Concentrations Of Guanidine Hydrochloride Research Articles

Guanidine Hydrochloride-Induced Hepatitis B Virus Capsid Disassembly Hysteresis.

Hepatitis B virus (HBV) displays remarkable self-assembly capabilities that interest the scientific community and biotechnological industries as HBV is leading to an annual mortality of up to 1 million people worldwide (especially in Africa and Southeast Asia). When the ionic strength is increased, hepatitis B virus-like particles (VLPs) can assemble from dimers of the first 149 residues of the HBV capsid protein core assembly domain (Cp149). Using solution small-angle X-ray scattering, we investigated the disassembly of the VLPs by titrating guanidine hydrochloride (GuHCl). Measurements were performed with and without 1 M NaCl, added either before or after titrating GuHCl. Fitting the scattering curves to a linear combination of atomic models of Cp149 dimer (the subunit) and T = 3 and T = 4 icosahedral capsids revealed the mass fraction of the dimer in each structure in all the titration points. Based on the mass fractions, the variation in the dimer-dimer association standard free energy was calculated as a function of added GuHCl, showing a linear relation between the interaction strength and GuHCl concentration. Using the data, we estimated the energy barriers for assembly and disassembly and the critical nucleus size for all of the assembly reactions. Extrapolating the standard free energy to [GuHCl] = 0 showed an evident hysteresis in the assembly process, manifested by differences in the dimer-dimer association standard free energy obtained for the disassembly reactions compared with the equivalent assembly reactions. Similar hysteresis was observed in the energy barriers for assembly and disassembly and the critical nucleus size. The results suggest that above 1.5 M, GuHCl disassembled the capsids by attaching to the protein and adding steric repulsion, thereby weakening the hydrophobic attraction.

Collection and transportation of SARS-CoV-2 and influenza A virus diagnostic samples: Optimizing the usage of guanidine-based chaotropic salts for enhanced biosafety and viral genome preservation

Many virus lysis/transport buffers used in molecular diagnostics, including the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, contain guanidine-based chaotropic salts, primarily guanidine hydrochloride (GuHCl) or guanidine isothiocyanate (GITC). Although the virucidal effects of GuHCl and GITC alone against some enveloped viruses have been established, standardized data on their optimum virucidal concentrations against SARS-CoV-2 and effects on viral RNA stability are scarce. Thus, we aimed to determine the optimum virucidal concentrations of GuHCl and GITC against SARS-CoV-2 compared to influenza A virus (IAV), another enveloped respiratory virus. We also evaluated the effectiveness of viral RNA stabilization at the determined optimum virucidal concentrations under high-temperature conditions (35°C) using virus-specific real-time reverse transcription polymerase chain reaction. Both viruses were potently inactivated by 1.0 M GITC and 2.5 M GuHCl, but the GuHCl concentration for efficient SARS-CoV-2 inactivation was slightly higher than that for IAV inactivation. GITC showed better viral RNA stability than GuHCl at the optimum virucidal concentrations. An increased concentration of GuHCl or GITC increased viral RNA degradation at 35°C. Our findings highlight the need to standardize GuHCl and GITC concentrations in virus lysis/transport buffers and the potential application of these guanidine-based salts alone as virus inactivation solutions in SARS-CoV-2 and IAV molecular diagnostics.

Nanopore single-molecule biosensor in protein denaturation analysis

Unclear issues in protein studies include but not limited to the stability and denaturation mechanism in the presence of denaturants. Herein, we report a dynamic monitoring approach based on nanopore single-molecule biosensor, which can detect the protein's folding and unfolding transitions by recording a nanopore ionic current. When gradually increasing the concentration of denaturant guanidine hydrochloride (GdmCl), sensitive responses were observed with lysozyme unfolding. The emergence of the featured biphasic-pulse demonstrated the existence of a stable intermediate. It was the first time to experimentally confirm the dynamic equilibrium between the intermediate and the native states at single molecule level, therefore consolidating the standpoint of lysozyme denaturation process following the three-state model. Additionally, we got more insights into the conformation about the intermediate as globular-like structure, larger gyration radius, and enhanced positive charge density. We considered that the manner of denaturant toward lysozyme adopts the “direct” model based on stronger electrostatic and van der Waals forces. Nanopore biosensor exhibited excellent sensitivity with a low detection concentration of 280 pM and reproducibility in analysing the folding intermediate of lysozyme.

