THERMAL STABILITY OF SYNTHETIC PEPTIDES MIMICKING THE  SEQUENCE OF THE REGION CONTAINING THE SKIP RESIDUES IN SQUID MYOSIN ROD

Myosin is the major protein in skeletal muscles including those of fish and shellfish. The characteristics of this protein are closely related to the biological function and the quality and physical properties of muscle<br />food. In the myosin rod (the coiled-coil region of myosin), several amino acid residues, known as skip residues, seem to destabilize the ordered structure (heptad repeat). These residues might be responsible for reducing thermal stability. Attempts were thus made to examine the role of these residues in the rod of squid myosin, based on the thermodynamic properties of synthetic peptides which have been designed to mimic the partial sequence of myosin heavy chain from the squid Todarodes pacificus mantle muscle. Five peptides, namely, with the sequence of Trp1343 -Ala1372 having the skip residue Glu1357 at the center (Peptide WT), without the skip residue (Peptide Δ), with the replacements of the skip residue (Glu) by Ile, Gln and Pro (Peptides E/I, E/Q, and E/P, respectively) to modify the helix forming propensity, were synthesized. The results obtained showed that the stability of the peptides as measured by circular dichroism spectrometry was in the order of Peptide Δ > Peptide WT > Peptide E/Q > Peptide E/P > Peptide E/I. It is suggested that the presence of the skip residues dexterously tunes the stability or flexibility of the coiled-coil structure, thus possibly regulating thick filament formation and further gel formation ability of myosin.

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The stability of the coiled-coil structure near to N-terminus influence the heat resistance of harpin proteins from Xanthomonas

BMC Microbiology ◽

10.1186/s12866-020-02029-6 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Yue Liu ◽

Xiaoyun Zhou ◽

Wenbo Liu ◽

Weiguo Miao

Keyword(s):

Thermal Stability ◽

Heat Resistance ◽

Coiled Coil ◽

Temperature Treatment ◽

High Temperature Treatment ◽

Amino Acid Residues ◽

Important Amino Acid ◽

C Terminus ◽

N Terminus ◽

The Stability

Abstract Background Heat resistance is a common characteristic of harpins, a class of proteins found in Gram-negative bacteria, which may be related to the stability of coiled-coil (CC) structure. The CC structure is a ubiquitous protein folding and assembly motif made of α-helices wrapping around each other forming a supercoil. Specifically, whether the stability of the CC structure near to N-terminus of four selected harpin proteins from Xanthomonas (hereafter referred to as Hpa1) would influence their characteristics of heat resistance was investigated. We used bioinformatics approach to predict the structure of Hpa1, used the performance of hypersensitive response (HR)-induction activity of Hpa1 and circular dichroism (CD) spectral analyses to detect the relationship between the stability of the CC structure of Hpa1 and heat resistance. Results Each of four-selected Hpa1 has two α-helical regions with one in their N-terminus that could form CC structure, and the other in their C-terminus that could not. And the important amino acid residues involved in the CC motifs are located on helices present on the surface of these proteins, indicating they may engage in the formation of oligo mericaggregates, which may be responsible for HR elicitation by harpins and their high thermal stability. Increased or decreased the probability of forming a CC could either induce a stronger HR response or eliminate the ability to induce HR in tobacco after high temperature treatment. In addition, although the four Hpa1 mutants had little effect on the induction of HR by Hpa1, its thermal stability was significantly decreased. The α-helical content increased with increasing temperature, and the secondary structures of Hpa1 became almost entirely α-helices when the temperature reached 200 °C. Moreover, the stability of the CC structure near to N-terminus was found to be positively correlated with the heat resistance of Hpa1. Conclusions The stability of the CC structure might sever as an inner drive for mediating the heat resistance of harpin proteins. Our results offer a new insight into the interpretation of the mechanism involved in the heat resistance of harpin protein and provide a theoretical basis for further harpin function investigations and structure modifications.

