dynamics simulations and CS data of the gpW protein demonstrates that some local native states defined by the local curvature and torsion of the main chain, mainly located in secondary structures, are coupled to each other whereas others, mainly located in flexible protein segments, are not. Consequently, CS's of some residues are faithful reporters of global protein unfolding, with heat-induced denaturation curves similar to the average global one, whereas other residues remain silent about the protein unfolded state. For the latter, the local deformation of the protein main chain, characterized by its local curvature and torsion, is not cooperatively coupled to global unfolding.

Ultrasound-assisted extraction ameliorates the physicochemical properties of defatted mulberry seed protein to promote lipid production in Schizochytrium sp. SR21

To improve protein extraction rate and the utilization of mulberry seed protein (MSP) for biorefinery purposes, ultrasound-assisted extraction was used to treating defatted mulberry seed protein (DMSP) for enhancing lipid production in Schizochytrium sp. SR21. Under ultrasound irradiation with solid-liquid ratio of 1:40, temperature of 60 °C, time of 9 min, and output power of 600 W, the extraction rate of DMSP reached 77.05%, which was significantly improved than the blank control (18.70%) and microwave treatment (22.50%). Moreover, the solubility and emulsibility of DMSP treated by ultrasound were significantly improved; the assay results measured by near-infrared Raman Spectroscopy showed that ultrasound-assisted treatment could reduce the α-helix in the protein (reduced by 9.75%); correspondingly, structures of β-sheet and β-turn increased 1.33% and 3.01%; it indicated that ultrasound treatment changed the spatial arrangement of protein main chain atoms. The antioxidant activity test and the amino acid composition of defatted mulberry seed protein hydrolysate (DMSPH) showed that ultrasound-assisted treatment enhanced the antioxidant activity of the protein hydrolysate and there was no significant difference in amino acid composition between the DMSP and yeast extract. The DMSPH was used as nitrogen source to cultivate Schizochytrium sp. SR21 which showed that DMSPH promoted lipid production and the DHA content was 35.55%, which was better than the results of cultured with traditional nitrogen source; it indicates that DMSP can be used as nitrogen source in culture of microorganisms and further explanation of ultrasound-assisted treatment of modified MSP as an alternative nitrogen source achieved the goal of biorefinery.

Characterization of Libyan Cobra (Naja haje) Venom using Fluorescence and UV-Visible Spectroscopy

Snake venoms act as a preparative to defend animals against predators and helps in immobilizing and digestion of prey. Venoms consist of a combination of enzymes and toxins, such as metalloproteases, phospholipase A2, L-amino acid oxidase and toxins, including cytotoxins and neurotoxins. In addition to their toxicity, venom components exhibit several pharmacological activities and can be used as templates for drug design. The Libyan cobra venom was studied using UV-visible and fluorescence spectroscopic techniques. The cobra protein main chain absorbs light in the region of 240-340nm. The aromatic side-chains of cobra venom contain tyrosine, tryptophan and phenylalanine which are responsible for the absorbance in this region. Cobra venom provides intrinsic fluorescence emissions due to excitation of tryptophan residues, with some contribution from phenylalanine and tyrosine emissions. In addition, disulphide bridges contribute considerable absorption in this wavelength range. The main fluorescence obtained is due to tryptophan which has a wavelength of maximum absorption at 280 nm and an emission peak ranging from 310 to 350 nm. UV-visible absorption and fluorescence spectroscopic techniques are sensitive and rapid to study cobra venom in order to better comprehend the performance of this venom.

Electron density based analysis of N–H⋯OC hydrogen bonds and electrostatic interaction energies in high-resolution secondary protein structures: insights from quantum crystallographic approaches

Limiting values of the topological parameters and the electrostatic interaction energies to establish the presence of true N–H⋯OC H-bonds in protein main-chain have been identified using quantitative and qualitative analyses of electron densities.

The Distinct Conformational Landscapes of 4S-Substituted Prolines That Promote an endo Ring Pucker.

4-Substitution on proline directly impacts protein main chain conformational preferences. The structural effects of N-acyl substitution and of 4-substitution were examined by NMR spectroscopy and X-ray crystallography on minimal molecules with a proline 4S-nitrobenzoate. The effects of N-acyl substitution on conformation were attenuated in the 4S-nitrobenzoate context, due to the minimal role of the n→π* interaction in stabilizing extended conformations. By X-ray crystallography, an extended conformation was observed for most molecules. The formyl derivative adopted a δ conformation that is observed at the i+2 position of β-turns. Computational analysis indicated that the structures observed crystallographically represent the inherent conformational preferences of 4S-substituted prolines with electron-withdrawing 4-position substituents. The divergent conformational preferences of 4R- and 4S-substituted prolines suggest their wider structure-specific application in molecular design. In particular, the proline endo ring pucker favored by 4S-substituted prolines uniquely promotes the δ conformation [(ϕ, ψ) ≈(-80°, 0°)] found in β-turns. In contrast to other acyl capping groups, the pivaloyl group strongly promoted trans amide bond and polyproline II helix conformation, with a close n→π* interaction in the crystalline state, despite the endo ring pucker, suggesting its special capabilities in promoting compact conformations in ϕ due to its strongly electron-donating character.

Secondary Forces in Protein Folding.

A complete inventory of the forces governing protein folding is critical for productive protein modeling, including structure prediction and de novo design, as well as understanding protein misfolding diseases of clinical significance. The dominant contributors to protein folding include the hydrophobic effect and conventional hydrogen bonding, along with Coulombic and van der Waals interactions. Over the past few decades, important additional contributors have been identified, including C-H···O hydrogen bonding, n→π* interactions, C5 hydrogen bonding, chalcogen bonding, and interactions involving aromatic rings (cation-π, X-H···π, π-π, anion-π, and sulfur-arene). These secondary contributions fall into two general classes: (1) weak but abundant interactions of the protein main chain and (2) strong but less frequent interactions involving protein side chains. Though interactions with high individual energies play important roles in specifying nonlocal molecular contacts and ligand binding, we estimate that weak but abundant interactions are likely to make greater overall contributions to protein folding, particularly at the level of secondary structure. Further research is likely to illuminate additional roles of these noncanonical interactions and could also reveal contributions yet unknown.