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The Journal of Physical Chemistry. B Jun 2020Thermal protein unfolding resembles a global (two-state) phase transition. At the local scale, protein unfolding is, however, heterogeneous and probe dependent. Here, we...
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 C 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 CC 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.
Topics: Amino Acid Sequence; Protein Conformation; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Protein Unfolding; Thermodynamics
PubMed: 32392067
DOI: 10.1021/acs.jpcb.0c01230 -
Food Chemistry Jun 2022Osteopontin (OPN) is a multifunctional whey protein with numerous health benefits and is recommended to supplement Infant Milk Formula (IMF) with it. For the first time,...
Osteopontin (OPN) is a multifunctional whey protein with numerous health benefits and is recommended to supplement Infant Milk Formula (IMF) with it. For the first time, the denaturation kinetics and storage stability of bovine OPN were studied in reconstituted IMF. The effect of thermal treatments (63-90 °C) and High Pressure Processing (HPP) were investigated. OPN showed higher retention (around 90%) after both LTLT (Low Temperature Long Time) and HTST (High Temperature Short Time) treatments, however higher temperature and longer treatment induced substantial denaturation indicating UHT may not suit OPN-added items. Significant denaturation occurred at higher pressure (>400 MPa), and more pronounced OPN denaturation was observed after HPP at elevated temperature. OPN remained stable during4 °Cstorage for 14 days. This investigation deduced thermal stability of OPN as α-Lac > OPN > LF, whereas the pressure resistance observed as α-Lac > LF > OPN > β-Lg.
Topics: Animals; Cattle; Humans; Infant; Infant Formula; Kinetics; Milk; Milk Proteins; Osteopontin; Protein Denaturation; Whey Proteins
PubMed: 35063849
DOI: 10.1016/j.foodchem.2022.132138 -
International Journal of Biological... Jan 2021The nano-conjugation of proteins is an active area of research due to potential biomedical and nanotechnological applications. Many protein-nanoconjugates were designed...
The nano-conjugation of proteins is an active area of research due to potential biomedical and nanotechnological applications. Many protein-nanoconjugates were designed for various applications, such as drug delivery, molecular imaging, and liquid biopsy etc. However, the challenges remain to ensure protein stability and to retain the conformational state of the protein intact upon nano-conjugation. In this communication we have reported the status of stability and refolding ability of Au-NP conjugated zDHFR protein. The effect of nano-conjugation of zDHFR on the thermal stability and it's refolding from thermally denatured state have been extensively studied. Zebrafish Dihydrofolate reductase (zDHFR) is an essential enzyme which acts as a crucial part in synthesis of purine, thymidylate and various amino acids in cells. We have nano-conjugated zDHFR protein with Au-nanoparticles and studies were conducted for thermally denatured Au-NP conjugated zDHFR and compared with the non-conjugated protein. Refolding experiment of heat denatured Au-NP conjugated zDHFR was carried out to check the status of refolding and the result was compared with the non-conjugated protein. Our observation reveals that nano-conjugation stabilises the zDHFR protein against thermal denaturation. Furthermore, the nano-conjugation promotes refolding process of thermally unfolded DHFR such that the yield of refolding substantially increases.
Topics: Animals; Chemical Phenomena; Gene Expression; Gold; Kinetics; Metal Nanoparticles; Nanostructures; Protein Denaturation; Protein Folding; Protein Refolding; Protein Stability; Recombinant Proteins; Tetrahydrofolate Dehydrogenase; Thermodynamics; Zebrafish
PubMed: 33181215
DOI: 10.1016/j.ijbiomac.2020.11.053 -
Polyols, increasing global stability of cytochrome , destabilize the thermal unfolding intermediate.Journal of Biomolecular Structure &... 2022Studies on the intermediate states of proteins provide essential information on folding pathway and energy landscape of proteins. Osmolytes, known to alter the stability...
Studies on the intermediate states of proteins provide essential information on folding pathway and energy landscape of proteins. Osmolytes, known to alter the stability of proteins, might also affect the structure and energy states of folding intermediates. This was examined using cytochrome (Cyt) as a model protein which forms a spectroscopically detectable intermediate during thermal denaturation transition. Most of the secondary structure and the native heme-ligation were intact in the intermediate state of the protein. Denaturants, urea and guanidinium hydrochloride, and ionic salt destabilizes the intermediate and drive the protein to follow two-state transition. The effect of polyol class of osmolytes, glycol, glycerol, erythritol, xylitol and sorbitol (with OH-groups two to six), on the intermediate was studied using Soret absorbance and far-UV circular dichroism. With the increasing concentration of any of the polyols, the transition-midpoint temperature () and the enthalpy change (Δ) for native to intermediate transition were decreased. This indicated that the intermediate was destabilized by the polyols. However, the polyols increased the overall stability of the protein by increasing and Δ for intermediate to unfolded transition, except for glycol which destabilized the protein. These results show that the polyols could alter the energy state of the intermediate, and the effect of lower and higher polyols might be different on the stability and folding pathway of the protein.Communicated by Ramaswamy H. Sarma.
