-
Protein Science : a Publication of the... Dec 2022Protein folding and unfolding is a complex process, underscored by the many proteotoxic diseases associated with misfolded proteins. Mapping pathways from a native...
Protein folding and unfolding is a complex process, underscored by the many proteotoxic diseases associated with misfolded proteins. Mapping pathways from a native structure to an unfolded protein or vice versa, identifying the intermediates, and defining the role of sequence and structure en route remain outstanding problems in the field. It is even more challenging to capture the events at atomistic resolution. X-ray diffraction has so far been used to understand how urea interacts with and unfolds two stable globular proteins. Here, we present the case study on PSMD10 , a prototype for a moderately stable, non-globular repeat protein, long and rigid, with its termini located at either end. We define structural changes in the time dimension using low urea concentrations to arrive at the following conclusions. (a) Unfolding is rapidly initiated at the C-terminus, slowly at the N-terminus, and proceeds inwards from both ends. (b) C-terminus undergoes an α to 3 helix transition, representing the structure of a potential early unfolding intermediate before disorder sets in. (c) Distinct and progressive changes in the electrostatic landscape of PSMD10 were observed, indicative of conformational changes in the seemingly inflexible motif involved in protein-protein interaction. We believe this unique study will open up the field for better and bolder queries and increase the choice of model proteins for a better understanding of the challenging problems of protein folding, protein interactions, protein degradation, and diseases associated with misfolding.
Topics: Protein Denaturation; Urea; Ankyrin Repeat; Protein Folding; X-Ray Diffraction; Protein Conformation; Protein Unfolding
PubMed: 36382986
DOI: 10.1002/pro.4515 -
Biochemistry Jun 2023Caspases are evolutionarily conserved cysteinyl proteases that are integral in cell development and apoptosis. All apoptotic caspases evolved from a common ancestor into...
Caspases are evolutionarily conserved cysteinyl proteases that are integral in cell development and apoptosis. All apoptotic caspases evolved from a common ancestor into two distinct subfamilies with either monomeric (initiators) or dimeric (effectors) oligomeric states. The regulation of apoptosis is influenced by the activation mechanism of the two subfamilies, but the evolution of the well-conserved caspase-hemoglobinase fold into the two subfamilies is not well understood. We examined the folding landscape of monomeric caspases from two coral species over a broad pH range of 3-10.5. On an evolutionary timescale, the two coral caspases diverged from each other approximately 300 million years ago, and they diverged from human caspases about 600 million years ago. Our results indicate that both proteins have overall high stability, ∼15 kcal mol, near the physiological pH range (pH 6-8) and unfold via two partially folded intermediates, I and I, that are in equilibrium with the native and the unfolded state. Like the dimeric caspases, the monomeric coral caspases undergo a pH-dependent conformational change resulting from the titration of an evolutionarily conserved site. Data from molecular dynamics simulations paired with limited proteolysis and MALDI-TOF mass spectrometry show that the small subunit of the monomeric caspases is unstable and unfolds prior to the large subunit. Overall, the data suggest that all caspases share a conserved folding landscape, that a conserved allosteric site can be fine-tuned for species-specific regulation, and that the subfamily of stable dimers may have evolved to stabilize the small subunit.
Topics: Humans; Protein Folding; Caspases; Protein Denaturation
PubMed: 37337671
DOI: 10.1021/acs.biochem.3c00004 -
Biochemical and Biophysical Research... Oct 2021The pathogenesis of heart failure with preserved ejection fraction (HFpEF) in obese diabetic patients has been implicated in metainflammation. Increased expression of...
The pathogenesis of heart failure with preserved ejection fraction (HFpEF) in obese diabetic patients has been implicated in metainflammation. Increased expression of inducible nitric oxide synthase (iNOS) and dysfunction of the unfolded protein response (UPR), especially inositol-requiring enzyme 1α-X-box binding protein 1 (IRE1α-Xbp1s) signaling in the heart, have been associated with HFpEF. We investigated the effect of imeglimin, a potential new treatment for type 2 diabetes, on the pathogenesis of HFpEF. We induced obesity, impaired glucose tolerance, and cardiac hypertrophy with fibrosis, fat accumulation, and diastolic dysfunction in wild-type mice with a high-fat diet (HFD) and the nitric oxide synthase (NOS) inhibitor l-NAME for 16 weeks. Treatment with imeglimin starting at 10 weeks not only improved their abnormal systemic glucose metabolism and visceral obesity but also their cardiac abnormalities. We found that imeglimin suppressed the upregulation of iNOS, and restored the expression of Xbp1s and the expression of the E3 ubiquitin ligase STIP1 homology and U-box-containing protein 1 (STUB1), which is responsible for the degradation of Forkhead box protein O1 (FoxO1), a direct transcriptional target of Xbp1s. It also suppressed the excessive transcriptional activity of FoxO1, which is located downstream of Xbp1s and is involved in the form development of HFpEF and cardiac adipogenesis. Imeglimin also restored the expression of Glutathione peroxidase 4 (GPX4), which protects cells against excess lipid peroxidation and governs a novel form of programmed cell death, called ferroptosis.
