Active Site Of Protein Research Articles

Molecular Thermodynamic Origin of Substrate Promiscuity in the Enzyme Laccase: Toward a Broad-Spectrum Degrader of Dye Effluents.

Industrial dye effluents have emerged as significant health hazard. Laccases found in white rot fungi can degrade an assortment of dyes. Here, we explore the molecular thermodynamic origin of the substrate promiscuity in laccases using a combination of steady-state UV-visible absorption spectroscopy, molecular docking, and molecular dynamics (MD) simulation studies on the interaction of laccase with five dye molecules with varying charge, size, and shape. The spectroscopic studies confirm that all of these dyes can be degraded by laccase. Using MD simulations, we have demonstrated the presence of various distinct conformations of a loop in the protein active site that can accommodate the wide range of dye molecules. We have also shown that the diverse selection of dye molecules may exhibit surprisingly similar binding affinity due to cancellation of different thermodynamic factors. Our results highlight the potential of laccase as a multipurpose degrader for industrial dye effluents.

De novo design of BMP mimics.

In traditional structure-guided drug designs, scientists often design drugs to target the active site of proteins of interest with detailed knowledge about the active site. However, despite the success rate, targeting the active site often leads to off-target side effects. With the development of protein binder design in the Baker Lab, one may imagine rewiring this drug development process by using de novo designed binders to regulate cell signaling pathways. Here I will present my efforts toward designing agonists/antagonists targeting different cell signaling pathways. I will focus on our efforts in developing bone morphogenetic protein 2/9/10 (BMP2/9/10) mimics, which are a group of growth factors to constitute a group of morphogenetic and angiogenetic signals. Recombinant human BMPs are used in orthopedics including oral surgery and spinal surgery. This project aims to specifically activate/inhibit BMP signaling pathways of interest by designing protein binders targeting specific BMP receptors, therefore, providing new therapeutics.

Quantifying the evolvability of allostery at different protein surfaces.

Allosteric regulation requires the cooperative action of multiple amino acid residues to relay a signal from a distal surface site to the protein active site. It is an open problem to understand how new regulation evolves through a Darwinian process of single mutation and selection. Yet a better understanding of how allostery emerges is critical to our ability to engineer new custom biosensors. Are some protein surfaces more prone to evolve into allosteric sites than others? Or is the capacity to evolve allostery homogenously distributed across a protein's entire surface? Recent work has identified that allosteric regulation is more easily engineered at certain protein surfaces termed “allosteric hotspots.” These hotspots occur at surface positions that co-evolve with enzyme active sites. Allosteric hotspots have been used to engineer new allosteric regulation of dihydrofolate reductase (DHFR) by the light-oxygen-voltage-sensing domain (LOV2). When LOV2 is fused to allosteric hotspots on DHFR, DHFR activity is weakly regulated by light (1.3-fold). However, fusing LOV2 to non-hotspot positions does not result in allosteric control. The low level of regulation observed in DHFR/LOV2 fusions presents an opportunity to study how allostery can be optimized by evolution. In my work, I am using random mutagenesis combined with a high-throughput screen to evolve new allosteric regulation. I am performing forward evolution of allostery for a weakly allosteric, hotspot-connected fusion, and for a non-allosteric, non-hotspot-connected fusion. I am monitoring the distribution of mutational effects on allostery for each construct over rounds of selection. With this approach, I will quantify the evolvability of allosteric regulation at different protein surface sites. I hypothesize that allosteric hotspots will have a higher prevalence of allostery-tuning mutations, and that these mutations will result in higher levels of allosteric regulation.

Molecular docking analysis of oxazole compounds with the heme-binding protein from Porphyromonas gingivalis.

Porphyromonas gingivalis, a peripathogen, has several methods to impede or modify the protective mechanisms of the teeth. Targeting the inhibition of the heme protein will prevent the organism from multiplying and inhibit the virulence mechanism. The literature derived oxazole compounds (1-5) were docked against the protein's active site, and the results show that the selected oxazole derivatives exhibit better interaction compared to clinically proven drugs.

