Side Chains Of Residues Research Articles

Investigating the Determinants of Substrate Binding Orientation within the E. coli Hsp70 Chaperone Protein

Molecular chaperone proteins play a critical role in maintenance of the cell’s proteome through protein quality control. Heat shock protein 70 chaperones (Hsp70s) are an evolutionarily conserved and diverse family of chaperone proteins that assist with proper protein folding and prevention of aggregation in the cell. Previous research has shown that the substrate‐binding domain of the E. coli Hsp70, DnaK, contains binding pockets of low stringency extending bilaterally from a highly hydrophobic central pocket. The SBD interacts with short hydrophobic sequences of substrates by “reading” the sequence. The SBD will then bind the substrate in one of two orientations: an N‐C, or forward, orientation, or a C‐N, reverse, orientation. Research done thus far on substrate binding has not revealed an exact mechanism that determines orientation. In this study, peptides possessing the same sequence with opposing backbone directions were used to test the significance of backbone interactions with the SBD as well as residue side chains and their interactions with the SBD. The analysis of the bound orientation of these peptides is accomplished by disulfide cross‐linking using peptides containing an N‐terminal Cys and SBD proteins with a Cys introduced in such a way where it can be reached in only one orientation. The results indicated slight energetic favorable biases for the interactions of residue side chains and the SBD pockets. The results also place a high importance on leucine residues in substrates as a determinant of optimal pocket ‐ residue fit and binding orientation. Altogether, the data suggest that binding occurs in the orientation that supports the most optimal fit of residue side chains into binding pockets, which reveals further intricacies and selectivity involved in substrate binding for Hsp70s.

Fluorescent Hydrogel Based on Self-assembling Acridonylalanine-phenylalanine

Abstract A new dipeptide with an acridone chromophore, namely 2-acridonylalanine-phenylalanine (AAP) was synthesized, and a fluorescent hydrogel was produced under physiological pH conditions by the AAP self-assembly process. The hydrogel exhibited a blue emission and reasonable thermoresponsiveness. Therefore, dipeptides containing an acridone side chain of the first residue were found to be effective hydrogelators.

Computational Modeling of Conformer Stability in Benenodin-1, a Thermally Actuated Lasso Peptide Switch.

Benenodin-1 is a thermally actuated lasso peptide rotaxane switch with two primary translational isomers that differ in the relative position of the residue Gln15. The conversion from one conformer to the other involves substantial enthalpy-entropy compensation: one conformer is energetically favored and the other is entropically favored. Here, we take a multi-scale quantum mechanical (QM) and classical molecular dynamic (MD) approach to reveal residue-specific sources of these differences in stability. QM reveals that the two benenodin-1 conformers involve distinct hydrogen bonding networks, with the enthalpically favored conformer having more intra-peptide hydrogen bonds between the Gln15 side chain and nearby residues. The evaluation of configurational entropy over the MD-sampled geometries reveals that the entropically favored conformer has enhanced conformational flexibility. By computing the by-residue-sum entropies, we identify the role of Gln15 and neighboring Glu14 in mediating the entropic variation during the switching process. These computational insights help explain the effects of Glu14Ala and Gln15Ala mutations on the conformational population of benenodin-1 observed experimentally.

PASS2.7: a database containing structure-based sequence alignments and associated features of protein domain superfamilies from SCOPe.

Sequence alignments are models that capture the structural, functional and evolutionary relationships between proteins. Structure-guided sequence alignments are helpful in the case of distantly related proteins with poor sequence identity, thus rendering routine sequence alignment methods ineffective. Protein Alignment organized as Structural Superfamilies or PASS2 database provides such sequence alignments of protein domains within a superfamily as per the Structural Classification of Proteins extended (SCOPe) database. The current update of PASS2 (i.e. PASS2.7) is following the latest release of SCOPe (2.07) and we provide data for 14 323 protein domains that are <40% identical and are organized into 2024 superfamilies. Several useful features derived from the alignments, such as conserved secondary structural motifs, HMMs and residues conserved across the superfamily, are also reported. Protein domains that are deviant from the rest of the members of a superfamily may compromise the quality of the alignment, and we found this to be the case in ∼7% of the total superfamilies we considered. To improve the alignment by objectively identifying such ‘outliers’, in this update, we have used a k-means-based unsupervised machine learning method for clustering superfamily members, where features provided were length of domains aligned, Cα-RMSD derived from the rigid-body superposition of all members and gaps contributed to the alignment by each domain. In a few cases, we have split the superfamily as per the clusters predicted and provided complete data for each cluster. A new feature included in this update is absolutely conserved interactions (ACIs) between residue backbones and side chains, which are obtained by aligning protein structure networks using structure-guided sequence alignments of superfamilies. ACIs provide valuable information about functionally important residues and the structure–function relationships of proteins. The ACIs and the corresponding conserved networks for backbone and sidechain have been marked on the superimposed structure separately.Database URLThe updated version of the PASS2 database is available at http://caps.ncbs.res.in/pass2/.