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Expression in Escherichia coli of fragments of the coiled-coil rod domain of rabbit myosin: influence of different regions of the molecule on aggregation and paracrystal formation

Journal of Cell Science ◽

10.1242/jcs.99.4.823 ◽

1991 ◽

Vol 99 (4) ◽

pp. 823-836

Author(s):

S.J. Atkinson ◽

M. Stewart

Keyword(s):

Escherichia Coli ◽

Ionic Strength ◽

Coiled Coil ◽

Periodic Variation ◽

Heptad Repeat ◽

C Terminus ◽

N Terminus ◽

Low Ionic Strength ◽

Myosin Rod

We have expressed in Escherichia coli a cDNA clone corresponding broadly to rabbit light meromyosin (LMM) together with a number of modified polypeptides and have used this material to investigate the role of different aspects of molecular structure on the solubility properties of LMM. The expressed material was characterized biochemically and structurally to ensure that it retained the coiled-coil conformation of the native molecule. Full-length recombinant LMM retained the general solubility properties of myosin and, although soluble at high ionic strength, precipitated when the ionic strength was reduced below 0.3 M. Constructs in which the ‘skip’ residues (that disrupt the coiled-coil heptad repeat) were deleted had solubility properties indistinguishable from the wild type, which indicated that the skip residues did not play a major role in determining the molecular interactions involved in assembly. Deletions from the N terminus of LMM did not alter the solubility properties of the expressed material, but deletion of 92 residues from the C terminus caused a large increase in solubility at low ionic strength, indicating that a determinant important for interaction between LMM molecules was located in this region. The failure of deletions from the molecule's N terminus to alter its solubility radically suggested that the periodic variation of charge along the myosin rod may not be as important as proposed for determining the strength of binding between molecules and thus the solubility of myosin.

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The C Terminus of the B5 Receptor for Herpes Simplex Virus Contains a Functional Region Important for Infection

Journal of Virology ◽

10.1128/jvi.79.12.7431-7437.2005 ◽

2005 ◽

Vol 79 (12) ◽

pp. 7431-7437 ◽

Cited By ~ 8

Author(s):

Pilar Perez-Romero ◽

A. Oveta Fuller

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Synthetic Peptides ◽

Point Mutations ◽

Coiled Coil ◽

Heptad Repeat ◽

Single Amino Acid ◽

Functional Region ◽

C Terminus ◽

Simplex Virus

ABSTRACT The expression of a previously uncharacterized human hfl-B5 cDNA confers susceptibility for herpes simplex virus (HSV) to porcine cells and fulfills criteria as an HSV entry receptor (A. Perez, Q.-X. Li, P. Perez-Romero, G. DeLassus, S. R. Lopez, S. Sutter, N. McLaren, and A. Oveta Fuller, J. Virol. 79:7419-7430, 2005). Heptad repeats found in the B5 C terminus are predicted to form an α-helix for coiled coil structure. We used mutagenesis and synthetic peptides with wild-type and mutant sequences to examine the function of the heptad repeat motif in HSV binding and entry into porcine cells that express B5 and for infection of naturally susceptible human HEp-2 cells. B5 with point mutations predicted to disrupt the putative C-terminal coiled coil failed to mediate HSV binding and entry into porcine cells. Synthetic peptides that contain the single amino acid changes lose the blocking activity of HSV entry. We concluded that the C terminus of B5 contains a functional region that is important for the B5 receptor to mediate events in HSV entry. Structural evidence that this functional region forms coiled coil structures is under investigation. Blocking of HSV interaction with the C-terminal region of the B5 receptor is a new potential target site to intervene in the virus infection of human cells.

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Structural and thermodynamic analysis of factors governing the stability and thermal folding/unfolding of SazCA

PLoS ONE ◽

10.1371/journal.pone.0249866 ◽

2021 ◽

Vol 16 (4) ◽

pp. e0249866

Author(s):

Shashi Kumar ◽

Parag A. Deshpande

Keyword(s):

