Time-dependent Conformational Changes Research Articles

Can Duvelisib and Eganelisib work for both cancer and COVID-19? Molecular-level insights from MD simulations and enhanced samplings.

SARS-CoV-2 has caused severe illness and anxiety worldwide, evolving into more dreadful variants capable of evading the host's immunity. Cytokine storms, led by PI3Kγ, are common in cancer and SARS-CoV-2. Naturally, there is a yearning to see whether any drugs could alleviate cytokine storms for both. Upon investigation, we identified two anticancer drugs, Duvelisib and Eganelisib, that could also work against SARS-CoV-2. This report is the first to decipher their synergic therapeutic effectiveness against COVID-19 and cancer with molecular insights from atomistic simulations. In addition to PI3Kγ, these drugs exhibit specificity for the main protease among all SARS-CoV-2 targets, with significant negative binding free energies and small time-dependent conformational changes of the complexes. Complexation makes active sites and secondary structures highly mechanically stiff, with barely any deformation. Replica simulations estimated large pulling forces in enhanced sampling to dissociate the drugs from Mpro's active site. Furthermore, the radial distribution function (RDF) demonstrated that the therapeutic molecules were closest to the His41 and Cys145 catalytic dyad residues. Finally, analyses implied Duvelisib and Eganelisib as promising dual-purposed anti-COVID and anticancer drugs, potentially targeting Mpro and PI3Kγ to stop virus replication and cytokine storms concomitantly. We also distinguished hotspot residues imparting significant interactions.

Phase Separation and Aggregation of a Globular Folded Protein Small Ubiquitin-like Modifier 1 (SUMO1).

Liquid-liquid phase separation (LLPS) plays a crucial role in cellular organization, primarily driven by intrinsically disordered proteins (IDPs) leading to the formation of biomolecular condensates. A folded protein SUMO that post-translationally modifies cellular proteins has recently emerged as a regulator of LLPS. Given its compact structure and limited flexibility, the precise role of SUMO in condensate formation remains to be investigated. Here, we show the rapid phase separation of SUMO1 into micrometer-sized liquid-like condensates in inert crowders under physiological conditions. Subsequent time-dependent conformational changes and aggregation are probed by label-free methods (tryptophan fluorescence and Raman spectroscopy). Remarkably, experiments on a SUMO1 variant lacking the N-terminal disordered region further corroborate the role of its structured part in phase transitions. Our findings highlight the potential of folded proteins to engage in LLPS and emphasize further investigation into the influence of the SUMO tag on IDPs associated with membrane-less assemblies in cells.

Probing concentration and time dependent conformational changes in poly acrylic acid stabilized magnetic nanoemulsion using magnetic chaining-based inter-droplet force measurement

Emulsions are stabilized with surfactants, polymers, or polyelectrolytes to extend their shelf-life and colloidal stability. Electro-steric interaction in polyelectrolyte stabilized emulsions makes them ideal systems for probing the conformational dynamics. Here, magnetic chaining technique is exploited for inter-droplet force measurement in poly acrylic acid (PAA) stabilized magnetic nanoemulsions, and to probe the conformational changes of the stabilizing moieties as a function of time and PAA concentration. Force measurement studies indicate an increased inter-droplet spacing for higher concentration of PAA due to an increase in osmotic pressure with concentration. Further, for a fixed concentration, the inter-droplet spacing decreases with time due to the slow diffusion of the polymer chains resulting in conformational changes. This is the first experimental report on conformational dynamics of PAA stabilized oil-in-water magnetic nanoemulsions. The obtained results are beneficial for understanding the nature of colloidal interaction forces in polyelectrolyte stabilized nanoemulsions.

Characterizing aggregate growth and morphology of alanine-rich polypeptides as a function of sequence chemistry and solution temperature from scattering, spectroscopy, and microscopy

The aggregation behavior and stability of a series of alanine-rich peptides, which are included as components of peptide-polymer conjugates, were characterized using a combination of biophysical techniques. Light scattering techniques were used to monitor changes in peptide morphology and size distributions as a function of time and temperature. The results show large particles immediately upon dissolution in buffer. At room temperature, these particles relaxed to reach a mostly monomeric peptide state, while at higher temperatures, they grew to form aggregates. Circular dichroism spectroscopy (CD) was used to monitor temperature- and time-dependent conformational changes as a function of peptide sequence and incubation time. CD measurements reveal that all of the sequences are helical at low temperatures with transitions to non-helical conformation with increased temperature. Samples incubated at room temperature were able to recover their original helicity. At increased temperature, the shorter and longer peptide sequences showed notable changes in conformation, and were not able to recover their original helicity after 72 h. After incubation for up to one week, β-sheet conformations were observed in these two cases, while only α-helical conformation loss was observed for the peptide of intermediate molecular weight. Transmission electron microscopy measurements reveal the formation of fibrils after 72 h of incubation at 60 °C for all samples, in agreement with the scattering measurements. Additional quenching experiments show that peptide aggregation can be stalled when solutions are cooled to room temperature.