NMR Assignment of Methyl Groups in Immobilized Proteins Using Multiple-Bond 13C Homonuclear Transfers, Proton Detection, and Very Fast MAS.

In nuclear magnetic resonance spectroscopy of proteins, methyl protons play a particular role as extremely sensitive reporters on dynamics, allosteric effects, and protein–protein interactions, accessible even in high-molecular-weight systems approaching 1 MDa. The notorious issue of their chemical shift assignment is addressed here by a joint use of solid-state 1H-detected methods at very fast (nearly 100 kHz) magic-angle spinning, partial deuteration, and high-magnetic fields. The suitability of a series of RF schemes is evaluated for the efficient coherence transfer across entire 13C side chains of methyl-containing residues, which is key for establishing connection between methyl and backbone 1H resonances. The performance of ten methods for recoupling of either isotropic 13C–13C scalar or anisotropic dipolar interactions (five variants of TOBSY, FLOPSY, DIPSI, WALTZ, RFDR, and DREAM) is evaluated experimentally at two state-of-the-art magic-angle spinning (55 and 94.5 kHz) and static magnetic field conditions (18.8 and 23.5 T). Model isotopically labeled compounds (alanine and Met-Leu-Phe tripeptide) and ILV-methyl and amide-selectively protonated, and otherwise deuterated chicken α-spectrin SH3 protein are used as convenient reference systems. Spin dynamics simulations in SIMPSON are performed to determine optimal parameters of these RF schemes, up to recently experimentally attained spinning frequencies (200 kHz) and B 0 field strengths (28.2 T). The concept of linearization of 13C side chain by appropriate isotope labeling is revisited and showed to significantly increase sensitivity of methyl-to-backbone correlations. A resolution enhancement provided by 4D spectroscopy with non-uniform (sparse) sampling is demonstrated to remove ambiguities in simultaneous resonance assignment of methyl proton and carbon chemical shifts.

Structure and mechanism for iterative amide N-methylation in the biosynthesis of channel-forming peptide cytotoxins

SignificanceThe channel-forming proteusins are bacterial helical peptides that allow permeation of positively charged ions to influence membrane potential and cellular physiology. We biochemically characterize the effect of two critical posttranslational modifications on the secondary structure of the peptide substrate. We determine how a methyl group can be added to the side chains of D-Asn residues in a peptide substrate and show how flanking residues influence selectivity. These studies should foster the development of small-molecule peptide ion channels as therapeutics.

HPLC-free method of synthesis of isotopically labeled deoxyfructosylated peptides.

The biomarker strategy, based on multiple specific glycation sites in plasma proteins, could essentially increase the efficiency of glycemic control and disease prediction. Besides glycated albumin being a potential biomarker of early states of diabetes mellitus and control of short-term, it has been shown that the glycation of fibrinogen may also impact the formation of the fibrin network, while quantification of glycation of the CD59 protein allows for early detection of glucose intolerance in pregnant women. A different level of glycation of individual lysine residues in proteins has a crucial influence on the stages of the disease. The quantification of new biomarkers of different stages of diabetes requires appropriate isotope-labeled analogs that may improve biomarker search by providing more accurate quantitative data and by more robust detection/quantitation of low-abundance biomarkers. In the presented work, we proposed a fast and simple protocol for the synthesis of isotopically labeled and bi-labeled deoxyfructosylated peptide based on a combination of microwave-assisted synthesis and boronic affinity chromatography using functionalized resin (PhB-Lys(PhB)-ChemMatrix® Rink resin) developed by us. Our method is focused on the synthesis of glycated peptides identified in glycated albumin (GA) after enzymatic hydrolysis catalyzed by trypsin after arginine residues. Thereby, the standard peptides comprised [13C6]-deoxyfructose attached to lysine residue side chain, a dabcyl moiety for determination of standard amounts, and a cleavable linker. Moreover, we applied bi-labeled deoxyfructosylated peptide to determine the concentration of appropriate analog in a sample of human serum albumin glycated in vitro.

