Mechanism Of Protein Binding Research Articles

Tissue distribution of emerging per- and polyfluoroalkyl substances in wild fish species from Qiantang river, east China: Comparison of 6:2 Cl-PFESA with PFOS

This study argued for the first time that 6:2 chlorinated polyfluorinated ether sulfonate (6:2 Cl-PFESA) and perfluorooctanesulfonic acid (PFOS) might have different tissue distribution mechanisms in wild fish species. Nine emerging and legacy per- and polyfluoroalkyl substances (PFASs) were detected in the water and wild fish tissues samples collected from the Qiantang River. Perfluorooctanoic acid (213 ng/L) was the predominant PFAS contaminant, and the other contaminants included perfluorohexanoate (19 ng/L), perfluorobutanoate (199 ng/L) and hexafuoropropylene oxide dimer acid (55 ng/L), which are the main fluorinated alternatives used in various industries located along the Qiantang River. Furthermore, PFOS (742 ng/g) and 6:2 Cl-PFESA (9.0 ng/g) were the predominant PFAS contaminants detected in the fish tissue samples. The differences in the potential molecular mechanism of the tissue distribution of PFOS and 6:2 Cl-PFESA in wild fish species are discussed. Additionally, we hypothesize that phospholipid partitioning is the primary mechanism underlying the tissue distribution of PFOS, and that a specific protein-binding mechanism is involved in the tissue distribution of 6:2 Cl-PFESA.

Physicochemical and Adsorption Properties of Nanoporous Activated Biocarbons from Thermochemical Treatment of Horsetail Herb.

In this study, the adsorption characteristics of novel activated biocarbons prepared from horsetail herb (a popular and troublesome weed) by physical activation (using carbon dioxide) and chemical one (using phosphoric(V) acid) in the process of simultaneous proteins immobilization in multicomponent solutions were examined. The carbon materials were characterized in terms of their porous structure, acidic-basic properties, and surface morphology. The binding mechanisms of such proteins as bovine serum albumin (BSA) and lysozyme (LSZ), differing in internal stability, were determined alone and in their blends. This was done based on the comprehensive analysis of the results of adsorption/desorption, surface, electrokinetic and stability measurements. These experiments were carried out over a wide pH range of 3-11. They included the following issues: (1) determination of the protein adsorbed/desorbed amounts on/from a surface of activated biocarbons; (2) study of the kinetics of these processes; (3) examination of the macromolecules impact on the surface charge density and zeta potential of the carbon materials; and (4) determination of the suspension stability and size of aggregates formed in the examined systems. The analysis of the obtained results indicated the differences in the binding mechanism of both proteins that is of key importance for their simultaneous immobilization on activated biocarbons surface in the soil environment.

Fatty acid binding to the human transport proteins FABP3, FABP4, and FABP5 from a Ligand’s perspective

Fatty acid binding proteins (FABPs) are a family of amphiphilic transport proteins with high diversity in terms of their amino acid sequences and binding preferences. Beyond their main biological role as cytosolic fatty acid transporters, many aspects regarding their binding mechanism and functional specializations in human cells remain unclear. In this work, the binding properties and thermodynamics of FABP3, FABP4 and FABP5 were analyzed under various physical conditions. For this purpose, the FABPs were loaded with fatty acids bearing fluorescence or spin probes as model ligands, comparing their binding affinities via microscale thermophoresis (MST) and continuous-wave electron paramagnetic resonance (CW EPR) spectroscopy. The CW EPR spectra of non-covalently bound 5- and 16-DOXYL stearic acid (5/16-DSA) deliver in-depth information about the dynamics and chemical environments of ligands inside the binding pockets of the FABPs. EPR spectral simulations allow the construction of binding curves, revealing two different binding states (‘intermediately’ and ‘strongly’ bound). The proportion of bound 5/16-DSA depends strongly on the FABP concentration and the temperature, but with remarkable differences between the three isoforms. Additionally, the more dynamic state (‘intermediately bound’) seems to dominate at body temperature with thermodynamic preference. The ligand binding studies were supplemented by aggregation studies via dynamic light scattering (DLS) and bioinformatic analyses. Beyond the remarkably fine-tuned binding properties exhibited by each FABP, which were discernible with our EPR-centered approach, the results of this work attest the power of simple spectroscopic experiments to provide new insights into the ligand binding mechanisms of proteins in general on a molecular level.

AlphaFold2 Predictions of Conformational Ensembles and Atomistic Simulations of the SARS-CoV-2 Spike XBB Lineages Reveal Epistatic Couplings between Convergent Mutational Hotspots that Control ACE2 Affinity.