Thermal Stability ◽

Free Energy ◽

Protein Stability ◽

Explicit Knowledge ◽

Fundamental Problem ◽

Hydrophobic Surface ◽

Amino Acid Residues ◽

Different Temperatures ◽

The Stability ◽

Dynamics Simulations

Molecular basis of protein stability at different temperatures is a fundamental problem in protein science that is substantially far from being accurately and quantitatively solved as it requires an explicit knowledge of the temperature dependence of folding free energy of amino acid residues. In the present study, we attempted to gain insights into the thermodynamic stability of SazCA and its implications on protein folding/unfolding. We report molecular dynamics simulations of water solvated SazCA in a temperature range of 293-393 K to study the relationship between the thermostability and flexibility. Our structural analysis shows that the protein maintains the highest structural stability at 353 K and the protein conformations are highly flexible at temperatures above 353 K. Larger exposure of hydrophobic surface residues to the solvent medium for conformations beyond 353 K were identified from H-bond analysis. Higher number of secondary structure contents exhibited by SazCA at 353 K corroborated the conformations at 353 K to exhibit the highest thermal stability. The analysis of thermodynamics of protein stability revealed that the conformations that denature at higher melting temperatures tend to have greater maximum thermal stability. Our analysis shows that 353 K conformations have the highest melting temperature, which was found to be close to the experimental optimum temperature. The enhanced protein stability at 353 K due the least value of heat capacity at unfolding suggested an increase in folding. Comparative Gibbs free energy analysis and funnel shaped energy landscape confirmed a transition in folding/unfolding pathway of SazCA at 353 K.

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Residues in the gp41 Ectodomain Regulate HIV-1 Envelope Glycoprotein Conformational Transitions Induced by gp120-Directed Inhibitors

Journal of Virology ◽

10.1128/jvi.02219-16 ◽

2016 ◽

Vol 91 (5) ◽

Cited By ~ 28

Author(s):

Beatriz Pacheco ◽

Nirmin Alsahafi ◽

Olfa Debbeche ◽

Jérémie Prévost ◽

Shilei Ding ◽

...

Keyword(s):

Amino Acid ◽

Conformational Changes ◽

Envelope Glycoprotein ◽

Coiled Coil ◽

Bound State ◽

Heptad Repeat ◽

Amino Acid Residues ◽

Entry Inhibitors ◽

Gp41 Ectodomain ◽

Hiv 1

ABSTRACT Interactions between the gp120 and gp41 subunits of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer maintain the metastable unliganded form of the viral spike. Binding of gp120 to the receptor, CD4, changes the Env conformation to promote gp120 interaction with the second receptor, CCR5 or CXCR4. CD4 binding also induces the transformation of Env into the prehairpin intermediate, in which the gp41 heptad repeat 1 (HR1) coiled coil is assembled at the trimer axis. In nature, HIV-1 Envs must balance the requirements to maintain the noncovalent association of gp120 with gp41 and to evade the host antibody response with the need to respond to CD4 binding. Here we show that the gp41 HR1 region contributes to gp120 association with the unliganded Env trimer. Changes in particular amino acid residues in the gp41 HR1 region decreased the efficiency with which Env moved from the unliganded state. Thus, these gp41 changes decreased the sensitivity of HIV-1 to cold inactivation and ligands that require Env conformational changes to bind efficiently. Conversely, these gp41 changes increased HIV-1 sensitivity to small-molecule entry inhibitors that block Env conformational changes induced by CD4. Changes in particular gp41 HR1 amino acid residues can apparently affect the relative stability of the unliganded state and CD4-induced conformations. Thus, the gp41 HR1 region contributes to the association with gp120 and regulates Env transitions from the unliganded state to downstream conformations. IMPORTANCE The development of an efficient vaccine able to prevent HIV infection is a worldwide priority. Knowledge of the envelope glycoprotein structure and the conformational changes that occur after receptor engagement will help researchers to develop an immunogen able to elicit antibodies that block HIV-1 transmission. Here we identify residues in the HIV-1 transmembrane envelope glycoprotein that stabilize the unliganded state by modulating the transitions from the unliganded state to the CD4-bound state.

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A Region of the Myosin Rod Important for Interaction With Paramyosin in Caenorhabditis elegans Striated Muscle

Genetics ◽

10.1093/genetics/156.2.631 ◽

2000 ◽

Vol 156 (2) ◽

pp. 631-643

Author(s):

Pamela E Hoppe ◽

Robert H Waterston

Keyword(s):

Caenorhabditis Elegans ◽

Heavy Chain ◽

Molecular Interaction ◽

Striated Muscle ◽

Genetic Interaction ◽

Specific Interaction ◽

Thick Filament ◽

Parallel Arrangement ◽

Myosin Rod

Abstract The precise arrangement of molecules within the thick filament, as well as the mechanisms by which this arrangement is specified, remains unclear. In this article, we have exploited a unique genetic interaction between one isoform of myosin heavy chain (MHC) and paramyosin in Caenorhabditis elegans to probe the molecular interaction between MHC and paramyosin in vivo. Using chimeric myosin constructs, we have defined a 322-residue region of the MHC A rod critical for suppression of the structural and motility defects associated with the unc-15(e73) allele. Chimeric constructs lacking this region of MHC A either fail to suppress, or act as dominant enhancers of, the e73 phenotype. Although the 322-residue region is required for suppression activity, our data suggest that sequences along the length of the rod also play a role in the isoform-specific interaction between MHC A and paramyosin. Our genetic and cell biological analyses of construct behavior suggest that the 322-residue region of MHC A is important for thick filament stability. We present a model in which this region mediates an avid interaction between MHC A and paramyosin in parallel arrangement in formation of the filament arms.