Hydrophobic/Hydrophilic Ratio of Amphiphilic Helix Mimetics Determines the Effects on Islet Amyloid Polypeptide Aggregation.

Amyloid depositions of human islet amyloid polypeptides (hIAPP) are associated with type II diabetes (T2D) impacting millions of people globally. Accordingly, strategies against hIAPP aggregation are essential for the prevention and eventual treatment of the disease. Helix mimetics, which modulate the protein-protein interaction by mimicking the side chain residues of a natural α-helix, were found to be a promising strategy for inhibiting hIAPP aggregation. Here, we applied molecular dynamics simulations to investigate two helix mimetics reported to have opposite effects on hIAPP aggregation in solution, the oligopyridylamide-based scaffold 1e promoted, whereas naphthalimide-appended oligopyridylamide scaffold DM 1 inhibited the aggregation of hIAPP in solution. We found that 1e promoted hIAPP aggregation because of the recruiting effects through binding with the N-termini of hIAPP peptides. In contrast, DM 1 with a higher hydrophobic/hydrophilic ratio effectively inhibited hIAPP aggregation by strongly binding with the C-termini of hIAPP peptides, which competed for the interpeptide contacts between amyloidogenic regions in the C-termini and impaired the fibrillization of hIAPP. Structural analyses revealed that DM 1 formed the core of hIAPP-DM 1 complexes and stabilized the off-pathway oligomers, whereas 1e formed the corona outside the hIAPP-1e complexes and remained active in recruiting free hIAPP peptides. The distinct interaction mechanisms of DM 1 and 1e, together with other reported potent antagonists in the literature, emphasized the effective small molecule-based amyloid inhibitors by disrupting peptide interactions that should reach a balanced hydrophobic/hydrophilic ratio, providing a viable and generic strategy for the rational design of novel anti-amyloid nanomedicine.

The phytohormone abscisic acid modulates protein carbonylation in Arabidopsis thaliana.

Protein carbonylation is a post-translational modification associated with the reactive oxygen species. It results from the direct oxidation of the side chains of Lys, Arg, Pro, and Thr residues by hydroxyl radical HO• or the addition of reactive carbonyl species including α,β-unsaturated aldehydes and oxylipins to the side chain of Cys, His, and Lys. Recent findings indicated that the phytohormone abscisic acid (ABA) induces the production of α,β-unsaturated aldehydes that modulate the effect of ABA on stomatal closure. This indicated that α,β-unsaturated aldehydes might mediate ABA signaling. In this study, we investigated the ABA-induced protein carbonylation events by profiling the carbonylated proteome extracted from Arabidopsis thaliana leaves after ABA treatment. The carbonylated proteins were enriched by affinity chromatography and subjected to liquid chromatography-tandem mass spectrometry. We identified 180 carbonylated proteins. Of these, 26 proteins became carbonylated upon ABA treatment, whereas 163 proteins that were carbonylated in untreated samples were no longer detected in the ABA-treated samples, which points to dynamic control of protein carbonylation by ABA in A. thaliana. A few regulatory stress-related proteins and enzymes involved in the biosynthesis of the aspartate family of amino acids were overrepresented in the list of proteins, which the carbonylation status changed between untreated and ABA-treated samples. These results indicated that ABA triggers a change in the pattern of protein carbonylation in A. thaliana. This change is independent of the commonly seen increased levels of carbonylated proteins in the plants subjected to deadly stress conditions.

Extended β-Strands Contribute to Reversible Amyloid Formation.