Differentiating between the sequence of structural events on alternative pathways of folding of a heterodimeric protein.

Distinguishing between competing pathways of folding of a protein, on the basis of how they differ in their progress of structure acquisition, remains an important challenge in protein folding studies. A previous study had shown that the heterodimeric protein, double chain monellin (dcMN) switches between alternative folding pathways upon a change in guanidine hydrochloride (GdnHCl) concentration. In the current study, the folding of dcMN has been characterized by the pulsed hydrogen exchange (HX) labeling methodology used in conjunction with mass spectrometry. Quantification of the extent to which folding intermediates accumulate and then disappear with time of folding at both low and high GdnHCl concentrations, where the folding pathways are known to be different, shows that the folding mechanism is describable by a triangular three-state mechanism. Structural characterization of the productive folding intermediates populated on the alternative pathways has enabled the pathways to be differentiated on the basis of the progress of structure acquisition that occurs on them. The intermediates on the two pathways differ in the extent to which the α-helix and the rest of the β-sheet have acquired structure that is protective against HX. The major difference is, however, that β2 has not acquired any protective structure in the intermediate formed on one pathway, but it has acquired significant protective structure in the intermediate formed on the alternative pathway. Hence, the sequence of structural events is different on the two alternative pathways.

Clinical relevance of pancreatic fistula in pancreas transplantation: a Comprehensive Complication Index (CCI)-based analysis of postoperative outcomes and predictors of graft survival

Guanidine hydrochloride and urea have different effects on the surface properties of lysozyme solutions due to different interaction mechanisms between the protein and denaturants: an increase in the guanidine hydrochloride concentration leads to noticeable changes in the dynamic surface elasticity, while the main effect of urea consists in a significant drop of the static surface tension.

Influence of denaturants on amyloid β42 aggregation kinetics.

Amyloid formation is linked to devastating neurodegenerative diseases, motivating detailed studies of the mechanisms of amyloid formation. For Aβ, the peptide associated with Alzheimer’s disease, the mechanism and rate of aggregation have been established for a range of variants and conditions in vitro and in bodily fluids. A key outstanding question is how the relative stabilities of monomers, fibrils and intermediates affect each step in the fibril formation process. By monitoring the kinetics of aggregation of Aβ42, in the presence of urea or guanidinium hydrochloride (GuHCl), we here determine the rates of the underlying microscopic steps and establish the importance of changes in relative stability induced by the presence of denaturant for each individual step. Denaturants shift the equilibrium towards the unfolded state of each species. We find that a non-ionic denaturant, urea, reduces the overall aggregation rate, and that the effect on nucleation is stronger than the effect on elongation. Urea reduces the rate of secondary nucleation by decreasing the coverage of fibril surfaces and the rate of nucleus formation. It also reduces the rate of primary nucleation, increasing its reaction order. The ionic denaturant, GuHCl, accelerates the aggregation at low denaturant concentrations and decelerates the aggregation at high denaturant concentrations. Below approximately 0.25 M GuHCl, the screening of repulsive electrostatic interactions between peptides by the charged denaturant dominates, leading to an increased aggregation rate. At higher GuHCl concentrations, the electrostatic repulsion is completely screened, and the denaturing effect dominates. The results illustrate how the differential effects of denaturants on stability of monomer, oligomer and fibril translate to differential effects on microscopic steps, with the rate of nucleation being most strongly reduced.

