scholarly journals Cold Denaturation of Proteins in the Absence of Solvent

2021 ◽  
Author(s):  
Emma Norgate ◽  
Rosie Upton ◽  
Bruno Bellina ◽  
Kjetil Hansen ◽  
Catherine Brookes ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Freezing Point ◽  
Collision Cross Section ◽  
Variable Temperature ◽  
Structural Rearrangement ◽  
Effect Of Temperature ◽  
Cold Denaturation ◽  
Ambient Temperatures ◽  
The Stability

The effect of temperature on the stability of proteins is well explored for high temperatures, but harder to track below the freezing point of water. This challenge is met with the use of variable temperature ion mobility mass spectrometry (VT IM-MS), which allows the structure of isolated, solvent free molecules to be measured at sub ambient temperatures in the form of their collision cross section (CCS). Here we monitor conformational changes that occur to two isotypes of monoclonal antibodies over a temperature range from 295 to 165 K. For each we observe a large increase in the magnitude of the CCS at 250K (-20 °C) substantially above that predicted. This loss of structure in the absence of bulk solvent is attributed to a change in the strength of stabilizing intermolecular interactions, causing rearrangement. At 190 K (-80 °C) the CCS distribution narrows which we attribute to better resolution. These findings indicate that in vacuo deep-freezing minimizes denaturation and maintains the gas phase native fold supporting this practice in vitro. Comparing the data for each isotype suggests that the disulfide bridging influences thermal structural rearrangement and taken together we show that this method provides unique insights to the phenomenon of cold denaturation.

Applicability of Instability Index for In vitro Protein Stability Prediction

2019 ◽  
Vol 26 (5) ◽  
pp. 339-347 ◽  
Author(s):  
Dilani G. Gamage ◽  
Ajith Gunaratne ◽  
Gopal R. Periyannan ◽  
Timothy G. Russell
Keyword(s):  
Protein Stability ◽  
Caulobacter Crescentus ◽  
Effect Of Temperature ◽  
Instability Index ◽  
Dipeptide Composition ◽  
Experimental Conditions ◽  
The Stability ◽  
Vitro Protein

Background: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. Objective: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. Methods: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. Results: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in vitro conditions. However, the II failed to accurately represent the stability of other tested proteins. Data indicate the influence of protein environmental factors on the autoproteolysis of proteins. Conclusion: Broader application of the II method for the prediction of protein stability under in vitro conditions is questionable as the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu.

Comparison of the effect of poly(N-vinyl caprolactam) and poly(N-isopropyl acrylamide) trimers on the stability of hydrated Na-montmorillonite: A molecular dynamics study

2020 ◽  
pp. 096739112093524
Author(s):  
Jiafang Xu ◽  
Moussa Camara ◽  
Hualin Liao ◽  
Hong Guo ◽  
Kouassi Louis Kra ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Conformational Changes ◽  
Stable State ◽  
Dynamics Simulation ◽  
Effect Of Temperature ◽  
Radius Of Gyration ◽  
Molecular Arrangement ◽  
Isopropyl Acrylamide ◽  
The Stability ◽  
The Impact

In the present study, we performed a molecular dynamics simulation of the intercalation of poly( N-isopropyl acrylamide) (NIPAM)3 and poly( N-vinyl caprolactam) (NVCL)3 trimers into Na-montmorillonite (Na-Mt) to evaluate their effects on the interlayer structure and the stability of hydrated Na-Mt. The impact of both trimers on the interlayer species and their dynamics properties at different temperatures in a canonical ensemble (NVT) were investigated. The results showed that the electrostatic forces exerted by Na cations on H2O molecules and the interlayer H2O molecular arrangement are not affected by the rise in temperature after adding both trimers. Trimer addition reinforced the structure of interlayer H2O molecules so that the effect of temperature increase on them became negligible. The structural dynamics evolution of the radius of gyration of both trimers showed the existence of conformation changes when temperature increased. These conformational changes are more complex in the case of (NVCL)3 than (NIPAM)3 due to its large monomers. Both trimers reduced the mobility of interlayer particles with a better inhibition effect obtained for (NVCL)3 compared to (NIPAM)3. The concentration profile of interlayers’ species showed the affinity of Na cations for clay mineral surfaces while H2O molecules moved away. Compared these two trimers, the most stable state of Na-Mt is achieved with (NVCL)3. These results could help highlight the inhibition properties of (NIPAM)3 and (NVCL)3 on hydrated Na-Mt and to predict its stability against changes in environmental conditions.

scholarly journals Development of Lyophilized Gemini Surfactant-Based Gene Delivery Systems: Influence of Lyophilization on the Structure, Activity and Stability of the Lipoplexes