Mutation Effects on Structure and Dynamics: Adaptive Evolution of the SARS-CoV-2 Main Protease.

The main protease of SARS-CoV-2 (Mpro) plays a critical role in viral replication; although it is relatively conserved, Mpro has nevertheless evolved over the course of the COVID-19 pandemic. Here, we examine phenotypic changes in clinically observed variants of Mpro, relative to the originally reported wild-type enzyme. Using atomistic molecular dynamics simulations, we examine effects of mutation on protein structure and dynamics. In addition to basic structural properties such as variation in surface area and torsion angles, we use protein structure networks and active site networks to evaluate functionally relevant characters related to global cohesion and active site constraint. Substitution analysis shows a continuing trend toward more hydrophobic residues that are dependent on the location of the residue in primary, secondary, tertiary, and quaternary structures. Phylogenetic analysis provides additional evidence for the impact of selective pressure on mutation of Mpro. Overall, these analyses suggest evolutionary adaptation of Mpro toward more hydrophobicity and a less-constrained active site in response to the selective pressures of a novel host environment.

In Silico Mining of Natural Products Atlas (NPAtlas) Database for Identifying Effective Bcl-2 Inhibitors: Molecular Docking, Molecular Dynamics, and Pharmacokinetics Characteristics.

The Bcl-2 protein has a vital function in controlling the programmed cell doom of mitochondria. If programmed cell death signals are obstructed, an imbalance between cell survival and death will occur, which is a significant reason for cancer. Therefore, the Bcl-2 protein was identified as a possible therapeutic target for carcinoma treatment. Herein, the Natural Products Atlas (NPAtlas) compounds were virtually screened, seeking potent inhibitors towards the Bcl-2 protein. The performance of AutoDock Vina software to predict the docking score and pose of the investigated compounds was first validated according to the available experimental data. Based on the validated AutoDock Vina parameters, the NPAtlas database was filtered against the Bcl-2 protein. The natural compounds with docking scores less than that of the venetoclax (calc. -10.6 kcal/mol) were submitted to MD simulations, followed by MM-GBSA binding energy calculations. According to MM-GBSA//200 ns MD simulations, saquayamycin F (NPA002200) demonstrated promising binding affinity with a ΔGbinding value of -53.9 kcal/mol towards the Bcl-2 protein when compared to venetoclax (ΔGbinding = -50.6 kcal/mol). The energetical and structural analyses showed a great constancy of the saquayamycin F inside the Bcl-2 protein active site. Moreover, the ADMET and drug-likeness features of the saquayamycin F were anticipated, indicating its good oral bioavailability. According to in silico computations, saquayamycin F is proposed to be used as a therapeutic agent against the wild-type Bcl-2 protein and warrants further experimental assays.

Development of piperazine conjoined 1,2,3-triazolyl-γ-propyltriethoxysilanes: Fluorometric detection of Cr3+ ions and computational study