Characterization of structural and functional properties of soybean 11S globulin during the renaturation after the guanidine hydrochloride denaturation

In this study, we explored how 11S globulin undergoes the renaturation with the decrease of guanidine hydrochloride (GuHCl) concentration. To analyze the changes in the structural and functional properties of 11S globulin, circular dichroism spectroscopy (CD), intrinsic fluorescence, particle size, foaming capacity (FC), foam stability (FS), emulsifying activity index (EAI) and emulsion stability index (ESI) at different GuHCl concentrations (8.0 mol/L, 4.0 mol/L, 2.0 mol/L, 0.8 mol/L, and 0.4 mol/L) were performed. 11S globulin was completely denatured after being treated with 8.0 mol/L of GuHCl. The CD spectra and particle size analyses showed that the α-helix and β-sheet contents and particle size of the renaturation process for 11S globulin were gradually close to those of native state protein and the protein structure became more orderly. The α-helix content increased from 0.2 ± 0.1% to 7.8 ± 0.3% and the hydrophobic groups inside the 11S globulin were exposed, which increased the surface hydrophobicity of the 11S globulin and changed its conformation. Regarding the functional properties, when the GuHCl concentration was diluted to 2.0 mol/L, some functional properties were improved compared with those of native state protein. The FC and FS of 11S globulin were increased by 5 and 2.2 times respectively and the EAI and ESI were increased by 1.25 times respectively. This study will provide guidance for the control of renaturation degree during 11S globulin production process to improve functional properties.

Optimization of GuHCl extraction protocol on collagen-based binders in murals by response surface methodology

In this paper, response surface method (RSM) was used to study the effect of extraction parameters on the extracting efficiency of guanidine hydrochloride (GuHCl) on residual protein from mural model samples. The central composite design (CCD) was applied to find the optimal combination of variables, including GuHCl concentration ( x : 1.5-2.5 M), extraction time ( y : 90-180 min), temperature ( z : 50-70 °C) and ultrasound power ( α : 120-280 W) during the extraction. Multiple regression analysis was employed to fit the experimental data into a second-order polynomial equation, and appropriate statistical methods were used for analysis. The optimal extraction conditions were determined as follows: GuHCl concentration of 1.89 M, extraction time of 132 min, temperature of 57 °C, and ultrasonic power of 210 W. The extraction efficiency was 16.685 % under the optimal conditions, which was in agreement with the theoretically predictive value of 17.707 %. The extraction process under optimized parameters had no significant impact on the collagenous structures, characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), ultraviolet (UV) absorption spectroscopy and circular dichroism (CD) spectroscopy.

An Evaluation of the Stability of Splicing Protein Dib1 Through Circular Dichroism Spectroscopy

Pre‐messenger RNA (pre‐mRNA) splicing is the process in which introns, noncoding regions of DNA, are removed and exons, coding regions of DNA, are ligated to produce a mature mRNA transcript. This process is mediated by the spliceosome, a dynamic macromolecular complex composed of five small nuclear ribonucleoproteins and other protein factors. At the catalytic center of the splicing machinery is the evolutionarily conserved yeast protein, Dib1, composed of 143 amino acids essential for splicing and cell viability. Dib1 and its human ortholog, hDim1, have demonstrated peptidase activity, specifically autocleavage of the last thirteen and fourteen amino acids, respectively, of their carboxy‐terminal tails. The mechanism and function of this autocleavage activity of the carboxy‐terminal tail is uncharacterized.Previous yeast growth assays comparing mutants to wildtype Dib1 have established that deletion of fifteen and sixteen amino acids off the carboxy‐terminal tail results in temperature‐sensitivity of yeast cell growth at 37ºC. Since Dib1 and splicing are essential for cell viability, the temperature‐sensitive mutants suggest that truncations to the carboxy‐terminal tail of Dib1 may result in defective splicing. Thus, the carboxy‐terminal tail may play a direct role in splicing or destabilize splicing complexes. To address these two possible hypotheses, we are investigating the effects of the carboxy‐terminal truncations on Dib1 stability. Protein purification of wild type and a series of truncated Dib1 mutants were performed. Circular dichroism spectroscopy employing increasing concentrations of guanidinium hydrochloride, a protein denaturant, was used to determine the stability of Dib1 and its truncated mutants. Overall, our findings show that Dib1 stability is directly linked to the length of the tail region and lead us to speculate that the tail is critical for stabilization of the spliceosomal complex.