The assembly of proteins into fibrillar amyloid structures was once considered to be pathologic and essentially irreversible. Recent studies reveal amyloid-like structures that form reversibly, derived from protein low-complexity domains which function in cellular metabolism. Here, by comparing atomic-level structures of reversible and irreversible amyloid fibrils, we find that the β-sheets of reversible fibrils are enriched in flattened (as opposed to pleated) β-sheets formed by stacking of extended β-strands. Quantum mechanical calculations show that glycine residues favor extended β-strands which may be stabilized by intraresidue interactions between the amide proton and the carbonyl oxygen, known as C5 hydrogen-bonds. Larger residue side chains favor shorter strands and pleated sheets. These findings highlight a structural element that may regulate reversible amyloid assembly.

Electrostatically induced pKa shifts in oligopeptides: the upshot of neighboring side chains

pKa values of homorepeat hexapeptides with a 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) chromophore attached at the peptide C termini, through an asparagine derivative (Dbo), namely His6-Dbo (H6), Lys6-Dbo (K6), and Arg6-Dbo (R6), were determined by a novel fluorescence-based method. The fluorescence lifetime of Dbo in the peptides (τ) was measured as a function of pH. The side chains collide with Dbo intramolecularly and quench it efficiently only when they are deprotonated (i.e., pH ≥ side chain pKa). The pKa values of the H6, K6, and R6 peptides, attributable to side chain ionization, were found to be depressed compared to the pKa values of the His, Lys, and Arg residues in their free amino acid forms. We further looked into the structural changes of the peptides by molecular dynamics (MD) simulations; the peptides were structurally more expanded when their side chains are protonated. The structural expansion of the peptides reflects an electrostatic repulsion between the protonated side chain residues, which also accounts for the observed decrease in pKa values, which corresponds to a facilitated deprotonation, assisted by electrostatic repulsion.

Comparative Genomic Analyses of the Genus Nesterenkonia Unravels the Genomic Adaptation to Polar Extreme Environments.

The members of the Nesterenkonia genus have been isolated from various habitats, like saline soil, salt lake, sponge-associated and the human gut, some of which are even located in polar areas. To identify their stress resistance mechanisms and draw a genomic profile across this genus, we isolated four Nesterenkonia strains from the lakes in the Tibetan Plateau, referred to as the third pole, and compared them with all other 30 high-quality Nesterenkonia genomes that are deposited in NCBI. The Heaps’ law model estimated that the pan-genome of this genus is open and the number of core, shell, cloud, and singleton genes were 993 (6.61%), 2782 (18.52%), 4117 (27.40%), and 7132 (47.47%), respectively. Phylogenomic and ANI/AAI analysis indicated that all genomes can be divided into three main clades, named NES-1, NES-2, and NES-3. The strains isolated from lakes in the Tibetan Plateau were clustered with four strains from different sources in the Antarctic and formed a subclade within NES-2, described as NES-AT. Genome features of this subclade, including GC (guanine + cytosine) content, tRNA number, carbon/nitrogen atoms per residue side chain (C/N-ARSC), and amino acid composition, in NES-AT individuals were significantly different from other strains, indicating genomic adaptation to cold, nutrient-limited, osmotic, and ultraviolet conditions in polar areas. Functional analysis revealed the enrichment of specific genes involved in bacteriorhodopsin synthesis, biofilm formation, and more diverse nutrient substance metabolism genes in the NES-AT clade, suggesting potential adaptation strategies for energy metabolism in polar environments. This study provides a comprehensive profile of the genomic features of the Nesterenkonia genus and reveals the possible mechanism for the survival of Nesterenkonia isolates in polar areas.

Recent Advances in Sorbicillinoids from Fungi and Their Bioactivities (Covering 2016-2021).