Chromium is essential for some biochemical processes, and excess is a big concern that shows adverse effects on human health and the environment. Therefore, it is urgent to design new sensors to detect chromium ions rapidly. The present study discusses the synthesis of piperazine conjoined 1,2,3-triazolyl-γ-propyltriethoxysilanes (4a-4b) and development of 4a as fluorescence turn-on sensor for the detection of Cr3+ ions. The mechanistic insights reveal to the restricted CN rotation and inhibited intramolecular charge transfer (ICT) process. In addition, Job's plot and Benesi-Hildebrand plot justify the 1:1 binding affinity with a binding constant of 9.96 × 105 M−1 for [ligand 4a + Cr3+] complex and the limit of detection for Cr3+ ions is observed as 6.06 × 10-8 M. The fluorescence spectral changes, 1H NMR spectra and DFT studies provide evidences for ligand 4a and Cr3+ ions interactions. Further, the reversibility of the ligand 4a from [ligand 4a + Cr3+] complex on the addition of EDTA can be used in the construction of molecular logic gate where Cr3+ and EDTA are considered as inputs and the fluorescence intensity at 398 nm as output. Further, compounds 4a-4b were then evaluated for their antibacterial activity against bacterial strains (Escherichia coliand Staphylococcus aureus), revealing a modest activity. The binding mode of ligand 4a to Staphylococcus aureus (PDB ID − 3U2K) and Escherichia coli (PDB ID − 5Z4O) was investigated using an in-silico molecular docking technique, which revealed that the triazole ring and silanyl group are involved in hydrogen bonding with proteins and may be the cause of the ligand's antibacterial activity. The ligand 4a demonstrated a high affinity for binding within the active sites of proteins with binding energies of −7.97 kcal/mol (3U2K) and −8.68 kcal/mol (5Z4O).

Design, synthesis, molecular dynamic simulation studies, and antibacterial evaluation of new spirocyclic aminopyrimidines

The rise of new bacterial pathogens that ascribed as multi-drug resistant became a major concern, particularly in the medical sectors. In the current study, we designed novel molecular hybrids based on spirocyclic pyrimidine which feature either triazolo ring or various functionalities such as alkyl, hydrazino, azomethine motifs. The newly synthesized spirocyclic pyrimidines were screened for their bactericidal activities against some bacterial pathogens such as Salmonella typhimurium and Proteus vulgaris in addition to the multi-drug resistant Pseudomonas aeruginosa and Staphylococcus aureus (MRSA). Compounds coded 4, 6, and 8e were found to possess potent bactericidal activities compared to the standard drugs (Ciprofloxacin and Cephradine). Interestingly, compound 6 exhibited the highest antibacterial activity with low MIC value close to that obtained for ciprofloxacin. Molecular dynamic simulations and docking studies were investigated to check the performance of our compounds upon binding to the protein active sites as well as their interactions and stabilities through the simulation process.

Ehretia Species Phytoconstituents as Potential Lead Compounds against Klebsiella pneumoniae Carbapenemase: A Computational Approach.

The evolution of antibiotic-resistant carbapenemase has negatively impacted the management of critical healthcare-associated infections. K. pneumoniae carbapenemase-2- (KPC-2-) expressing bacteria have developed resistance to conventional therapeutic options, including those used as a last resort for life-threatening diseases. In this study, Ehretia species phytoconstituents were screened for their potential to inhibit KPC-2 protein using in silico approaches. Molecular docking was used to identify strong KPC-2 protein binding phytoconstituents retrieved from the literature. The best-docked conformation of the ligands was selected based on their glide energy and binding interactions. To determine their binding free energies, these hit compounds were subjected to molecular mechanics with generalized born and surface area (MM-GBSA) in the PRIME module. Pharmacological assessments of the ligands were performed to evaluate their drug-likeness. Molecular dynamic (MD) simulations were used to analyze the conformational stability of the selected druglike compounds within the active site of the KPC-2 protein. Overall, a total of 69 phytoconstituents were compiled from the literature. Fourteen of these compounds exhibited a stronger binding affinity for the protein target than the reference drugs. Four of these top hit compounds, DB09, DB12, DB28, and DB66, revealed the highest efficacy in terms of drug-likeness properties. The MD simulation established that among the druglike compounds, DB66 attained stable conformations after 150 ns simulation in the active site of the protein. We concluded that DB66 from Ehretia species could play a significant role in therapeutic efforts against KPC-2-expressing bacteria.

Small-Molecule Phosphine Activation of Protein Function in Zebrafish Embryos with an Expanded Genetic Code.