Multiplexed Exchange-PAINT imaging reveals ligand-dependent EGFR and Met interactions in the plasma membrane

Signal transduction by receptor tyrosine kinases (RTKs) involves complex ligand- and time-dependent changes in conformation and modification state. High resolution structures are available for individual receptors dimers, but less is known about receptor clusters that form in plasma membranes composed of many different RTKs with the potential to interact. We report the use of multiplexed super-resolution imaging (Exchange-PAINT) followed by mean-shift clustering and random forest analysis to measure the precise distributions of five receptor tyrosine kinases (RTKs) from the ErbB, IGF-1R and Met families in breast cancer cells. We find that these receptors are intermixed nonhomogenously on the plasma membrane. Stimulation by EGF does not appear to induce a change in the density of EGFR in local clusters but instead results in formation of EGFR-Met and EGFR-ErbB3 associations; non-canonical EGFR-Met interactions are implicated in resistance to anti-cancer drugs but have not been previously detected in drug-naïve cells.

Thermodynamic Analysis of the Geldanamycin-Hsp90 Interaction in a Whole Cell Lysate Using a Mass Spectrometry-Based Proteomics Approach.

Geldanamycin is a natural product with well-established and potent anti-cancer activities. Heat shock protein 90 (Hsp90) is the known target of geldanamycin, which directly binds to Hsp90's N-terminal ATP binding domain and inhibits Hsp90's ATPase activity. The affinity of geldanamycin for Hsp90 has been measured in multiple studies. However, there have been large discrepancies between the reported dissociation constants (i.e., Kd values), which have ranged from low nanomolar to micromolar. Here the stability of proteins from rates of oxidation (SPROX) technique was used in combination with an isobaric mass tagging strategy to measure the binding affinity of geldanamycin to unpurified Hsp90 in an MCF-7 cell lysate. The Kd values determined here were dependent on how long geldanamycin was equilibrated with the lysate prior to SPROX analysis. The Kd values determined using equilibration times of 0.5 and 24 h were 1 and 0.03 μM, respectively. These Kd values, which are similar to those previously reported in a geldanamycin-Hsp90 binding study that involved the use of a fluorescently labeled geldanamycin analogue, establish that the slow-tight binding behavior previously observed for the fluorescently labeled geldanamycin analogue is not an artifact of the fluorescent label, but rather an inherent property of the geldanamycin-Hsp90 binding interaction. The slow-tight binding property of this complex may be related to time-dependent conformational changes in Hsp90 and/or to time-dependent chemical changes in geldanamycin, both of which have been previously proposed to explain the slow-tight binding behavior of the geldanamycin-Hsp90 complex. Graphical Abstract ᅟ.

In vivo regulation of human CrkII by cyclophilin A and FK506-binding protein

Members of the Crk family of adaptor proteins are key players in signal transduction from a variety of cell surface receptors. CrkI and CrkII are two alternative-spliced forms of a single gene which possess an N-terminal SH2 domain and an SH3 domain that mediate interaction with other proteins. CrkII possesses an additional C-terminal linker region plus an extra SH3 domain, which does not interact with other proteins, but operates as regulatory moiety. Utilizing human Jurkat T cells, we demonstrate that CrkII-SH3N binding of C3G is inhibited by cyclosporin A (CsA) plus FK506 that inhibit the cyclophilin A (CypA) and FK506 binding protein (FKBP) peptidyl-prolyl cis-trans isomerases (PPIases; also termed immunophilins), respectively. Jurkat T cells were found to express ∼5-fold lower levels of CrkI protein compared to CrkII, but the efficiency of C3G binding by CrkI was ∼5-fold higher than that of CrkII, suggesting that the majority of cellular CrkII proteins adopt a conformation that is inaccessible for C3G. Treatment of Jurkat T cells with CsA plus FK506 led to a time-dependent conformational change in overexpressed human CrkII1-236 protein-containing FRET-based biosensor, supporting the accumulation of cis conformers of human CrkII1-236 in the presence of PPIase inhibitors. Our data suggest that the Gly219-Pro-Tyr motif in the human CrkII linker region serves as the recognition and isomerization site of PPIases, and raise the possibility that CsA and FK506 might interfere with selected effector T cell functions via a CrkII-, but not CrkI-dependent mechanisms.