2012 ◽  
Vol 15 (4) ◽  
pp. 548 ◽  
Author(s):  
Waleed Mohammed-Saeid ◽  
Deborah Michel ◽  
Anas El-Aneed ◽  
Ronald E Verrall ◽  
Nicholas H Low ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Gemini Surfactant ◽  
Gemini Surfactants ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Physiochemical Properties ◽  
Cationic Gemini Surfactants ◽  
The Stability ◽  
In Vitro Transfection

Purpose. Cationic gemini surfactants have been studied as non-viral vectors for gene therapy. Clinical applications of cationic lipid/DNA lipoplexes are restricted by their instability in aqueous formulations. In this work, we investigated the influence of lyophilization on the essential physiochemical properties and in vitro transfection of gemini surfactant-lipoplexes. Additionally, we evaluated the feasibility of lyophilization as a technique for preparing lipoplexes with long term stability. Methods. A gemini surfactant [12-7NH-12] and plasmid DNA encoding for interferon-γ were used to prepare gemini surfactant/pDNA [P/G] lipoplexes. Helper lipid DOPE [L] was incorporated in all formulation producing a [P/G/L] system. Sucrose and trehalose were utilized as stabilizing agents. To evaluate the ability of lyophilization to improve the stability of gemini surfactant-based lipoplexes, four lyophilized formulations were stored at 25˚C for three months. The formulations were analyzed at different time-points for physiochemical properties and in vitro transfection. Results. The results showed that both sucrose and trehalose provided anticipated stabilizing effect. The transfection efficiency of the lipoplexes increased 2-3 fold compared to fresh formulations upon lyophilization. This effect can be attributed to the improvement of DNA compaction and changes in the lipoplex morphology due to the lyophilization/rehydration cycles. The physiochemical properties of the lyophilized formulations were maintained throughout the stability study. All lyophilized formulations showed a significant loss of gene transfection activity after three months of storage. Nevertheless, no significant losses of transfection efficiency were observed for three formulations after two months storage at 25 ˚C. Conclusion. Lyophilization significantly improved the physical stability of gemini surfactant-based lipoplexes compared to liquid formulations. As well, lyophilization improved the transfection efficiency of the lipoplexes. The loss of transfection activity upon storage is most probably due to the conformational changes in the supramolecular structure of the lipoplexes as a function of time and temperature rather than to DNA degradation. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.

HLA Binding Characteristics and Generation of Cytotoxic Lymphocytes against Peptides Derived from Oncogenic Proteins

1997 ◽  
Vol 83 (5) ◽  
pp. 847-855 ◽  
Author(s):  
Carla Bertazzoni ◽  
Edoardo Marchesi ◽  
Said Dermime ◽  
Fernando Ravagnani ◽  
Giorgio Parmiani ◽  
...  
Keyword(s):  
Cellular Response ◽  
Calf Serum ◽  
Effect Of Temperature ◽  
Binding Motif ◽  
Thymidine Uptake ◽  
Binding Motifs ◽  
Peptide Complex ◽  
Hla Binding ◽  
The Stability

Aims and background Structurally altered proteins (derived from chromosomal translocations or gene mutations) can be considered tumor specific antigens and represent an attractive target for a T-cell mediated response. T lymphocytes recognize antigens in the form of peptides bound to HLA-mole-cules. Materials and methods Peptides derived from oncogenic proteins were screened fro the presence of HLA binding motifs; actual binding were evaluated by HLA stabilization experiments using transfectants and specific anti-HLA antibodies. Specific lymphocytes were induced by in vitro peptide sensitization and screened by thymidine uptake or cellular cytotoxic assays. Results We identified peptides derived from EWS/FLI-1 fusion protein and from mutated K-RAS protein (encompassing respectively the fusion point and the mutation at position 12) that showed binding motif for HLA-Cw*0702 and HLA-A3 respectively. The actual binding of these peptides was analysed in a stabilization assay. We detected binding for the EWS/FLI-I peptide and for 5 RAS peptides (1 wild type and 4 mutated). The effect of temperature, β2-microglobulin (β2-m) and fetal calf serum (FCS) on the binding and the stability of the HLA/peptide complex was studied. A low temperature (26°C) increased the binding both in HLA-A3 and HLA-Cw*0702, while FCS reduced it. β2-m increased the binding to the HLA-A3 molecule but did not influence the binding to the HLA-Cw*0702. The stability of already formed complexed was somewhat different in the HLA-A3 and HLA-Cw*0702 system: both were more stable at 26°C than at 37°C but while the β2-m and FCS did not influence the stability of the HLA-A3/peptide complex, they seemed to cause opposite effects in the HLA-Cw*0702 system (β2-m stabilized and FCS destabilized the complex). Finally, we were able to generate a specific CD8+ CTL line against a K-RAS mutated peptide. Conclusions Although binding motifs and actual HLA binding can be detected in several cases, the generation of a cellular response is infrequent, confirming that HLA binding is necessary but not sufficient to obtain an in vitro response. Further optimization of culture conditions, type of Antigen Presenting Cells (APC), peptides, use of stabilizers like β2-m are still needed.