Sorbicillinoids are a family of hexaketide metabolites with a characteristic sorbyl side chain residue. Sixty-nine sorbicillinoids from fungi, newly identified from 2016 to 2021, are summarized in this review, including their structures and bioactivities. They are classified into monomeric, dimeric, trimeric, and hybrid sorbicillinoids according to their basic structural features, with the main groups comprising both monomeric and dimeric sorbicillinoids. Some of the identified sorbicillinoids have special structures such as ustilobisorbicillinol A, and sorbicillasins A and B. The majority of sorbicillinoids have been reported from fungi genera such as Acremonium, Penicillium, Trichoderma, and Ustilaginoidea, with some sorbicillinoids exhibiting cytotoxic, antimicrobial, anti-inflammatory, phytotoxic, and α-glucosidase inhibitory activities. In recent years, marine-derived, extremophilic, plant endophytic, and phytopathogenic fungi have emerged as important resources for diverse sorbicillinoids with unique skeletons. The recently revealed biological activities of sorbicillinoids discovered before 2016 are also described in this review.

Complexation of specific residues by carboxylatopillar[6]arene for improving the zymolytic stability of arginine-containing peptides.

A general strategy for improving the zymolytic stability against proteases is reported. Carboxylatopillar[6]arene (CP6A) could effectively bind arginine and arginine-containing peptides, thereby improving the stability of angiotensin peptides in the presence of trypsin by the complexation of the side chain of the arginine residue.

Improved Handling of Peptide Segments Using Side Chain-Based "Helping Hand" Solubilizing Tools.

Maintaining high, or even sufficient, solubility of every peptide segment in chemical protein synthesis (CPS)remains a critical challenge; insolubility of just a single peptide segment can thwart a total synthesis venture. Multiple approaches have been used to address this challenge, most commonly by employing a chemical tool to temporarily improve peptide solubility. In this chapter, we discuss chemical tools for introducing semipermanent solubilizing sequences (termed helping hands) at the side chains of Lys and Glu residues. We describe the synthesis, incorporation by Fmoc-SPPS, and cleavage conditions for utilizing these two tools. For Lys sites, we discuss the Fmoc-Ddap-OH dimedone-based linker, which is achiral, synthesized in one step, can be introduced directly at primary amines, and is removed using hydroxylamine (or hydrazine). For Glu sites, we detail the new Fmoc-SPPS building block, Fmoc-Glu(AlHx)-OH, which can be prepared in an efficient process over two purifications. Solubilizing sequences are introduced directly on-resin and later cleaved with palladium-catalyzed transfer under aqueous conditions to restore a native Glu side chain. These two chemical tools are straightforward to prepare and implement, and we anticipate continued usage in "difficult" peptide segments following the protocols described herein.

Advanced approaches of developing targeted covalent drugs.

In recent years, the development of targeted covalent inhibitors has gained popularity around the world. Specific groups (electrophilic warheads) form irreversible bonds with the side chain of nucleophilic amino acid residues, thus changing the function of biological targets such as proteins. Since the first targeted covalent inhibitor was disclosed in the 1990s, great efforts have been made to develop covalent ligands from known reversible leads or drugs by addition of tolerated electrophilic warheads. However, high reactivity and "off-target" toxicity remain challenging issues. This review covers the concept of targeted covalent inhibition to diseases, discusses traditional and interdisciplinary strategies of cysteine-focused covalent drug discovery, and exhibits newly disclosed electrophilic warheads majorly targeting the cysteine residue. Successful applications to address the challenges of designing effective covalent drugs are also introduced.

Chemical synthesis of human selenoprotein F and elucidation of its thiol-disulfide oxidoreductase activity.

Selenoprotein F (SelF) is an endoplasmic reticulum-residing eukaryotic protein that contains a selenocysteine (Sec) residue. It has been suggested to be involved in a number of physiological processes by acting as a thiol-disulfide oxidoreductase, but the exact role has remained unclear due to the lack of a reliable production method. We document herein a robust synthesis of the human SelF through a three-segment two-ligation semisynthesis strategy. Highlighted in this synthetic route are the use of a mild desulfurization process to protect the side-chain of the Sec residue from being affected and the simultaneous removal of acetamidomethyl and p-methoxybenzyl protection groups by PdCl2, thus facilitating the synthesis of multi-milligrams of homogenous SelF. The reduction potential of SelF was determined and the thiol-disulfide oxidoreductase activity was further supported by its ability to catalyze the reduction and isomerization of disulfide bonds.