Probing Conformational Change of Bovine Serum Albumin-Dextran Conjugates under Controlled Dry Heating.

The time-dependent conformational change of bovine serum album (BSA) during Maillard reaction with dextran under controlled dry heating has been studied by small-angle X-ray scattering, fluorescence spectroscopy, dynamic light scattering, and circular dichroism analysis. Through the research on the radii of gyration (Rg), intrinsic fluorescence, and secondary structure, conjugates with dextran coating were found to inhibit BSA aggregation and preserve the secondary structure of native BSA against long-time heat treatment during Maillard reaction. The results suggested that the hydrophilic dextran was conjugated to the compact protein surface and enclosed it and more dextran chains were attached to BSA with the increase of the heating time. The study presented here will be beneficial to the understanding of the conformational evolution of BSA molecules during the dry-heating Maillard reaction and to the control of the protein-polysaccharide conjugate structure.

Evidence of conformational changes in oil molecules with protein aggregation and conformational changes at oil–‘protein solution’ interface

Time-dependent conformational changes of proteins and oil molecules at oil–‘protein solution’ interface were studied using ATR (Attenuated Total Reflection)-FTIR spectroscopic technique for the case of Bacillus subtilis extracellular proteins (BSEPs) and bovine serum albumin (BSA) in hexane–‘protein solution’ system. The IR spectra collected on the protein aggregate – film – formed at the hexane–‘protein solution’ interface demonstrated time-dependent conformational changes of the proteins through changes in the shapes and positions of the H2O–‘amide I’ cross peaks and the amide II peaks as a function of time (0–90th minute). Hexane–protein intermolecular association in the film was evident as the CH stretching vibration peaks of hexane were present along with the amide peaks in all the spectra collected over a period of 90min. Conformational changes of the hexane molecules, along with that of the proteins, were observed via variations (broadening and red/blue shifts) in the CH stretching vibration peaks of the CH3 and the CH2 groups of hexane. The red/blue shifts of the CH stretching vibration peaks of hexane were different with BSEPs and BSA, further indicating that the conformational changes of hexane molecules being protein specific. As similar to the protein types considered here, at oil–‘protein solution’ interfaces, conformational changes of the oil molecules appear to be a regular phenomenon.

Reconstructing Global Conformational Dynamics of a Multi-Domain Protein

Time-dependent large-amplitude conformational changes of a single protein can be directly probed via single-molecule Forster-type resonance energy transfer (smFRET), typically one pair of dyes at a time. Monitoring complex motions within a multi-domain protein requires additional distance constraints that could be provided by separate measurements using multiple dye pairs installed at various locations. To infer the possible global conformations and to extract the associated conformational dynamics, we develop a generalizable resampling scheme that integrates high-resolution smFRET data and computational modeling. Time-stamped distances from multiple dye labeling points are statistically resampled and all the possible relative positions of individual domains are mapped geometrically. The choice of a global conformation conforming to all distance constraints is then made given the computed energy profiles. Principal component analysis is applied to such resampled single-molecule trajectories to extract the key conformational dynamics. We apply this resampling scheme to the human insulin-degrading enzyme (IDE), a metallopeptidase potentially implicated in type-2 diabetes. Structurally, IDE possesses two equal-sized N- and C-terminal domains, IDE-N and IDE-C, respectively. IDE is found in the compact closed conformation among all available X-ray structures; yet it processively breaks down insulin to several fragments at non-specific sites. Application of this proposed resampling approach allows one to reconstruct and to discover the unexpected global shearing and grinding motions between IDE-N and IDE-C. This resampling scheme should be applicable for illustrating conformational dynamics of other protein systems.

Time domain simulations of chemical bonding effects in surface-enhanced spectroscopy

The atom-centered density-matrix propagation method is used to illustrate how time-dependent conformational changes affect the electronic structure and derived spectroscopic properties of a prototypical finite metal cluster-bound π-conjugated organic complex, Ag7-benzenethiol. We establish that there is considerable conformational flexibility to the model structure, even at relatively low temperatures, which influences the predicted spectroscopic properties. Namely, the computed electron densities, dipoles, and polarizabilities are all dictated by torsional motion which controls the coupling between the π-framework of the chemisorbed molecular system and the cluster.