Physicochemical and biological properties of experimental dental adhesives doped with a guanidine-based polymer: an in vitro study.

The objective of this study is to formulate experimental dental adhesives with different polyhexamethylene guanidine hydrochloride concentrations (PHMGH) and evaluate their physical, chemical, and biological properties. The experimental adhesives were formulated with 0 (control, GCTRL), 0.5 (G0.5%), 1 (G1%), or 2 (G2%) wt.% into the adhesive. The adhesives were analyzed for degree of conversion (DC%), softening in solvent (ΔKHN%), ultimate tensile strength (UTS), microtensile bond strength (μTBS) immediately and after 1 year of aging, antibacterial activity, and cytotoxicity. There were no differences among groups for DC%, ΔKHN%, and UTS (p > 0.05%). There were no differences between each PHMGH-doped adhesive compared to GCTRL in the immediate μ-TBS (p > 0.05). Adhesives with at least 1 wt.% of PHMGH presented better stability of μ-TBS. PHMGH-doped adhesives showed improved longitudinal μ-TBS compared to GCTRL (p < 0.05). Lower Streptococcus mutans biofilm formation was observed for PHMGH-doped adhesives (p < 0.05). There was lower viability of planktonic S. mutans in the media in contact with the samples when at least 1 wt.% of PHGMGH was incorporated (p < 0.05). The formulated adhesives showed no cytotoxicity against pulp cells (p > 0.05). The adhesive with 2 wt.% of PHMGH showed the highest antibacterial activity, without affecting the physicochemical properties and cytotoxicity, besides conferring stability for the dental adhesion. PHMGH, a positively charged polymer, conveyed antibacterial activity to dental adhesives. Furthermore, it did not negatively affect the essential physicochemical and biocompatibility properties of the adhesives. More importantly, the incorporation of PHMGH provided stability for the μ-TBS compared to the control group without this additive.

Disassembly of Single Virus Capsids Monitored in Real Time with Multicycle Resistive-Pulse Sensing.

Virus assembly and disassembly are critical steps in the virus lifecycle; however, virus disassembly is much less well understood than assembly. For hepatitis B virus (HBV) capsids, disassembly of the virus capsid in the presence of guanidine hydrochloride (GuHCl) exhibits strong hysteresis that requires additional chemical energy to initiate disassembly and disrupt the capsid structure. To study disassembly of HBV capsids, we mixed T = 4 HBV capsids with 1.0-3.0 M GuHCl, monitored the reaction over time by randomly selecting particles, and measured their size with resistive-pulse sensing. Particles were cycled forward and backward multiple times to increase the observation time and likelihood of observing a disassembly event. The four-pore device used for resistive-pulse sensing produces four current pulses for each particle during translocation that improves tracking and identification of single particles and increases the precision of particle-size measurements when pulses are averaged. We studied disassembly at GuHCl concentrations below and above denaturing conditions of the dimer, the fundamental unit of HBV capsid assembly. As expected, capsids showed little disassembly at low GuHCl concentrations (e.g., 1.0 M GuHCl), whereas at higher GuHCl concentrations (≥1.5 M), capsids exhibited disassembly, sometimes as a complex series of events. In all cases, disassembly was an accelerating process, where capsids catastrophically disassembled within a few 100 ms of reaching critical stability; disassembly rates reached tens of dimers per second just before capsids fell apart. Some disassembly events exhibited metastable intermediates that appeared to lose one or more trimers of dimers in a stepwise fashion.