A role for the beta-subunit in the expression of functional Na+-K+-ATPase in Xenopus oocytes

1989 ◽  
Vol 257 (5) ◽  
pp. C851-C858 ◽  
Author(s):  
K. Geering ◽  
I. Theulaz ◽  
F. Verrey ◽  
M. T. Hauptle ◽  
B. C. Rossier
Keyword(s):  
Plasma Membrane ◽  
Conformational Changes ◽  
Structural Rearrangement ◽  
Cellular Systems ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Functional Maturation ◽  
Unique System

In all cellular systems studied so far, the catalytic alpha- and the glycosylated beta-subunit of Na+-K+-ATPase are coordinately synthesized and are assembled into stoichiometric alpha, beta-complexes. In contrast to these data, in this study we show that the fully grown oocyte of Xenopus laevis synthesizes much less beta-subunit than alpha-subunit. The alpha-subunit produced in excess over the beta-subunit is membrane associated but highly trypsin sensitive and can be compared with the immature alpha-subunit population identified in epithelial cells immediately after synthesis (K. Geering, J. P. Kraehenbuhl, and B.C. Rossier, J. Cell Biol. 105: 2613-2619, 1987). The Xenopus oocyte thus turns out to be a unique system to study the functional role of the beta-subunit. Injection of beta-subunit-specific mRNA transcribed in vitro from a beta-cDNA clone (derived from Xenopus kidney, A6 cells) into oocytes results in translation of a glycosylated beta-subunit. The synthesis of this exogenous beta-subunit increases significantly the proportion of trypsin-resistant oocyte alpha-subunits able to perform cation-dependent conformational changes. In addition, 25-65% more ouabian binding sites are expressed at the plasma membrane in beta-mRNA-injected oocytes. In contrast, newly synthesized alpha-subunit translated after injection of size-fractionated mRNA enriched in alpha-mRNA remains trypsin sensitive as the oocyte alpha-subunit. These data suggest that association of the beta-subunit to the alpha-subunit provokes a structural rearrangement of the alpha-subunit that might be a first step toward the functional maturation of the Na+-K+-ATPase and its expression at the plasma membrane.

scholarly journals Computational Insights into the Unfolding of a Destabilized Superoxide Dismutase 1 Mutant

Biology ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1240
Author(s):  
Stepan Timr ◽  
Fabio Sterpone
Keyword(s):  
Superoxide Dismutase ◽  
Conformational Changes ◽  
State Of The Art ◽  
Model System ◽  
Superoxide Dismutase 1 ◽  
Ambient Temperatures ◽  
Conformational Landscape ◽  
The Stability ◽  
Lateral Sclerosis

In this work, we investigate the β-barrel of superoxide dismutase 1 (SOD1) in a mutated form, the isoleucine 35 to alanine (I35A) mutant, commonly used as a model system to decipher the role of the full-length apoSOD1 protein in amyotrophic lateral sclerosis (ALS). It is known from experiments that the mutation reduces the stability of the SOD1 barrel and makes it largely unfolded in the cell at 37 degrees Celsius. We deploy state-of-the-art computational machinery to examine the thermal destabilization of the I35A mutant by comparing two widely used force fields, Amber a99SB-disp and CHARMM36m. We find that only the latter force field, when combined with the Replica Exchange with Solute Scaling (REST2) approach, reproduces semi-quantitatively the experimentally observed shift in the melting between the original and the mutated SOD1 barrel. In addition, we analyze the unfolding process and the conformational landscape of the mutant, finding these largely similar to those of the wildtype. Nevertheless, we detect an increased presence of partially misfolded states at ambient temperatures. These states, featuring conformational changes in the region of the β-strands β4−β6, might provide a pathway for nonnative aggregation.