Forces Driving the Attachment of Staphylococcus epidermidis to Fibrinogen-Coated Surfaces

Cell surface proteins of bacteria play essential roles in mediating the attachment of pathogens to host tissues and, therefore, represent key targets for anti-adhesion therapy. In the opportunistic pathogen Staphylococcus epidermidis , the adhesion protein SdrG mediates attachment of bacteria to the blood plasma protein fibrinogen (Fg) through a binding mechanism that is not yet fully understood. We report the direct measurement of the forces driving the adhesion of S. epidermidis to Fg-coated substrates using single-cell force spectroscopy. We found that the S. epidermidis -Fg adhesion force is of ~150 pN magnitude and that the adhesion strength and adhesion probability strongly increase with the interaction time, suggesting that the adhesion process involves time-dependent conformational changes. Control experiments with mutant bacteria lacking SdrG and substrates coated with the Fg β(6-20) peptide, instead of the full Fg protein, demonstrate that these force signatures originate from the rupture of specific bonds between SdrG and its peptide ligand. Collectively, our results are consistent with a dynamic, multi-step ligand-binding mechanism called "dock, lock, and latch".

Time resolved SAXS and RNA folding

Small angle X-ray scattering provides low resolution structural information about macromolecules in solution. When coupled with rapid mixing methods, SAXS reports time-dependent conformational changes of RNA induced by the addition of Mg(2+) to trigger folding. Thus time-resolved SAXS provides unique information about the global or overall structures of transient intermediates populated during folding. Notably, SAXS provides information about the earliest folding events, which can evade detection by other methods.

Linear Response of Biomolecules To External Perturbations: Revisit Induce-fit

Atoms in proteins, viewed as interlinked hubs, communicate with each other in complying with governed physical forces. The deviations of their positions from the mean, known as fluctuations, are essential in mediating functionally relevant biological processes maintaining one's daily life. The coupled fluctuations between pairs of atoms, the fluctuation covariance, can be determined analytically by Normal Mode Analysis, or numerically by MD simulations. Our study considers how this network of atoms reacts in response to external perturbations. Effects of such perturbations are exemplified by ligand- or (another) protein-induced conformational changes as well as the appreciated structural distortion of crystalline structures from its solution conformers. We assume the response of the system has linear departure from the mean under small perturbations on the Hamiltonian at equilibrium state. The formulated linear response theories, either time-dependent or -independent [1], says that the positional change of a given atom i is the accumulative sum of fluctuation covariance ij, at unperturbed state, multiplied by the force exerted on atom j. The time-dependent response function determined from MD simulation of carbonmonoxy myoglobin is used to track time-dependent conformational changes upon photo-dissociation of CO. The consequently obtained understanding of perturbation propagation is compared with experimental results. Also, the structural distortion of X-ray-characterized ubiquitin from its solution conformer can be well explained by the induce-fit theory, in a wider sense, while population-shift does not account for such a deviation.[1] Ikeguchi M, Ueno J, Sato M, and Kidera A. (2005) Phys Rev Lett, 94, 078102.

Role of Glass-Transition on Fluid Transport in Porous Food Materials

Many food materials such as pasta, potatoes, starch, corn, soybeans, food gels etc. exhibit glass transition in the range of industrial temperature and fluid content values during transport processes such as drying, sorption, cooking, frying etc. Glass transition plays a critical role in affecting the fluid distribution profiles inside the food and average rate of fluid uptake by the whole food matrix. Yet this phenomenon is not included in most modeling studies that are primarily based upon Fick's law of diffusion. The purpose of this communication is to elucidate that Fick's law is valid only when the food is in glassy or rubbery states. Near glass-transition, the relaxation time of polymers is of the order of diffusion time. Thus, time-dependent conformational changes in food biopolymers add an extra stress term to fluid flow. Keeping track of this stress term is also the key to predict quality changes (related to food texture, physical state and crust formation) in foods via modeling. Based upon the average moisture and temperature at a given instant, even if the entire food matrix exists in a specific mechanical state in an average sense, its different sub-regions may be glassy, rubbery or in glass transition state. For example, during sorption, as a fluid front penetrates through a glassy food matrix, regions ahead of the front will be glassy. Regions in the proximity of the moving front would be undergoing glass transition and the regions already saturated by the fluid would be in a rubbery state. In the proximity of the moving front, where glass-transition is taking place, the fluid flow behavior is non-Fickian. During drying of biopolymers these regions will be in reverse order with the outer surface in a glassy state, center in a rubbery state and the middle region in transition state. Here a continuum mechanics based modeling approach is presented that can be used to predict fluid profiles and normalized stress profiles in all three states (glassy, rubbery and transition).