Topics: Protein Denaturation; Cytochromes c; Polymers; Thermodynamics; Circular Dichroism; Glycols; Protein Folding
PubMed: 34308796
DOI: 10.1080/07391102.2021.1956593 -
Journal of Molecular Modeling Aug 2021Microsecond timescale explicit-solvent atomistic simulations were carried out to investigate how anionic surfactants modulate protein structure and dynamics. We found...
Microsecond timescale explicit-solvent atomistic simulations were carried out to investigate how anionic surfactants modulate protein structure and dynamics. We found that lysozyme undergoes near-complete denaturation at the high concentration (> 0.1 M) of sodium pentadecyl sulfonate (SPDS), while only partial denaturation occurs at the concentration slightly below 0.1 M. In large part, protein denaturation is structurally manifested by disappearance of helical segments and loss of tertiary interactions. The computational prediction of the extent of burial of cysteine residues was experimentally validated by measuring the accessibility of the respective sulfhydryl groups. Overall, our work indicates an interesting synergy between electrostatic and hydrophobic contributions to lysozyme's denaturation process by anionic surfactants. In fact, first disulfide bridges and hydrogen bonds from protein surface to SPDS head groups loosen the protein globule followed by fuller denaturation via insertion of the surfactant's hydrophobic tails into the protein core.
Topics: Molecular Dynamics Simulation; Muramidase; Protein Denaturation; Sulfonic Acids; Surface-Active Agents
PubMed: 34432183
DOI: 10.1007/s00894-021-04882-2 -
International Journal of Cosmetic... Jun 2024Hair ageing is accompanied by hair fibres becoming irregularly shaped causing them to stick out in irregular directions or have more curliness and being spread out. This...
OBJECTIVE
Hair ageing is accompanied by hair fibres becoming irregularly shaped causing them to stick out in irregular directions or have more curliness and being spread out. This is believed to be due to changes within the hair fibre structure which occur with ageing, and one of the causes of these changes could be an increase in the number of protein carbonyl groups present in the hair. The aim of this study is to investigate the internal denaturation of hair related to protein carbonyls in attempt to gain new insight into age-related changes that occur in hair.
METHODS
The degree of carbonylation of the hair structural protein as determined by fluorescent labelling and Western blotting analysis was used to investigate the primary structure of hair protein. The amount of helix, a common conformation in the secondary structure of proteins, in hair in groups of women with different ages was also analysed using infrared microscopy coupled with multivariate curve resolution (MCR). From the results of this, an image of the two-dimensional distribution of the α-helices was generated for the hair taken from each age group. Also, high-pressure differential scanning calorimetry (HPDSC) of the hair in water was performed on the hair taken from each age group to determine the peak temperature of endothermic effect and the enthalpy of denaturation.
RESULTS
We found that the amino group content in hair proteins decreased and Type II keratin, one of the subunits of intermediate filament, was more carbonylated with age. The results of the MCR indicated eight separate components, including components of the secondary structure of proteins, such as α helices and β sheets. Two-dimensional images of the hair cross-sections revealed that the presence of α helices decreased with age. In addition, data from the HPDSC showed that the enthalpy associated with the denaturing temperature also significantly decreased with age.
CONCLUSION
These results suggest that there is a negative correlation between age and structural integrity of the helix segment in intermediate filament. The results of this study also show that there is a positive correlation between age-related hair denaturation and protein carbonyls.
Topics: Hair; Humans; Protein Denaturation; Female; Adult; Calorimetry, Differential Scanning; Middle Aged; Aging; Protein Carbonylation; Young Adult; Blotting, Western
PubMed: 38083986
DOI: 10.1111/ics.12934 -
Biochemistry Mar 2020-designed protein domains are increasingly being applied in biotechnology, cell biology, and synthetic biology. Therefore, it is imperative that these proteins be robust...
-designed protein domains are increasingly being applied in biotechnology, cell biology, and synthetic biology. Therefore, it is imperative that these proteins be robust to superficial changes; i.e., small changes to their amino acid sequences should not cause gross structural changes. In turn, this allows properties such as stability and solubility to be tuned without affecting structural attributes like tertiary fold and quaternary interactions. Reliably designed proteins with predictable behaviors may then be used as scaffolds to incorporate function, e.g., through the introduction of features for small-molecule, metal, or macromolecular binding, and enzyme-like active sites. Generally, achieving this requires the starting protein fold to be well understood. Herein, we focus on designing α-helical coiled coils, which are well studied, widespread, and often direct protein-protein interactions in natural systems. Our initial investigations reveal that a previously designed parallel, homotetrameric coiled coil, CC-Tet, is not robust to sequence changes that were anticipated to maintain its structure. Instead, the alterations switch the oligomeric state from tetramer to trimer. To improve the robustness of designed homotetramers, additional sequences based on CC-Tet were produced and characterized in solution and by X-ray crystallography. Of these updated sequences, one is robust to truncation and to changes in surface electrostatics; we call this CC-Tet*. Variants of the general CC-Tet* design provide a set of homotetrameric coiled coils with unfolding temperatures in the range from 40 to >95 °C. We anticipate that these will be of use in applications requiring robust and well-defined tetramerization domains.