In this study, we combined AlphaFold-based atomistic structural modeling, microsecond molecular simulations, mutational profiling, and network analysis to characterize binding mechanisms of the SARS-CoV-2 spike protein with the host receptor ACE2 for a series of Omicron XBB variants including XBB.1.5, XBB.1.5+L455F, XBB.1.5+F456L, and XBB.1.5+L455F+F456L. AlphaFold-based structural and dynamic modeling of SARS-CoV-2 Spike XBB lineages can accurately predict the experimental structures and characterize conformational ensembles of the spike protein complexes with the ACE2. Microsecond molecular dynamics simulations identified important differences in the conformational landscapes and equilibrium ensembles of the XBB variants, suggesting that combining AlphaFold predictions of multiple conformations with molecular dynamics simulations can provide a complementary approach for the characterization of functional protein states and binding mechanisms. Using the ensemble-based mutational profiling of protein residues and physics-based rigorous calculations of binding affinities, we identified binding energy hotspots and characterized the molecular basis underlying epistatic couplings between convergent mutational hotspots. Consistent with the experiments, the results revealed the mediating role of the Q493 hotspot in the synchronization of epistatic couplings between L455F and F456L mutations, providing a quantitative insight into the energetic determinants underlying binding differences between XBB lineages. We also proposed a network-based perturbation approach for mutational profiling of allosteric communications and uncovered the important relationships between allosteric centers mediating long-range communication and binding hotspots of epistatic couplings. The results of this study support a mechanism in which the binding mechanisms of the XBB variants may be determined by epistatic effects between convergent evolutionary hotspots that control ACE2 binding.

Exploration of the Binding Mechanism of Cyclic Dinucleotide Analogs to Stimulating Factor Proteins and the Implications for Subsequent Analog Drug Design.

Cyclic dinucleotides (CDNs) are cyclic molecules consisting of two nucleoside monophosphates linked by two phosphodiester bonds, which act as a second messenger and bind to the interferon gene stimulating factor (STING) to activate the downstream signaling pathway and ultimately induce interferon secretion, initiating an anti-infective immune response. Cyclic dinucleotides and their analogs are lead compounds in the immunotherapy of infectious diseases and tumors, as well as immune adjuvants with promising applications. Many agonists of pathogen recognition receptors have been developed as effective adjuvants to optimize vaccine immunogenicity and efficacy. In this work, the binding mechanism of human-derived interferon gene-stimulating protein and its isoforms with cyclic dinucleotides and their analogs was theoretically investigated using computer simulations and combined with experimental results in the hope of providing guidance for the subsequent synthesis of cyclic dinucleotide analogs.

Astraoleanosides E–P, oleanane-type triterpenoid saponins from the aerial parts of Astragalus membranaceus Bunge and their β-glucuronidase inhibitory activity

Historically, Astragalus membranaceus Bunge has been used as a beneficial medicinal plant, particularly in the Asian traditional medical systems, for the treatment of various human diseases such as stomach ulcers, diarrhea, and respiratory issues associated with phlegm. In this study, a phytochemical characterization of the aerial parts of A. membranaceusled to the isolation of 29 oleanane-type triterpenoid saponins, including 11 new compounds named astraoleanosides E–P (6–9, 13, 14, 18–22), as well as 18 known ones. The structures of these compounds were elucidated using nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry. Among them, astraoleanoside H (9) and cloversaponin III (15) demonstrated the most potent β-glucuronidase inhibitory activities, with IC50 values of 21.20 ± 0.75 and 9.05 ± 0.47 µM, respectively, compared to the positive control d-saccharic acid 1,4-lactone (IC50 = 20.62 ± 1.61 µM). Enzyme kinetics studies were then conducted to investigate the type of inhibition exhibited by these active compounds. In addition, the binding mechanism, key interactions, binding stability, and dynamic behavior of protein–ligand complexes were investigated through in silico approaches, such as molecular docking and molecular dynamics simulations. These findings highlight the promising potential of triterpenoid saponins from A. membranaceus as lead compounds for β-glucuronidase inhibitors, offering new possibilities for the development of therapeutic agents targeting various diseases where β-glucuronidase plays a crucial role.