Temperature and Guanidine Hydrochloride Effects on the Folding Thermodynamics of WW Domain and Variants.

We used simulations based on an all-atom Go model to calculate the folding temperatures (Tfs) and free energies (ΔGs) of two variants of the WW domain, which is a small all-β-sheet protein. The results, without adjusting any parameter, are in good agreement with experiments, thus validating the simulations. We then used the molecular transfer model to predict the changes in the ΔGs and Tfs as the guanidine hydrochloride concentration is varied. The predictions can be readily tested in experiments.

Pathologic tau conformer ensembles induce dynamic, liquid-liquid phase separation events at the nuclear envelope

BackgroundThe microtubule-associated protein tau forms aggregates in different neurodegenerative diseases called tauopathies. Prior work has shown that a single P301L mutation in tau gene, MAPT, can promote alternative tau folding pathways that correlate with divergent clinical diagnoses. Using progressive chemical denaturation, some tau preparations from the brain featured complex transitions starting at low concentrations of guanidine hydrochloride (GdnHCl) denaturant, indicating an ensemble of differently folded tau species called conformers. On the other hand, brain samples with abundant, tangle-like pathology had simple GdnHCl unfolding profile resembling the profile of fibrillized recombinant tau and suggesting a unitary conformer composition. In studies here we sought to understand tau conformer progression and potential relationships with condensed liquid states, as well as associated perturbations in cell biological processes.ResultsAs starting material, we used brain samples from P301L transgenic mice containing tau conformer ensembles that unfolded at low GdnHCl concentrations and with signatures resembling brain material from P301L subjects presenting with language or memory problems. We seeded reporter cells expressing a soluble form of 4 microtubule-binding repeat tau fused to GFP or YFP reporter moieties, resulting in redistribution of dispersed fluorescence signals into focal assemblies that could fuse together and move within processes between adjacent cells. Nuclear envelope fluorescent tau signals and small fluorescent inclusions behaved as a demixed liquid phase, indicative of liquid-liquid phase separation (LLPS); these droplets exhibited spherical morphology, fusion events and could recover from photobleaching. Moreover, juxtanuclear tau assemblies were associated with disrupted nuclear transport and reduced cell viability in a stable cell line. Staining for thioflavin S (ThS) became more prevalent as tau-derived inclusions attained cross-sectional area greater than 3 μm2, indicating (i) a bipartite composition, (ii) in vivo progression of tau conformers, and (iii) that a mass threshold applying to demixed condensates may drive liquid-solid transitions.ConclusionsTau conformer ensembles characterized by denaturation at low GdnHCl concentration templated the production of condensed droplets in living cells. These species exhibit dynamic changes and develop in vivo, and the larger ThS-positive assemblies may represent a waystation to arrive at intracellular fibrillar tau inclusions seen in end-stage genetic tauopathies.

Protein Stability Depends Critically on the Surface Hydrogen-Bonding Network: A Case Study of Bid Protein.

Understanding how proteins retain structural stability is not only of fundamental importance in biophysics but also critical to industrial production of antibodies and vaccines. Protein stability is known to depend mainly on two effects: internal hydrophobicity and H-bonding between the protein surface and solvent. A challenging task is to identify their individual contributions to a protein. Here, we investigate the structural stability of the apoptotic Bid protein in solutions containing various concentrations of guanidinium hydrochloride and urea using a combination of recently developed methods including the QTY (glutamine, threonine, and tyrosine) code and electron spin resonance-based peak-height analysis. We show that when the internal hydrophobicity of Bid is broken down using the QTY code, the surface H-bonding alone is sufficient to retain the structural stability intact. When the surface H-bonding is disrupted, Bid becomes sensitive to the temperature-dependent internal hydrophobicity such that it exhibits a reversible cold unfolding above water's freezing point. Using the combined approach, we show that the free-energy contributions of the two effects can be more reliably obtained. The surface H bonds are more important than the other effect in determining the structural stability of Bid protein.

