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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 -
Protein Science : a Publication of the... Dec 2015We present the software CDpal that is used to analyze thermal and chemical denaturation data to obtain information on protein stability. The software uses standard...
We present the software CDpal that is used to analyze thermal and chemical denaturation data to obtain information on protein stability. The software uses standard assumptions and equations applied to two-state and various types of three-state denaturation models in order to determine thermodynamic parameters. It can analyze denaturation monitored by both circular dichroism and fluorescence spectroscopy and is extremely flexible in terms of input format. Furthermore, it is intuitive and easy to use because of the graphical user interface and extensive documentation. As illustrated by the examples herein, CDpal should be a valuable tool for analysis of protein stability.
Topics: Circular Dichroism; Computational Biology; Models, Molecular; Protein Denaturation; Protein Stability; Proteins; Software; Spectrometry, Fluorescence; Temperature
PubMed: 26402034
DOI: 10.1002/pro.2809 -
Protein Science : a Publication of the... Jan 2009Trehalose is a ubiquitous molecule that occurs in lower and higher life forms but not in mammals. Till about 40 years ago, trehalose was visualized as a storage... (Review)
Review
Trehalose is a ubiquitous molecule that occurs in lower and higher life forms but not in mammals. Till about 40 years ago, trehalose was visualized as a storage molecule, aiding the release of glucose for carrying out cellular functions. This perception has now changed dramatically. The role of trehalose has expanded, and this molecule has now been implicated in a variety of situations. Trehalose is synthesized as a stress-responsive factor when cells are exposed to environmental stresses like heat, cold, oxidation, desiccation, and so forth. When unicellular organisms are exposed to stress, they adapt by synthesizing huge amounts of trehalose, which helps them in retaining cellular integrity. This is thought to occur by prevention of denaturation of proteins by trehalose, which would otherwise degrade under stress. This explanation may be rational, since recently, trehalose has been shown to slow down the rate of polyglutamine-mediated protein aggregation and the resultant pathogenesis by stabilizing an aggregation-prone model protein. In recent years, trehalose has also proved useful in the cryopreservation of sperm and stem cells and in the development of a highly reliable organ preservation solution. This review aims to highlight the changing perception of the role of trehalose over the last 10 years and to propose common mechanisms that may be involved in all the myriad ways in which trehalose stabilizes protein structures. These will take into account the structure of trehalose molecule and its interactions with its environment, and the explanations will focus on the role of trehalose in preventing protein denaturation.
Topics: Carbohydrates; Protein Conformation; Protein Denaturation; Proteins; Solutions; Stress, Physiological; Trehalose; Water
PubMed: 19177348
DOI: 10.1002/pro.3 -
Biophysical Journal Mar 2011Urea is a commonly used protein denaturant, and it is of great interest to determine its interaction with various protein groups to elucidate the molecular basis of its...
Urea is a commonly used protein denaturant, and it is of great interest to determine its interaction with various protein groups to elucidate the molecular basis of its effect on protein stability. Using the Trp-cage miniprotein as a model system, we report what we believe to be the first computation of changes in the preferential interaction coefficient of the protein upon urea denaturation from molecular-dynamics simulations and examine the contributions from the backbone and the side-chain groups. The preferential interaction is obtained from reversible folding/unfolding replica exchange molecular-dynamics simulations of Trp-cage in presence of urea, over a wide range of urea concentration. The increase in preferential interaction upon unfolding is dominated by the side-chain contribution, rather than the backbone. Similar trends are observed in simulations using two different force fields, Amber94 and Amber99sb, for the protein. The magnitudes of the side-chain and backbone contributions differ in the two force fields, despite containing identical protein-solvent interaction terms. The differences arise from the unfolded ensembles sampled, with Amber99sb favoring conformations with larger surface area and lower helical content. These results emphasize the importance of the side-chain interactions with urea in protein denaturation, and highlight the dependence of the computed driving forces on the unfolded ensemble sampled.
Topics: Dose-Response Relationship, Drug; Hydrogen Bonding; Molecular Dynamics Simulation; Peptides; Protein Denaturation; Protein Folding; Temperature; Urea
PubMed: 21402035
DOI: 10.1016/j.bpj.2011.01.028 -
Journal of Synchrotron Radiation Mar 2009Many advances in the understanding of radiation damage to protein crystals, particularly at cryogenic temperatures, have been made in recent years, but with this comes... (Review)
Review
Many advances in the understanding of radiation damage to protein crystals, particularly at cryogenic temperatures, have been made in recent years, but with this comes an expanding literature, and, to the new breed of protein crystallographer who is not really interested in X-ray physics or radiation chemistry but just wants to solve a biologically relevant structure, the technical nature and breadth of this literature can be daunting. The purpose of this paper is to serve as a rough guide to radiation damage issues, and to provide references to the more exacting and detailed work. No attempt has been made to report precise numbers (a factor of two is considered satisfactory), and, since there are aspects of radiation damage that are demonstrably unpredictable, the 'worst case scenario' as well as the 'average crystal' are discussed in terms of the practicalities of data collection.