MOLECULAR DOCKING AND BIOACTIVITY STUDIES OF COVALENT INHIBITORS TARGETING RDRP OF SARS-COV-2

The objective of the study was to examine the compounds which inhibit the enzyme RNA-Dependent RNA-Polymerase (RdRp) of SARS-CoV-2. In this study, we collected 120 similar compounds of Remdesivir, Favipiravir, Novobiocin, and Cortisone inhibitors from PubChem and docked them within the RdRp enzyme using Patchdock/ Firedock server. Seven compounds were screened based on binding affinity and binding mode analysis and further studied the covalent and hydrogen interaction networks using Ligplot+ software. Moreover, the pharmacore properties and bioactivity of the compounds were analyzed with molinspiration cheminformatics software. In this study, we identified the compounds 56832851, 134443512, and 5745 have more binding affinity and mostly covalent/H-bonding interactions occurred with side chain residues Asp161 (B)-Trp187 (B)-Thr367 (A)-Arg377 (A)-Asn387 (A)-Glu588 (A) of RdRp. Furthermore, these three compounds showed up drug-likeness and biologically active, though further in-vitro investigations are needed

Characterizing fenestration size in sodium channel subtypes and their accessibility to inhibitors

Voltage-gated sodium channels (Nav) underlie the electrical activity of nerve and muscle cells. Humans have nine different subtypes of these channels, which are the target of small-molecule inhibitors commonly used to treat a range of conditions. Structural studies have identified four lateral fenestrations within the Nav pore module that have been shown to influence Nav pore blocker access during resting-state inhibition. However, the structural differences among the nine subtypes are still unclear. In particular, the dimensions of the four individual fenestrations across the Nav subtypes and their differential accessibility to pore blockers is yet to be characterized. To address this, we applied classical molecular dynamics simulations to study the recently published structures of Nav1.1, Nav1.2, Nav1.4, Nav1.5, and Nav1.7. Although there is significant variability in the bottleneck sizes of the Nav fenestrations, the subtypes follow a common pattern, with wider DI-II and DIII-IV fenestrations, a more restricted DII-III fenestration, and the most restricted DI-IV fenestration. We further identify the key bottleneck residues in each fenestration and show that the motions of aromatic residue sidechains govern the bottleneck radii. Well-tempered metadynamics simulations of Nav1.4 and Nav1.5 in the presence of the pore blocker lidocaine also support the DI-II fenestration being the most likely access route for drugs. Our computational results provide a foundation for future in vitro experiments examining the route of drug access to sodium channels. Understanding the fenestrations and their accessibility to drugs is critical for future analyses of diseases mutations across different sodium channel subtypes, with the potential to inform pharmacological development of resting-state inhibitors and subtype-selective drug design.

New insights of falcipain 2 structure from Plasmodium falciparum 3D7 strain

Malaria identifies as a tropical hallmark, conforming to the burgeoning notion of escalating drug resistance among virulent strains, with the burdensome Plasmodium falciparum under its wing. The cysteine protease Falcipain-2 (FP2) is released in the parasite's food vacuole in the trophozoite stage and contributes to disease progression through its hemoglobinase activity. In the present study, we have determined the crystal structure of FP2 from a drug resistant P. falciparum 3D7 strain. FP2-3D7 sequence has detected four amino acid variants, R12K, E14 N, P100T and G102D, in the mature domain of the protease, when compared with other reported structures. FP2-3D7 protease has been crystallized in the presence of two inhibitors E−64 and Iodoacetamide, which diffracted up to 3.5 Å and 3.4 Å respectively. Structural analyses of the mature domain helped unveil two solvent-exposed pockets with bound ligands where one is structurally homologous to the allosteric binding site of human Cathepsin-K and thus, could be exploited for designing allosteric modifiers of FP2. The structure has also revealed (poly)ethylene glycol molecules along the catalytic cleft, providing interesting insights for exploring FP2 as a chemotherapeutic target and for PEGylation in drug delivery. The side-chains of P2 and P3 residues of E−64 also adopt different rotamer conformations, compared with previously reported structure, emphasizing strain-specific multiple binding-modes of active-site targeted inhibitors. Docking studies, along with normal mode analyses, highlight the mode of hemoglobin binding and the active/inactive switch in hemoglobinase activity, conjecturing the formation of a stable dimeric state with a symmetry related copy in crystal packing.