Conditional control of protein function in a living model organism is an important tool for studying the effects of that protein during development and disease. In this chapter, we walk through the steps to generate a small-molecule-activatable enzyme in zebrafish embryos through the incorporation of a noncanonical amino acid into the protein active site. This method can be applied to many enzyme classes, which we highlight with temporal control of a luciferase and a protease. We demonstrate that strategic placement of the noncanonical amino acid completely blocks enzyme activity, which is then promptly restored after addition of the nontoxic small molecule inducer to the embryo water.

Role of distal arginine residue in the mechanism of heme nitrite reductases.

Heme nitrite reductases reduce NO2- by 1e-/2H+ to NO or by 6e-/8H+ to NH4+ which are key steps in the global nitrogen cycle. Second-sphere residues, such as arginine (with a guanidine head group), are proposed to play a key role in the reaction by assisting substrate binding and hydrogen bonding and by providing protons to the active site for the reaction. The reactivity of an iron porphyrin with a NO2- covalently attached to a guanidinium arm in its 2nd sphere was investigated to understand the role of arginine residues in the 2nd sphere of heme nitrite reductases. The presence of the guanidinium residue allows the synthetic ferrous porphyrin to reduce NO2- and produce a ferrous nitrosyl species ({FeNO}7), where the required protons are provided by the guanidinium group in the 2nd sphere. However, in the presence of additional proton sources in solution, the reaction of ferrous porphyrin with NO2- results in the formation of ferric porphyrin and the release of NO. Spectroscopic and kinetic data indicated that re-protonation of the guanidine group in the 2nd sphere by an external proton source causes NO to dissociate from a ferric nitrosyl species ({FeNO}6) at rates similar to those observed for enzymatic sites. This re-protonation of the guanidine group mimics the proton recharge mechanism in the active site of NiR. DFT calculations indicated that the lability of the Fe-NO bond in the {FeNO}6 species is derived from the greater binding affinity of anions (e.g. NO2-) to the ferric center relative to neutral NO due to hydrogen bonding and electrostatic interaction of these bound anions with the protonated guanidium group in the 2nd sphere. The reduced {FeNO}7 species, once formed, is not affected significantly by the re-protonation of the guanidine residue. These results provide direct insight into the role of the 2nd sphere arginine residue present in the active sites of heme-based NiRs in determining the fate of NO2- reduction. Specifically, the findings using the synthetic model suggest that rapid re-protonation of these arginine residues may trigger the dissociation of NO from the {FeNO}6, which may also be the case in the protein active site.

Masked cerulenin enables a dual-site selective protein crosslink.

Protein-reactive natural products such as the fungal metabolite cerulenin are recognized for their value as therapeutic candidates, due to their ability to selectively react with catalytic residues within a protein active site or a complex of protein domains. Here, we explore the development of fatty-acid and polyketide-synthase probes by synthetically modulating cerulenin's functional moieties. Using a mechanism-based approach, we reveal unique reactivity within cerulenin and adapt it for fluorescent labeling and crosslinking of fatty-acid and iterative type-I polyketide synthases. We also describe two new classes of silylcyanohydrin and silylhemiaminal masked crosslinking probes that serve as new tools for activity and structure studies of these biosynthetic pathways.

Molecular modeling of the interactions of Curcuma longa compounds with VEGFR towards colorectal cancer drug development

Curcuma longa, a member of Curcuma Zingiberaceae family is said to possess anti-carcinogenic, anti-oxidant, and anti-inflammatory properties. Mechanistic studies on its effectiveness against carcinogenesis are still under investigation. This study assessed the molecular interactions of C. longa compounds with VEGFR. Computational methods such as molecular docking, calculation of binding free energy, induced fit docking, Molecular dynamic simulation,PPI targets and KEGGS enrichment pathways were employed to discover approximately 117 bioactive compounds against VEGFR in designing novel anticancer drugs. The five top-scoring C. longa compounds out of the 117 bioactive compounds are 2-Hydroxymethyl anthraquinone (HMA), Riboflavin, Quercetin, Vitamin E, and Dicinnamoylmethane (−8.515 kcal/mol to −7.293 kcal/mol) with higher docking scores. These compounds had stable conformation within the flexible protein's active site. The molecular interactions of these compounds with surrounding amino acids in the ATP binding gouge of VEGFR2 and their drug-like qualities with excellent safety profile make them potential inhibitors of VEGFR2 and future drug development against colorectal cancer.