Estrogen receptor beta target genes in estrogen receptor alpha negative cells

Members of the Crk family of adaptor proteins are key players in signal transduction from a variety of cell surface receptors. CrkI and CrkII are two alternative-spliced forms of a single gene which possess an N-terminal SH2 domain and an SH3 domain that mediate interaction with other proteins. CrkII possesses an additional C-terminal linker region plus an extra SH3 domain, which does not interact with other proteins, but operates as regulatory moiety. Utilizing human Jurkat T cells, we demonstrate that CrkII-SH3N binding of C3G is inhibited by cyclosporin A (CsA) plus FK506 that inhibit the cyclophilin A (CypA) and FK506 binding protein (FKBP) peptidyl-prolyl cis-trans isomerases (PPIases; also termed immunophilins), respectively. Jurkat T cells were found to express ∼5-fold lower levels of CrkI protein compared to CrkII, but the efficiency of C3G binding by CrkI was ∼5-fold higher than that of CrkII, suggesting that the majority of cellular CrkII proteins adopt a conformation that is inaccessible for C3G. Treatment of Jurkat T cells with CsA plus FK506 led to a time-dependent conformational change in overexpressed human CrkII1-236 protein-containing FRET-based biosensor, supporting the accumulation of cis conformers of human CrkII1-236 in the presence of PPIase inhibitors. Our data suggest that the Gly219-Pro-Tyr motif in the human CrkII linker region serves as the recognition and isomerization site of PPIases, and raise the possibility that CsA and FK506 might interfere with selected effector T cell functions via a CrkII-, but not CrkI-dependent mechanisms.

Kinetics of conformational changes of fibronectin adsorbed onto model surfaces

Fibronectin (FN), a large glycoprotein found in body fluids and in the extracellular matrix, plays a key role in numerous cellular behaviours. We investigate FN adsorption onto hydrophilic bare silica and hydrophobic polystyrene (PS) surfaces using Fourier transform infrared spectroscopy-attenuated total reflection (FTIR-ATR) in aqueous medium. Adsorption kinetics using different bulk concentrations of FN were followed for 2 h and the surface density of adsorbed FN and its time-dependent conformational changes were determined. When adsorption occurs onto the hydrophilic surface, FN molecules keep their native conformation independent of the adsorption conditions, but the amount of adsorbed FN increases with time and the bulk concentration. Although the protein surface density is the same on the hydrophobic PS surface, this has a strong impact on the average conformation of the adsorbed FN layer. Indeed, interfacial hydration changes induced by adsorption onto the hydrophobic surface lead to a decrease in unhydrated β-sheet content and cause an increase in hydrated β-strand and hydrated random domain content of adsorbed FN. This conformational change is mainly dependent on the bulk concentration. Indeed, at low bulk concentrations, the secondary structures of adsorbed FN molecules undergo strong unfolding, allowing an extended and hydrated conformation of the protein. At high bulk concentrations, the molecular packing reduces the unfolding of the stereoregular structures of the FN molecules, preventing stronger spreading of the protein.

Heritability, Platelet Function, and Aspirin

Platelets play a critical role in the pathophysiology of atherothrombotic disease. A pivotal event contributing to the understanding of platelet-dependent clot formation was the development of the platelet aggregometer in 1962.1 An aggregometer specifically measures the ability of platelets to adhere via glycoprotein IIb/IIIa (integrin αIIbβ3), and thousands of articles using this technique have been published, characterizing platelet function; however, the usefulness of these measurements remains unclear. Whereas the aggregometer and related techniques that measure platelet aggregation or glycoprotein expression have led to large amounts of data characterizing platelet function in various settings, the clinical importance of measurable differences in platelet function is still debated.2 The use of platelet function testing is established in rarer platelet abnormalities, such as the autosomal recessive bleeding disorder Glanzmann thrombasthenia,3 but no clear consensus has been reached on its usefulness for highly prevalent diseases caused by platelet-dependent thrombosis, such as myocardial infarction. A major factor for this discrepancy is that many of the platelet function defects that lead to bleeding are known to be caused by a single defect, whereas thrombosis in the setting of cardiovascular disease is presumed to be multifactorial. Article p 2490 The evolution of platelet function studies in various clinical settings has led to the realization that wide interindividual variability exists in the platelet activation response.4,5 What accounts for this variability? Only a few studies have systematically examined this question. Platelet function has been established as markedly dependent on the type of agonist used, the agonist concentration, and the concomitant use of antiplatelet therapy.6 In addition, in the large population-based Framingham Heart Study, O’Donnell and colleagues7 have demonstrated that heritable factors play a major role in determining platelet aggregation, as opposed to measured covariates. Less clear from the current literature is the direct …