A New, Heavier-Than-Water Silicone Oil: A Solution of Perfluorohexyloctane in Polydimethylsiloxane

2005 ◽  
Vol 15 (5) ◽  
pp. 627-637 ◽  
Author(s):  
Y. K. Kim ◽  
B. Günther ◽  
H. Meinert
Keyword(s):  
Silicone Oil ◽  
Interface Layer ◽  
Subretinal Space ◽  
Capillary Viscometer ◽  
Interface Tension ◽  
Ambient Temperatures ◽  
Surface And Interface ◽  
Low Viscosity ◽  
The Stability

Purpose To prepare and explore new solutions of semifluorinated alkane in silicone oil, which have a specific gravity slightly higher than silicone oil and vitreous fluid (referred to in the following as heavier-than-water silicone oils (HWSs), and to investigate, in vitro, whether HWSs can be used to plug retina holes, while allowing dehydration of the subretinal space. Methods HWS solutions were prepared with silicone oil 5000 and perfluorohexyloctane (F6H8). The stability was investigated under different conditions. The viscosity was determined by means of a capillary viscometer. The surface and interface tension were measured using the ring method. Results HWSS are insoluble in an aqueous medium. Densiron®68 (HWS 1.06) is a transparent homogeneous liquid which is slightly heavier (1.06 g/cm3) than water and has a refractive index close to that of vitreous liquid. Densiron®68 (HWS 1.06) has a low viscosity (1480 mPas) and interface tension (40.82 mN/m), making it an effective tamponade in the surgical treatment of an inferior detached retina. In addition, the interfaces between Densiron®68 and other perfluorocarbon liquids are clearly visible. However, the interface layer between Densiron®68 and water is not clear. Finally, all HWSs are stable over the long term at ambient temperatures, as well as physically and thermally resistant. Conclusions Due to its physiochemical properties, Densiron®68 could meet the requirements for a heavier-than-water tamponade.

Studies on the interaction of Escherichia coli endotoxin with erythrocyte membranes

1982 ◽  
Vol 60 (7) ◽  
pp. 977-985 ◽  
Author(s):  
David V. Godin ◽  
John M. Tuchek ◽  
Maureen E. Garnett
Keyword(s):  
Escherichia Coli ◽  
Membrane Lipids ◽  
Effect Of Temperature ◽  
Erythrocyte Membranes ◽  
Simple Relationship ◽  
In Vitro Effects ◽  
The Stability ◽  
Erythrocyte Stability

Escherichia coli lipopolysaccharide (endotoxin) alters the stability of erythrocytes to hypotonic lysis although the nature and magnitude of the effect varied with temperature and with the type of red blood cell examined. Evidence has been obtained suggesting a possible modulatory role of membrane lipids in governing the molecular consequences of membrane–endotoxin interaction. The marked effect of temperature on the stabilization of red cells by endotoxin was not attributable to variations in toxin binding and was not observed with more conventional structurally unrelated antihemolytic agents. Although chemical modifications which alter the toxicity of endotoxin in vivo also modify its ability to stabilize erythrocytes in vitro, no simple relationship between in vivo endotoxin toxicity and in vitro effects on erythrocyte stability was apparent. The critical dependence of endotoxin antihemolytic effects in vitro on molecular structure may offer a convenient means of assessing the homogeneity of these preparations before performing experiments in vivo.

Formation and Structure of Casein Micelles in Lactating Mammary Tissue

1970 ◽  
Vol 28 ◽  
pp. 150-151
Author(s):  
Robert J. Carroll ◽  
Marvin P. Thompson ◽  
Harold M. Farrell
Keyword(s):  
Size Distribution ◽  
G Protein ◽  
Milk Proteins ◽  
Mammary Tissue ◽  
Colloidal System ◽  
Casein Micelles ◽  
The Stability

Milk is an unusually stable colloidal system; the stability of this system is due primarily to the formation of micelles by the major milk proteins, the caseins. Numerous models for the structure of casein micelles have been proposed; these models have been formulated on the basis of in vitro studies. Synthetic casein micelles (i.e., those formed by mixing the purified αsl- and k-caseins with Ca2+ in appropriate ratios) are dissimilar to those from freshly-drawn milks in (i) size distribution, (ii) ratio of Ca/P, and (iii) solvation (g. water/g. protein). Evidently, in vivo organization of the caseins into the micellar form occurs in-a manner which is not identical to the in vitro mode of formation.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance

2019 ◽  
Vol 476 (21) ◽  
pp. 3141-3159 ◽  
Author(s):  
Meiru Si ◽  
Can Chen ◽  
Zengfan Wei ◽  
Zhijin Gong ◽  
GuiZhi Li ◽  
...  
Keyword(s):  
Stress Response ◽  
Ligand Binding ◽  
Corynebacterium Glutamicum ◽  
Conformational Changes ◽  
Cysteine Oxidation ◽  
Transcriptional Regulatory ◽  
Ligand Free ◽  
Redox Sensing

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

Sign in / Sign up

Export Citation Format

Share Document