Topics: Crystallization; Crystallography, X-Ray; Protein Conformation; Protein Denaturation; Proteins; Radiation Dosage; Specimen Handling; X-Rays
PubMed: 19240325
DOI: 10.1107/S0909049509004361 -
Chemistry and Physics of Lipids Apr 2013Because detergents are commonly used to solvate membrane proteins for structural evaluation, much attention has been devoted to assessing the conformational bias... (Review)
Review
Because detergents are commonly used to solvate membrane proteins for structural evaluation, much attention has been devoted to assessing the conformational bias imparted by detergent micelles in comparison to the native environment of the lipid bilayer. Here, we conduct six 500-ns simulations of a system with >600,000 atoms to investigate the spontaneous self assembly of dodecylphosphocholine detergent around multiple molecules of the integral membrane protein PagP. This detergent formed equatorial micelles in which acyl chains surround the protein's hydrophobic belt, confirming existing models of the detergent solvation of membrane proteins. In addition, unexpectedly, the extracellular and periplasmic apical surfaces of PagP interacted with the headgroups of detergents in other micelles 85 and 60% of the time, respectively, forming complexes that were stable for hundreds of nanoseconds. In some cases, an apical surface of one molecule of PagP interacted with an equatorial micelle surrounding another molecule of PagP. In other cases, the apical surfaces of two molecules of PagP simultaneously bound a neat detergent micelle. In these ways, detergents mediated the non-specific aggregation of folded PagP. These simulation results are consistent with dynamic light scattering experiments, which show that, at detergent concentrations ≥600 mM, PagP induces the formation of large scattering species that are likely to contain many copies of the PagP protein. Together, these simulation and experimental results point to a potentially generic mechanism of detergent-mediated protein aggregation.
Topics: Detergents; Hydrophobic and Hydrophilic Interactions; Membrane Proteins; Molecular Dynamics Simulation; Protein Denaturation; Protein Multimerization; Protein Stability; Protein Structure, Quaternary; Protein Structure, Secondary; Water
PubMed: 23466535
DOI: 10.1016/j.chemphyslip.2013.02.005 -
The Journal of Physical Chemistry... Apr 2023Protein denaturation is a ubiquitous process that occurs both and . While our molecular understanding of the denatured structures of proteins is limited, it is commonly...
Protein denaturation is a ubiquitous process that occurs both and . While our molecular understanding of the denatured structures of proteins is limited, it is commonly accepted that the loss of unique intramolecular contacts makes proteins larger. Herein, we report compaction of the immunoglobulin G1 (IgG1) protein upon acid denaturation. Small-angle X-ray scattering coupled with size exclusion chromatography revealed that IgG1 radii of gyration at pH 2 were ∼75% of those at a neutral pH. Scattering profiles showed a compact globular shape, supported by analytical ultracentrifugation. The acid denaturation of proteins with a decrease in size is energetically costly, and acid-induced compaction requires an attractive force for domain reorientation. Such intramolecular aggregation may be widespread in immunoglobulin proteins as noncanonical structures. Herein, we discuss the potential biological significance of these noncanonical structures of antibodies.
Topics: Protein Conformation; Protein Denaturation; Hydrogen-Ion Concentration; Immunoglobulin G
PubMed: 37093025
DOI: 10.1021/acs.jpclett.3c00258 -
Journal of Biomedical Optics Dec 2016We performed the feasibility study using speckle variance optical coherence tomography (SvOCT) to monitor the thermally induced protein denaturation and coagulation...
We performed the feasibility study using speckle variance optical coherence tomography (SvOCT) to monitor the thermally induced protein denaturation and coagulation process as a function of temperature and depth. SvOCT provided the depth-resolved image of protein denaturation and coagulation with microscale resolution. This study was conducted using egg white. During the heating process, as the temperature increased, increases in the speckle variance signal was observed as the egg white proteins coagulated. Additionally, by calculating the cross-correlation coefficient in specific areas, denaturized egg white conditions were successfully estimated. These results indicate that SvOCT could be used to monitor the denaturation process of various proteins.
Topics: Animals; Egg White; Feasibility Studies; Monitoring, Physiologic; Protein Aggregates; Protein Denaturation; Tomography, Optical Coherence
PubMed: 27942719
DOI: 10.1117/1.JBO.21.12.125004 -
Biophysical Journal Apr 2022Yfh1 is a yeast protein with the peculiar characteristic to undergo, in the absence of salt, cold denaturation at temperatures above the water freezing point. This...
Yfh1 is a yeast protein with the peculiar characteristic to undergo, in the absence of salt, cold denaturation at temperatures above the water freezing point. This feature makes the protein particularly interesting for studies aiming at understanding the rules that determine protein fold stability. Here, we present the phase diagram of Yfh1 unfolding as a function of pressure (0.1-500 MPa) and temperature 278-313 K (5-40°C) both in the absence and in the presence of stabilizers using Trp fluorescence as a monitor. The protein showed a remarkable sensitivity to pressure: at 293 K, pressures around 10 MPa are sufficient to cause 50% of unfolding. Higher pressures were required for the unfolding of the protein in the presence of stabilizers. The phase diagram on the pressure-temperature plane together with a critical comparison between our results and those found in the literature allowed us to draw conclusions on the mechanism of the unfolding process under different environmental conditions.
Topics: Cold Temperature; Hot Temperature; Iron-Binding Proteins; Protein Denaturation; Protein Folding; Saccharomyces cerevisiae; Thermodynamics; Frataxin
PubMed: 35278425
DOI: 10.1016/j.bpj.2022.03.010