In silico Structure Prediction, Molecular Docking, and Dynamic Simulation of Plasmodium falciparum AP2-I Transcription Factor.

Plasmodium falciparum Apicomplexan Apetala 2 Invasion (PfAP2-I) transcription factor (TF) is a protein that regulates the expression of a subset of gene families involved in P. falciparum red blood cell (RBC) invasion. Inhibiting PfAP2-I TF with small molecules represents a potential new antimalarial therapeutic target to combat drug resistance, which this study aims to achieve. The 3D model structure of PfAP2-I was predicted ab initio using ROBETTA prediction tool and was validated using Save server 6.0 and MolProbity. Computed Atlas of Surface Topography of proteins (CASTp) 3.0 was used to predict the active sites of the PfAP2-I modeled structure. Pharmacophore modeling of the control ligand and PfAP2-I modeled structure was carried out using the Pharmit server to obtain several compounds used for molecular docking analysis. Molecular docking and postdocking studies were conducted using AutoDock vina and Discovery studio. The designed ligands' toxicity predictions and in silico drug-likeness were performed using the SwissADME predictor and OSIRIS Property Explorer. The modeled protein structure from the ROBETTA showed a validation result of 96.827 for ERRAT, 90.2% of the amino acid residues in the most favored region for the Ramachandran plot, and MolProbity score of 1.30 in the 98th percentile. Five (5) best hit compounds from molecular docking analysis were selected based on their binding affinity (between -8.9 and -11.7 Kcal/mol) to the active site of PfAP2-I and were considered for postdocking studies. For the absorption, distribution, metabolism, elimination, and toxicity (ADMET) properties, compound MCULE-7146940834 had the highest drug score (0.63) and drug-likeness (6.76). MCULE-7146940834 maintained a stable conformation within the flexible protein's active site during simulation. The good, estimated binding energies, drug-likeness, drug score, and molecular dynamics simulation interaction observed for MCULE-7146940834 against PfAP2-I show that MCULE-7146940834 can be considered a lead candidate for PfAP2-I inhibition. Experimental validations should be carried out to ascertain the efficacy of these predicted best hit compounds.

Reorganization energy of electron transfer.

The theory of electron transfer reactions establishes the conceptual foundation for redox solution chemistry, electrochemistry, and bioenergetics. Electron and proton transfer across the cellular membrane provide all energy of life gained through natural photosynthesis and mitochondrial respiration. Rates of biological charge transfer set kinetic bottlenecks for biological energy storage. The main system-specific parameter determining the activation barrier for a single electron-transfer hop is the reorganization energy of the medium. Both harvesting of light energy in natural and artificial photosynthesis and efficient electron transport in biological energy chains require reduction of the reorganization energy to allow fast transitions. This review article discusses mechanisms by which small values of the reorganization energy are achieved in protein electron transfer and how similar mechanisms can operate in other media, such as nonpolar and ionic liquids. One of the major mechanisms of reorganization energy reduction is through non-Gibbsian (nonergodic) sampling of the medium configurations on the reaction time. A number of alternative mechanisms, such as electrowetting of active sites of proteins, give rise to non-parabolic free energy surfaces of electron transfer. These mechanisms, and nonequilibrium population of donor-acceptor vibrations, lead to a universal phenomenology of separation between the Stokes shift and variance reorganization energies of electron transfer.

Computationally guided bioengineering of the active site, substrate access pathway, and water channels of thermostable cytochrome P450, CYP175A1, for catalyzing the alkane hydroxylation reaction.