Topics: Animals; Heart Failure; Mice; Oxidative Stress; Protein Unfolding; Stroke Volume; Triazines
PubMed: 34375928
DOI: 10.1016/j.bbrc.2021.07.090 -
Inorganic Chemistry Jan 2022Human carbonic anhydrase II (HCA) is a robust metalloprotein and an excellent biological model system to study the thermodynamics of metal ion coordination. Apo-HCA...
Human carbonic anhydrase II (HCA) is a robust metalloprotein and an excellent biological model system to study the thermodynamics of metal ion coordination. Apo-HCA binds one zinc ion or two copper ions. We studied these binding processes at five temperatures (15-35 °C) using isothermal titration calorimetry, yielding thermodynamic parameters corrected for pH and buffer effects. We then sought to identify binding-induced structural changes. Our data suggest that binding at the active site organizes 6-8 residues; however, copper binding near the N-terminus results in a net of 6-7 residues. This surprising destabilization was confirmed using circular dichroism and protein stability measurements. Metal binding induced unfolding may represent an important regulatory mechanism, but it may be easily missed by NMR and X-ray crystallography. Thus, in addition to highlighting a highly novel binding-induced unfolding event, we demonstrate the value of calorimetry for studying the structural implications of metal binding.
Topics: Binding Sites; Calorimetry; Carbonic Anhydrase II; Carbonic Anhydrase Inhibitors; Coordination Complexes; Copper; Humans; Ions; Protein Unfolding; Zinc
PubMed: 34989562
DOI: 10.1021/acs.inorgchem.1c03271 -
Protein Science : a Publication of the... Dec 2020Protein aggregation is one of the most critical processes affecting protein solubility in various contexts-from protein therapeutics formulation to protein diseases. In...
Protein aggregation is one of the most critical processes affecting protein solubility in various contexts-from protein therapeutics formulation to protein diseases. In general, time-dependent changes in protein solubility are complex kinetically driven processes that often involve a triggering event that consists of a protein unfolding/misfolding followed by the assembling of aggregation-competent protein species. In this study, we have examined the relation between stability and time-dependent solubility of the recombinant human antibody light chain, hLC, which was found to form renal tubular casts in the multiple myeloma patient. To analyze the aggregation quantitatively, the hLC stability and protein solubility assays were performed in vitro at elevated temperatures. A differential acceleration of the processes at high temperatures enabled us to dissect observed kinetics of irreversible hLC unfolding and aggregation. We find that for hLC these processes have different molecularity and activation energy barriers. While the irreversible unfolding of hLC is a unimolecular step with a substantial activation energy barrier of 260 kJ/mol, the aggregation is rate-limited by the bimolecular reaction, which is characterized by a lower activation energy barrier of 40 kJ/mol. By the combination of experimental assays at different temperatures, different protein concentrations and kinetic modeling using ordinary differential equations, we were able to extrapolate time-dependent protein solubility to temperatures where both unfolding and aggregation processes are strongly kinetically coupled. Our study enables mechanism-based evaluation and interpretation of different physico-chemical factors contributing to the hLC unfolding and aggregation and their effect on the formation of extracellular protein deposits.
Topics: Humans; Immunoglobulin Light Chains; Kinetics; Multiple Myeloma; Neoplasm Proteins; Protein Aggregates; Protein Unfolding
PubMed: 33030218
DOI: 10.1002/pro.3968 -
Journal of the American Society For... Jun 2022Structural analysis by native ion mobility-mass spectrometry provides a direct means to characterize protein interactions, stability, and other biophysical properties of... (Comparative Study)
Comparative Study
Structural analysis by native ion mobility-mass spectrometry provides a direct means to characterize protein interactions, stability, and other biophysical properties of disease-associated biomolecules. Such information is often extracted from collision-induced unfolding (CIU) experiments, performed by ramping a voltage used to accelerate ions entering a trap cell prior to an ion mobility separator. Traditionally, to simplify data analysis and achieve confident ion identification, precursor ion selection with a quadrupole is performed prior to collisional activation. Only one charge state can be selected at one time, leading to an imbalance between the total time required to survey CIU data across all protein charge states and the resulting structural analysis efficiency. Furthermore, the arbitrary selection of a single charge state can inherently bias CIU analyses. We herein aim to compare two conformation sampling methods for protein gas-phase unfolding: (1) traditional quadrupole selection-based CIU and (2) nontargeted, charge selection-free and shotgun workflow, all ion unfolding (AIU). Additionally, we provide a new data interpretation method that integrates across all charge states to project collisional cross section (CCS) data acquired over a range of activation voltages to produce a single unfolding fingerprint, regardless of charge state distributions. We find that AIU in combination with CCS accumulation across all charges offers an opportunity to maximize protein conformational information with minimal time cost, where additional benefits include (1) an improved signal-to-noise ratios for unfolding fingerprints and (2) a higher tolerance to charge state shifts induced by either operating parameters or other factors that affect protein ionization efficiency.