Free drug percentage of moxidectin declines with increasing concentrations in the serum of marsupials

Moxidectin (MOX) is a macrocyclic lactone used to eliminate endo and ectoparasites in many mammalian species. It is notably the active ingredient of the anti-parasitic drug Cydectin®, manufactured by Virbac, and is frequently used to treat sarcoptic mange in Australian wildlife. Protein binding plays a significant role in the efficacy of a drug, as the unbound/free drug in plasma ultimately reflects the pharmacologically relevant concentration. This study aimed to investigate the free drug percentage of Moxidectin after in vitro spiking into the sera of four sarcoptic mange-susceptible Australian wildlife species; the koala (Phascolarctos cinereus), the bare-nosed wombat (Vombatus ursinus), the eastern grey kangaroo (Macropus giganteus), and the mountain brushtail possum (Trichosurus cunninghami). Three concentration points of MOX were tested for each individual: 20 pg/μL, 100 pg/μL and 500 pg/μL. Serum from five individuals of each species underwent an equilibrium dialysis followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The results showed an atypical concentration dependent binding across all species, where free drug percentage decreased as MOX concentration increased. In addition, wombats showed significantly lower free drug levels. These findings call for further research into the mechanisms of moxidectin protein binding to help understand MOX pharmacokinetics in marsupials.

Use of AlphaFold models to predict binding mechanisms of viral chemokine binding proteins

Use of AlphaFold models to predict binding mechanisms of viral chemokine binding proteins

Fly Ash-Based Na-X Zeolite Application in Separation Process of Bovine Serum Albumin from Aqueous Solution in the Presence of Organic Substances with Anionic Character.

The main purpose of the investigations was to explore the protein adsorption on porous materials, as well as to identify the mechanisms of protein attachment without and with other common environmental contaminants, such as drugs, polymers or surfactants. This study applied the Na-X zeolite for the adsorption of bovine serum albumin (BSA) from solutions with various pH values. Electrophoretic mobility measurements and potentiometric titrations were conducted in systems containing both protein and/or PAA (poly(acrylic acid) polymer/DCF (diclofenac) drug/SDS (sodium dodecyl sulfate) surfactant to investigate the protein binding mechanisms in the complex adsorbate systems. In addition, aggregate size and stability measurements were performed in the investigated systems. Based on the research results, it was possible to conclude that the protein adsorbed most preferably on the zeolite surface at a pH value close to its isoelectric point (pI) (102.15 mg/g), and protein adsorption was the lowest in the solutions with strongly alkaline (29.61 mg/g) or acidic (77.45 mg/g) pH values. Thus, the examined zeolitic material can be considered an effective adsorbent for protein removal from an aqueous solution.

Adsorption of SARS-CoV-2 Spike (N501Y) RBD to Human Angiotensin-Converting Enzyme 2 at a Lipid/Water Interface.

The receptor binding domain (RBD) of spike proteins plays a crucial role in the process of severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) attachment to the human angiotensin-converting enzyme 2 (ACE2). The N501Y mutation and later mutations introduced extra positive charges on the spike RBD and resulted in higher transmissibility, likely due to stronger binding with the highly negatively charged ACE2. Consequently, many studies have been devoted to understanding the molecular mechanism of spike protein binding with the ACE2 receptor. Most of the theoretical studies, however, have been done on isolated proteins. ACE2 is a transmembrane protein; thus, it is important to understand the interaction of spike proteins with ACE2 in a lipid matrix. In this study, the adsorption of ACE2 and spike (N501Y) RBD at a lipid/water interface was studied using the heterodyne-detected vibrational sum frequency generation (HD-VSFG) technique. The technique is a non-linear optical spectroscopy which measures vibrational spectra of molecules at an interface and provides information on their structure and orientation. It is found that ACE2 is effectively adsorbed at the positively charged 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP) lipid monolayer via electrostatic interactions. The adsorption of ACE2 at the DPTAP monolayer causes a reorganization of interfacial water (D2O) from the D-down to the D-up orientation, indicating that the originally positively charged DPTAP interface becomes negatively charged due to ACE2 adsorption. The negatively charged interface (DPTAP/ACE2) allows further adsorption of positively charged spike RBD. HD-VSFG spectra in the amide I region show differences for spike (N501Y) RBD adsorbed at D2O, DPTAP, and DPTAP/ACE2 interfaces. A red shift observed for the spectra of spike RBD/DPTAP suggests that spike RBD oligomers are formed upon contact with DPTAP lipids.

Odorant-Binding Protein 6 Contributes High Binding Affinity to Insecticides in a Parasitic Wasp Meteorus pulchricornis (Hymenoptera: Braconidae).