Understanding structure-function relationships in proteins is pivotal in their development as industrial biocatalysts. In this regard, rational engineering of protein active site access pathways and various tunnels and channels plays a central role in designing competent enzymes with high stability and enhanced efficiency. Here, we report the rational evolution of a thermostable cytochrome P450, CYP175A1, to catalyze the C-H activation reaction of longer-chain alkanes. A strategy combining computational tools with experiments has shown that the substrate scope and enzymatic activity can be enhanced by rational engineering of certain important channels such as the substrate entry and water channels along with the active site of the enzyme. The evolved enzymes showed an improved catalytic rate for hexadecane hydroxylation with high regioselectivity. The Q67L/Y68F mutation showed binding of the substrate in the active site, water channel mutation L80F/V220T showed improved catalytic activity through the peroxide shunt pathway and substrate entry channel mutation W269F/I270A showed better substrate accessibility to the active pocket. All-atom MD simulations provided the rationale for the inactivity of the wild-type CYP175A1 for hexadecane hydroxylation and predicted the above hot-spot residues to enhance the activity. The reaction mechanism was studied by QM/MM calculations for enzyme-substrate complexes and reaction intermediates. Detailed thermal and thermodynamic stability of all the mutants were analyzed and the results showed that the evolved enzymes were thermally stable. The present strategy showed promising results, and insights gained from this work can be applied to the general enzymatic system to expand substrate scope and improve catalytic activity.

Green synthesis of silver nanoparticles from saline tolerant microalgae and their antiviral properties against HIV-1

The microalgae were exposed to silver nitrate (AgNO3) solution and screened for their suitability for production of the nano-silver (nano-Ag). The HIV-1 gag p24 Elisa Assay was used to study the anti-HIV activity of biogenic silver nanoparticles from microalgae Nostoc sp., Lyngbya sp., and Phormidium sp. The antiviral activity of biogenic silver compounds can be explained by their ability to cross the lipophilic membranes and interact with proteins involved in apoptosis. The antiviral activity of silver ion assembled into the V3loop of HIV -GP120. CD4 cell with CXCR4 chemokine receptor binding with its ligand CXCL12 plays an important role in protecting the entry of HIV virus. The role of the extracellular loop from CXCR4 and its mechanism allowed for the binding of HIV glycoprotein. This active site of protein bind with target protein to form protein–protein-ligand interaction to carry out the signals to lead the various pathways such as CXCL12 or SDF-1 protein-mediated kinase, Ras-c-Raf-1-MEK1/2-ERK1/2 dependent pathway, and CD45 cell-mediated pathway. The sulfur-bearing residues located in the glycoprotein knobs would be attractive sites for nanoparticle interaction. Silver nanoparticles are blocked the protease activity of different HIV-1strains with overlooks their tropism. Modification in gp120 among HIV strains is the major determinant site of differing tropism among HIV strains.

Green synthesis of silver nanoparticles from saline tolerant microalgae and their antiviral properties against HIV-1

The microalgae were exposed to silver nitrate (AgNO3) solution and screened for their suitability for production of the nano-silver (nano-Ag). The HIV-1 gag p24 Elisa Assay was used to study the anti-HIV activity of biogenic silver nanoparticles from microalgae Nostoc sp., Lyngbya sp., and Phormidium sp. The antiviral activity of biogenic silver compounds can be explained by their ability to cross the lipophilic membranes and interact with proteins involved in apoptosis. The antiviral activity of silver ion assembled into the V3loop of HIV -GP120. CD4 cell with CXCR4 chemokine receptor binding with its ligand CXCL12 plays an important role in protecting the entry of HIV virus. The role of the extracellular loop from CXCR4 and its mechanism allowed for the binding of HIV glycoprotein. This active site of protein bind with target protein to form protein–protein-ligand interaction to carry out the signals to lead the various pathways such as CXCL12 or SDF-1 protein-mediated kinase, Ras-c-Raf-1-MEK1/2-ERK1/2 dependent pathway, and CD45 cell-mediated pathway. The sulfur-bearing residues located in the glycoprotein knobs would be attractive sites for nanoparticle interaction. Silver nanoparticles are blocked the protease activity of different HIV-1strains with overlooks their tropism. Modification in gp120 among HIV strains is the major determinant site of differing tropism among HIV strains.