Topics: Ion Mobility Spectrometry; Ions; Mass Spectrometry; Protein Conformation; Protein Unfolding; Proteins
PubMed: 35508074
DOI: 10.1021/jasms.2c00004 -
Chemical Science Mar 2023Confinement of molecules in a synthetic host can physically isolate even their unstable temporary structures, which has potential for application to protein transient...
Confinement of molecules in a synthetic host can physically isolate even their unstable temporary structures, which has potential for application to protein transient structure analysis. Here we report the NMR snapshot observation of protein unfolding and refolding processes by confining a target protein in a self-assembled coordination cage. With increasing acetonitrile content in CDCN/HO media (50 to 90 vol%), the folding structure of a protein sharply denatured at 83 vol%, clearly revealing the regions of initial unfolding. Unfavorable aggregation of the protein leading to irreversible precipitation is completely prevented because of the spatial isolation of the single protein molecule in the cage. When the acetonitrile content reversed (84 to 70 vol%), the once-denatured protein started to regain its original folded structure at 80 vol%, showing that the protein folding/unfolding process can be referred to as a phase transition with hysteresis behavior.
PubMed: 36937586
DOI: 10.1039/d2sc05879k -
The FEBS Journal Mar 2022Folding stability is a crucial feature of protein evolution and is essential for protein functions. Thus, the comprehension of protein folding mechanisms represents an...
Folding stability is a crucial feature of protein evolution and is essential for protein functions. Thus, the comprehension of protein folding mechanisms represents an important complement to protein structure and function, crucial to determine the structural basis of protein misfolding. In this context, thermal unfolding studies represent a useful tool to get a molecular description of the conformational transitions governing the folding/unfolding equilibrium of a given protein. Here, we report the thermal folding/unfolding pathway of VEGFR1D2, a member of the immunoglobulin superfamily by means of a high-resolution thermodynamic approach that combines differential scanning calorimetry with atomic-level unfolding monitored by NMR. We show how VEGFR1D2 folding is driven by an oxidatively induced disulfide pairing: the key event in the achievement of its functional structure is the formation of a small hydrophobic core that surrounds a disulfide bridge. Such a 'folding nucleus' induces the cooperative transition to the properly folded conformation supporting the hypothesis that a disulfide bond can act as a folding nucleus that eases the folding process.
Topics: Calorimetry, Differential Scanning; Circular Dichroism; Disulfides; Humans; Protein Denaturation; Protein Folding; Proteins; Thermodynamics
PubMed: 34689403
DOI: 10.1111/febs.16246 -
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 -
Molecules (Basel, Switzerland) Apr 2022In the development of therapeutic proteins, analytical assessment of structural stability and integrity constitutes an important activity, as protein stability and...
In the development of therapeutic proteins, analytical assessment of structural stability and integrity constitutes an important activity, as protein stability and integrity influence drug efficacy, and ultimately patient safety. Existing analytical methodologies solely rely on relative changes in optical properties such as fluorescence or scattering upon thermal or chemical perturbation. Here, we present an absolute analytical method for assessing protein stability, structure, and unfolding utilizing Taylor dispersion analysis (TDA) and LED-UV fluorescence detection. The developed TDA method measures the change in size (hydrodynamic radius) and intrinsic fluorescence of a protein during in-line denaturation with guanidinium hydrochloride (GuHCl). The conformational stability of the therapeutic antibody adalimumab and human serum albumin were characterized as a function of pH. The simple workflow and low sample consumption (40 ng protein per data point) of the methodology make it ideal for assessing protein characteristics related to stability in early drug development or when having a scarce amount of sample available.
Topics: Guanidine; Humans; Hydrodynamics; Protein Denaturation; Protein Folding; Protein Stability; Proteins; Serum Albumin, Human
PubMed: 35458703
DOI: 10.3390/molecules27082506