Effects of acid exposure on the conformation, stability, and aggregation of monoclonal antibodies

Exposure of antibodies to low pH is often unavoidable for purification and viral clearance. The conformation and stability of two humanized monoclonal antibodies (hIgG4-A and -B) directed against different antigens and a mouse monoclonal antibody (mIgG1) in 0.1M citrate at acidic pH were studied using circular dichroism (CD), differential scanning calorimetry (DSC), and sedimentation velocity. Near- and far-UV CD spectra showed that exposure of these antibodies to pH 2.7-3.9 induced only limited conformational changes, although the changes were greater at the lower pH. However, the acid conformation is far from unfolded or so-called molten globule structure. Incubation of hIgG4-A at pH 2.7 and 3.5 at 4 degrees C over the course of 24 h caused little change in the near-UV CD spectra, indicating that the acid conformation is stable. Sedimentation velocity showed that the hIgG4-A is largely monomeric at pH 2.7 and 3.5 as well as at pH 6.0. No time-dependent changes in sedimentation profile occurred upon incubation at these low pHs, consistent with the conformational stability observed by CD. The sedimentation coefficient of the monomer at pH 2.7 or 3.5 again suggested that no gross conformational changes occur at these pHs. DSC analysis of the antibodies showed thermal unfolding at pH 2.7-3.9 as well as at pH 6.0, but with decreased melting temperatures at the lower pH. These results are consistent with the view that the antibodies undergo limited conformational change, and that incubation at 4 degrees C at low pH results in no time-dependent conformational changes. Titration of hIgG4-A from pH 3.5 to 6.0 resulted in recovery of native monomeric proteins whose CD and DSC profiles resembled those of the original sample. However, titration from pH 2.7 resulted in lower recovery of monomeric antibody, indicating that the greater conformational changes observed at this pH cannot be fully reversed to the native structure by a simple pH titration.

Antibodies to inactive conformations of glyceraldehyde-3-phosphate dehydrogenase inactivate the apo-and holoforms of the enzyme

Polyclonal antibodies produced after the immunization of a rabbit with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus were used to isolate two types of antibodies interacting with different non-native forms of the antigen. Type I antibodies were purified using Sepharose-bound apo-GAPDH that was treated with glutaraldehyde to stabilize the enzyme in the tetrameric form. Type II antibodies were isolated using immobilized denatured monomers of the enzyme. It was shown that the type I antibodies bound to the native holo- and apoforms of the enzyme with the ratio of one antibody molecule per GAPDH tetramer. While interacting with the native holoenzyme, the type I antibodies induce a time-dependent decrease in its activity by 80-90%. In the case of the apoenzyme, the decrease in the activity constitutes only 25%, this indicating that only one subunit of the tetramer is inactivated. Differential scanning calorimetry experiments showed that the formation of the complex between both forms of the enzyme and the type I antibodies resulted in a shift of the maximum of the thermal capacity curves (T(m) value) to lower temperatures. The extremely stable holoenzyme was affected to the greatest extent, the shift of the T(m) value constituting approximately 20 degrees C. We assume that the formation of the complex between the holo- or apo-GAPDH and the type I antibody results in time-dependent conformational changes in the enzyme molecule. Thus, the antibodies induce the structural rearrangements yielding the conformation that is identical to the structure of the antigen used for the selection of the antibodies (i.e., inactive). The interaction of the antibodies with the apo-GAPDH results in the inactivation of the subunit directly bound to the antibody. Virtually complete inactivation of the holoenzyme by the antibodies is likely due to the transmission of the conformational changes through the intersubunit contacts. The type II antibodies, which were selected using the immunosorbent with unfolded enzyme form, do not affect the activity of native holo- and apo-GAPDH, but prevent the reactivation of the denatured GAPDH, binding the denatured forms of the enzyme.