In Silico Screening 2019-Coronavirus Inhibitors by SARS Template-Based Molecular Docking on ANTI-HIV Drugs

Proteases or proteolytic enzymes are effective targets for developing antiviral drugs. The chymotrypsin-like cysteine protease (3CLpro), known as the main protease (Mpro) of severe acute respiratory syndrome coronavirus (SARS-CoV) plays an essential role in the proteolytic processing of viruses, and it is an effective target for anti-SARS drug development. An outbreak of novel coronavirus, named 2019-nCoV that occurred in Wuhan, China, has been identified as the cause of the disease and spread rapidly in multiple countries. There are no drugs approved to be an effective treatment for the 2019-nCoV yet. The result of sequence alignment revealed that the 2019-nCov Mpro shows 96% similarity with that of SARS-CoV 3CLpro (SARS Mpro). This is a potential discovery for developing 2019-nCov inhibitors. In order to find more potential inhibitors of the 2019-nCov Mpro protein, we selected SARS-CoV 3CLpro (PDB ID: 2GTB) as a template to perform molecular docking on 10 approved anti-HIV drugs. The docking results revealed that the SARS Mpro gave the highest binding affinity for saquinavir with a binding energy of -29.21 kcal/mol. Anti-HIV drugs darunavir (-23.43 kcal/mol), indinavir (-22.87 kcal/mol), and nelfinavir (-21.54 kcal/mol) also showed good binding modes with the active sites of protein, indicating that they may have the potential to be used as anti-COVID-19 clinical drugs. The observations would contribute more drug candidates that could interact with key residues of 2019-nCov Mpro similarly to the existing inhibitors against SARS-CoV 3CLpro.

ВЛИЯНИЕ ЗАМОРАЖИВАНИЯ-ОТТАИВАНИЯ НА ТЕМПЕРАТУРНОЕ ПОВЕДЕНИЕ ГЕМОГЛОБИНА

The use of blood preservation at low temperatures methods in medical practice makes it necessary to study the temperature effect on hemoglobin. The effect of freezing-thawing to a liquid nitrogen temperature of -196C and subsequent thawing on hemoglobin A was studied, using the methods of temperature-perturbation differential spectrophotometry and analysis of absorption spectra first derivatives. After the hemoglobin A solution freezing-thawing, a smoothing of the breaks in the S-shaped temperature intensity dependence at the maximum of the temperature-perturbation differential spectra at 286 nm is noted. This probably means that temperature-dependent conformational changes in the hemoglobin A molecule are smoothed out and become less noticeable. It should be noted that after freezing-thawing, this dependence S-shaped form is preserved, i.e. conformational changes take place, but they are less pronounced than before freezing-thawing. Thus, on the one hand, freezing the hemoglobin A solution leads to conformational changes in the hemoglobin A molecule affecting the polar regions, namely, to an increase in the polar regions availability to the solvent, which is consistent with the data on the protein globule partial unfolding, accompanied by the globin aromatic amino acids exposure to the solvent, and with the data that the freezing-thawing process leads in most cases to conformational changes in the protein, which consist in the molecule weakening and increasing the protein active sites accessibility. On the other hand, freezing of hemoglobin A solutions leads to compaction of the hemoglobin A molecule subunits. This can probably be explained by the fact that, presumably, an increase in the availability of the hemoglobin A molecule absorbing chromophores located in the polar regions to the solvent leads to the compaction of the hemoglobin molecule A other parts, not involved in increasing the amino acid residues availability to the solvent. That is, some parts of the hemoglobin A molecule become more accessible to the solvent, while others become compact.