Unfolding of Helical Poly(L-Glutamic Acid) in N,N-Dimethylformamide Probed by Pyrene Excimer Fluorescence (PEF).

The denaturation undergone by α–helical poly(L-glutamic acid) (PLGA) in N,N-dimethylformamide upon addition of guanidine hydrochloride (GdHCl) was characterized by comparing the fluorescence of a series of PLGA constructs randomly labeled with the dye pyrene (Py-PLGA) to that of a series of Py-PDLGA samples prepared from a racemic mixture of D,L-glutamic acid. The process of pyrene excimer formation (PEF) was taken advantage of to probe changes in the conformation of α–helical Py-PLGA. Fluorescence Blob Model (FBM) analysis of the fluorescence decays of the Py-PLGA and Py-PDLGA constructs yielded the average number (<Nblob>) of glutamic acids located inside a blob, which represented the volume probed by an excited pyrenyl label. <Nblob> remained constant for randomly coiled Py-PDLGA but decreased from ~20 to ~10 glutamic acids for the Py-PLGA samples as GdHCl was added to the solution. The decrease in <Nblob> reflected the decrease in the local density of PLGA as the α–helix unraveled in solution. The changes in <Nblob> with GdHCl concentration was used to determine the change in Gibbs energy required to denature the PLGA α–helix in DMF. The relationship between <Nblob> and the local density of macromolecules can now be applied to characterize the conformation of macromolecules in solution.

Unidirectional Water-Transport Antibacterial Trilayered Nanofiber-Based Wound Dressings Induced by Hydrophilic-Hydrophobic Gradient and Self-Pumping Effects

Excessive wound exudates often cause infection and hinder wound repair and regeneration. It is therefore urgently needed to develop an ideal wound dressing for pumping the excessive biofluids and inhibiting bacterial infection in the wound healing process. Herein, we demonstrate a facile and efficient strategy to fabricate the unidirectional water-transport antibacterial wound dressings by embedding macromolecular antimicrobials into a multilayer nanofibrous membrane featured with hydrophobic-to-hydrophilic gradient structure and self-pumping effect using an electrospinning technique. Thanks to these features, the obtained nanofibrous dressings achieved excellent unidirectional water-transport performance, which could drive wound exudates to flow spontaneously from inside to outside and prevent pumped biofluids rewetting the wounds. In addition, the designed trilayered dressing exhibited approximately 100% antibacterial ability against Staphylococcus aureus and Escherichia coli when the concentration of antibacterial agents polyhexamethylene guanidine hydrochloride (PHGC) was 0.06 wt%. Moreover, the trilayered dressings revealed excellent water absorption performance, air and moisture permeability, mechanical strength, biocompatibility, and low cell adhesion behavior, indicating the potential applications for the trilayered nanofibrous wound dressings in wound care.

Unidirectional water-transport antibacterial trilayered nanofiber-based wound dressings induced by hydrophilic-hydrophobic gradient and self-pumping effects

Excessive wound exudates often cause infection and hinder wound repair and regeneration. It is therefore urgently needed to develop an ideal wound dressing for pumping the excessive biofluids and inhibiting bacterial infection in the wound healing process. Herein, we demonstrate a facile and efficient strategy to fabricate the unidirectional water-transport antibacterial wound dressings by embedding macromolecular antimicrobials into a multilayer nanofibrous membrane featured with hydrophobic-to-hydrophilic gradient structure and self-pumping effect using an electrospinning technique. Thanks to these features, the obtained nanofibrous dressings achieved excellent unidirectional water-transport performance, which could drive wound exudates to flow spontaneously from inside to outside and prevent pumped biofluids rewetting the wounds. In addition, the designed trilayered dressing exhibited approximately 100% antibacterial ability against Staphylococcus aureus and Escherichia coli when the concentration of antibacterial agents polyhexamethylene guanidine hydrochloride (PHGC) was 0.06 wt%. Moreover, the trilayered dressings revealed excellent water absorption performance, air and moisture permeability, mechanical strength, biocompatibility, and low cell adhesion behavior, indicating the potential applications for the trilayered nanofibrous wound dressings in wound care.