Topics: Amino Acid Sequence; Crystallography, X-Ray; Models, Molecular; Protein Conformation, alpha-Helical; Protein Denaturation; Protein Domains; Protein Engineering; Protein Folding; Protein Structure, Quaternary; Protein Structure, Secondary; Proteins; Static Electricity; Thermodynamics
PubMed: 32133841
DOI: 10.1021/acs.biochem.0c00082 -
Analytical Chemistry Feb 2023The conventional quality control techniques for identifying the denaturation of biopharmaceuticals includes sodium dodecyl sulfate-polyacrylamide gel electrophoresis for...
The conventional quality control techniques for identifying the denaturation of biopharmaceuticals includes sodium dodecyl sulfate-polyacrylamide gel electrophoresis for identifying fragmentation, ion exchange chromatography and isoelectric focusing for identifying deamidation, reverse-phase high-performance liquid chromatography (HPLC) for identifying oxidation, and size-exclusion HPLC for identifying aggregation. These stability assessments require essential processes that are destructive to the product tested. All these techniques are lab based and require sample removal from a sealed storage vial, which can breach the sterility. In this work, we investigate the heat- and surfactant-induced denaturation of an in-vial-stored model protein, bovine serum albumin (BSA), by analyzing its intrinsic fluorescence without removing the sample from the vial. A lab-based bespoke setup which can do the measurement in vial is used to demonstrate the change in fluorescence polarization of the protein to determine the denaturation level. The results obtained are compared to circular dichroism and size-exclusion HPLC measurements. The results prove that in-vial fluorescence measurements can be performed to monitor protein denaturation. A cost-effective portable solution to provide a top-level overview of biopharmaceutical product stability from manufacture to the point of patient administration can be further developed using the same technique.
Topics: Humans; Protein Denaturation; Serum Albumin, Bovine; Hot Temperature; Fluorescence Polarization
PubMed: 36696963
DOI: 10.1021/acs.analchem.2c03912 -
Analytical Chemistry Mar 2020Size-exclusion chromatography employing aqueous mobile phases with volatile salts at neutral pH combined with electrospray-ionization mass spectrometry (SEC-ESI-MS) is a...
Size-exclusion chromatography employing aqueous mobile phases with volatile salts at neutral pH combined with electrospray-ionization mass spectrometry (SEC-ESI-MS) is a useful tool to study proteins in their native state. However, whether the applied eluent conditions actually prevent protein-stationary phase interactions, and/or protein denaturation, often is not assessed. In this study, the effects of volatile mobile phase additives on SEC retention and ESI of proteins were thoroughly investigated. Myoglobin was used as the main model protein, and eluents of varying ionic strength and pH were applied. The degree of interaction between protein and stationary phase was evaluated by calculating the SEC distribution coefficient. Protein-ion charge state distributions obtained during offline and online native ESI-MS were used to monitor alterations in protein structure. Interestingly, most of the supposedly mild eluent compositions induced nonideal SEC behavior and/or protein unfolding. SEC experiments revealed that the nature, ionic strength, and pH of the eluent affected protein retention. Protein-stationary phase interactions were effectively avoided using ammonium acetate at ionic strengths above 0.1 M. Direct-infusion ESI-MS showed that the tested volatile eluent salts seem to follow the Hofmeister series: no denaturation was induced using ammonium acetate (kosmotropic), whereas ammonium formate and bicarbonate (both chaotropic) caused structural changes. Using a mobile phase of 0.2 M ammonium acetate (pH 6.9), several proteins (i.e., myoglobin, carbonic anhydrase, and cytochrome c) could be analyzed by SEC-ESI-MS using different column chemistries without compromising their native state. Overall, with SEC-ESI-MS, the effect of nonspecific interactions between protein and stationary phase on the protein structure can be studied, even revealing gradual structural differences along a peak.
Topics: Animals; Chromatography, Gel; Heart; Horses; Hydrogen-Ion Concentration; Myoglobin; Protein Denaturation; Spectrometry, Mass, Electrospray Ionization
PubMed: 32107919
DOI: 10.1021/acs.analchem.9b04961 -
Computers in Biology and Medicine Mar 2023Non-covalent intramolecular interactions play a key role in the protein folding process. Aminoacidic mutations or changes in physiological conditions such as pH and/or...
Non-covalent intramolecular interactions play a key role in the protein folding process. Aminoacidic mutations or changes in physiological conditions such as pH and/or temperature variations can compromise intramolecular stability generating misfolding or unfolding proteins with consequent impairment of functionality and the triggering of pathological states. The intramolecular HINT scoring function recently implemented and validated, is proposed as a rapid and sensitive method for the evaluation of different conformational states characterizing destabilization processes. In this work, the stability of Transthyretin, whose denaturation is related to amyloid fibril formation, is evaluated by generating multiple structural mutated models under different pH conditions in comparison with experimental data. These results suggest that the HINT scoring function can be used for an accurate and rapid evaluation and computational prediction of the effects of structural changes on any protein system.
Topics: Prealbumin; Amyloid; Comprehension; Protein Denaturation; Protein Folding
PubMed: 36805224
DOI: 10.1016/j.compbiomed.2023.106667