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Optimization of Organic Fibres%[Kevlar/Arobocel/Acrylic] in NAO Brake Pad Application and its Effect on Thermal Stability & Friction Characteristics

Key Engineering Materials ◽

10.4028/www.scientific.net/kem.531-532.8 ◽

2012 ◽

Vol 531-532 ◽

pp. 8-12

Author(s):

M.A. Sai Balaji ◽

K. Kalaichelvan

Keyword(s):

Thermal Stability ◽

Weight Loss ◽

Minimum Weight ◽

Friction Material ◽

Brake Pad ◽

Temperature Zone ◽

Good Thermal Stability ◽

Friction Testing ◽

Brake Application ◽

The Stability

Organic fibres (Kevlar/ Arbocel / Acrylic) have good thermal stability, higher surface area and bulk density. The optimization of organic fibres percentage for thermal behaviour is considered using TGA. The temperature raise during brake application will be between 150-4000 C and this temperature zone is very critical to determine the fade characteristics during friction testing. Hence, three different friction composites are developed with the same formulation varying only the Kevlar, Arbocel and Acrylic fibres which are compensated by the inert filler namely the barites and are designated as NA01, NA02 and NA03 respectively. After the fabrication, the TGA test reveals that the composite NA03 has minimum weight loss. The friction coefficient test rig is then used to test the friction material as per SAE J661a standards. The results prove that the brake pad with minimum weight loss during TGA has higher friction stability. Thus, we can correlate the thermal stability with the stability of friction.

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Amino Acids 1055 to 1192 in the S2 Region of Severe Acute Respiratory Syndrome Coronavirus S Protein Induce Neutralizing Antibodies: Implications for the Development of Vaccines and Antiviral Agents

Journal of Virology ◽

10.1128/jvi.79.6.3289-3296.2005 ◽

2005 ◽

Vol 79 (6) ◽

pp. 3289-3296 ◽

Cited By ~ 59

Author(s):

Choong-Tat Keng ◽

Aihua Zhang ◽

Shuo Shen ◽

Kuo-Ming Lip ◽

Burtram C. Fielding ◽

...

Keyword(s):

Amino Acids ◽

Severe Acute Respiratory Syndrome ◽

Viral Entry ◽

Neutralizing Antibodies ◽

Antiviral Agents ◽

Host Cells ◽

Heptad Repeat ◽

Antigenic Determinants ◽

Amino Acid Residues ◽

S Protein

ABSTRACT The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) interacts with cellular receptors to mediate membrane fusion, allowing viral entry into host cells; hence it is recognized as the primary target of neutralizing antibodies, and therefore knowledge of antigenic determinants that can elicit neutralizing antibodies could be beneficial for the development of a protective vaccine. Here, we expressed five different fragments of S, covering the entire ectodomain (amino acids 48 to 1192), as glutathione S-transferase fusion proteins in Escherichia coli and used the purified proteins to raise antibodies in rabbits. By Western blot analysis and immunoprecipitation experiments, we showed that all the antibodies are specific and highly sensitive to both the native and denatured forms of the full-length S protein expressed in virus-infected cells and transfected cells, respectively. Indirect immunofluorescence performed on fixed but unpermeabilized cells showed that these antibodies can recognize the mature form of S on the cell surface. All the antibodies were also able to detect the maturation of the 200-kDa form of S to the 210-kDa form by pulse-chase experiments. When the antibodies were tested for their ability to inhibit SARS-CoV propagation in Vero E6 culture, it was found that the anti-SΔ10 antibody, which was targeted to amino acid residues 1029 to 1192 of S, which include heptad repeat 2, has strong neutralizing activities, suggesting that this region of S carries neutralizing epitopes and is very important for virus entry into cells.

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