Overexpression and refolding of human Cyclin D3. A reliable method or not?

Cyclin D3 is a regulatory protein associated in a variety of human tumors. Despite several studies involving Cyclin D1 and D2, there are few articles that investigate the role of Cyclin D3. Therefore, this study aimed to produce and characterize Cyclin D3 using the Escherichia coli expression system and a protein refolding protocol. The anionic detergent SDS was used to solubilize the protein, then the solution were kept at 4 °C to precipitate SDS. After removing the precipitate by centrifugation, the supernatant was applied to the Ni-NTA column to purify His- tagged Cyclin D3. The recombinant protein shown to be refolded in a denaturation study by fluorescence spectroscopy at increasing concentrations of guanidine hydrochloride. The protein secondary structure, evaluated by circular dichroism, is composed of 39 % α helix and 15 % β-strands. In addition, the study aimed to validate the refolding protocol used to obtain Cyclin D3 from E. coli inclusion bodies.

Elastic Properties of Guanidine Hydrochloride Solutions with Various Concentrations in the Gigahertz Frequency Range

The temperature dependences of the veloсity of hypersound (V(T)) in solutions with various concentrations of guanidine hydrochloride (GndHCl) are studied by Brillouin–Mandelstam light scattering spectroscopy in the temperature range from 263 to 353 K. It is shown that the temperature dependence of the sound velocity has a pronounced maximum at low concentrations of GndHCl. An increase in the concentration of GndHCl in a solution is accompanied by a shift of the maximum to the low-temperature region and an increase in the absolute values of the sound velocity, which means a decrease in the adiabatic compressibility over the entire range of studied temperatures. The temperature dependence of the adiabatic compressibility is constructed at low concentrations of GndHCl. The contribution of relaxation processes to the temperature behavior of the attenuation of hypersound in solutions of GndHCl is determined. It is shown that their behavior is activated thermally and described by the Arrhenius law. The values of the activation energy of relaxation processes are calculated. Possible mechanisms that underlie the observed phenomena are discussed.

Probing the Stepwise Unfolding of Bovine Serum Albumin Using 2D Correlation Raman Spectroscopic Analysis.

Protein denaturation involves a change in the protein structure with the loss of activity, which proceeds via various intermediates. The possible intermediate structures account largely for understanding the process of unfolding. Hence, considerable attention is required to characterize partially unfolded protein states and to gain more insight into the information about the sequence and steps involved in protein folding mechanisms. In this report, a stepwise unfolding of bovine serum albumin (BSA) with guanidine hydrochloride (GuHCl) has been investigated using Raman spectroscopy in the amide I and III regions. Two-dimensional (2D) correlation analysis has been applied to reveal information on the sequential order and the dynamic properties of interaction during the unfolding process. Raman spectral signatures in the amide I region revealed that there is no significant change in secondary structures up to 2 M concentration of GuHCl. However, 2D correlation analysis further supports the observation by inferring the strengthening of secondary structure at the expense of tertiary structure. At a higher concentration of GuHCl (2-4 M), there is an accumulation of random and β-sheet structures that is mediated by small connecting segments of helices. It further accelerates the unfolding of helices and a complete collapse of structure. These analyses establish the ability of Raman spectroscopy to estimate the ensemble of secondary structures present in proteins. The results reveal the mechanistic details of unfolding, characterizing structure of intermediates even at high concentrations, and understanding the evolution of various secondary structures with respect to each other during unfolding. Such observations can be helpful in understanding the factors affecting the shape and size of proteins during folding/unfolding.