Meteorus pulchricornis is a preponderant parasitic wasp of various lepidopteran pests. The extensive application of broad-spectrum insecticides usually causes serious threats to the olfactory recognition of nontarget insects such as parasitoid wasps. However, the binding mechanism of odorant-binding proteins (OBPs) to insecticides in parasitoid wasps remains unknown. Herein, we find that the MpulOBP6 protein had a strong binding affinity to three insecticides (phoxim, chlorpyrifos, and chlorfenapyr). Results of computational simulations revealed that the hydrophobic interaction contributed by a mass of nonpolar amino acid residues was the primary driving force in the formation and stabilization of MpulOBP6-insecticide complexes. Among them, four residues (Met75, Val84, Phe121, and Pro122) and two residues (Val84 and Phe111) play an essential role in the binding of MpulOBP6 to phoxim and chlorfenapyr, respectively. Our findings could be instrumental to elucidate the effects of insecticide application toward the olfactory recognition of nontarget insects in the processes of agricultural production.

An Attempt of Seeking Favorable Binding Free Energy Prediction Schemes Considering the Entropic Effect on Fis-DNA Binding.

Protein-DNA binding mechanisms in a complex manner are essential for understanding many biological processes. Over the past decades, numerous experiments and calculations have analyzed the specificity of protein-DNA binding. However, the accuracy of binding free energy prediction for multi-base DNA systems still needs to be improved. Fis is a DNA-binding protein that regulates various transcription and recombination reactions. In the present work, we tested several methods of predict binding free energy based on this system to find a favorable prediction scheme and explore the binding mechanism of Fis protein and DNA. Two solvent models (explicit and implicit solvent models) were chosen for the dynamics process, and the predicted binding free energy was more accurate under the explicit solvent model. When different Poisson-Boltzmann/Generalized Born (PB/GB) models were tested for DNA force fields (BSC1 and OL15), it was found that the binding free energy predicted by the selected OL15 force field performed better and the correlation between predicted and experimental values was improved with the increasing interior dielectric constant (Dk). Finally, using Dk = 8, the GBOBC1 model combined with interaction entropy (IE), which was calculated for entropic contribution (GBOBC1_IE_8), was screened out for the binding free energy prediction and analysis of the Fis-DNA system, and the validity of the method was further verified by testing the Cren7-DNA system. By performing conformational analysis of the minor groove, it was found that mutation of the DNA central sequence A/T to C/G and deletion of the guanine 2-amino group would change the minor groove width and thus affect the formation of the major groove, altering the interaction and atomic contact between the protein and the major groove, thus changing the binding affinity of Fis and DNA. Hopefully, the series of tests in this work can shed some light on the related studies of protein and DNA systems.

Standard binding free-energy calculation of glycophorin a dimer in non-isotropic media sheds light on the transmembrane alpha-helices association mechanism.

The transmembrane (TM) protein domain recognition and association processes happening in lipid bilayers are pivotal for understanding membrane protein biogenesis. The most straightforward system to study the transmembrane protein binding mechanism is the glycophorin A (GpA) dimeric TM domain. The GpA is a glycoprotein ubiquitous to the human erythrocyte membrane, which forms a non-covalent dimer through the reversible association of its membrane-spanning domain sequences, creating a crossing angle between two alpha-helices. In our study, the membrane protein complex is primarily located in the phosphatidylcholine (POPC) lipid bilayer, surrounded by water. Applying a rigorous theoretical framework of the standard binding free-energy calculation that combines molecular dynamics and potential-of-mean-force calculations, the so-called “geometrical route”, we investigated this complex in non-isotropic media. Our analysis shows that at large separation distances, the helices are pushed together by the lipids, which triggers the interhelical interactions to occur. This observation is in agreement with the “two-stage” model of integral membrane protein folding. Additionally, we observed that the monomers, while reversibly associated, go through a meta-stable state, where the GpA alpha-helices form a different shape compared to the initial one. From the theoretical point of view, we suggest that for the membrane proteins in the non-isotropic environment, the standard binding free-energy estimation via the geometrical route should be analyzed from another perspective compared to those of protein-protein complexes in water media. We revised the separation PMF evaluation, assuming that the lipid bilayer forms a pseudo-cylindrical zone for the reversible separation of the dimer. The methodology employed herein successfully addresses the challenging transmembrane protein-protein binding problem while offering promising perspectives for the binding affinity characterization of different protein complexes in lipid bilayers.

Exploring the interaction of myricetin with human alpha-2-macroglobulin: biophysical and in-silico analysis.

Myricetin (MYR) is a bioactive secondary metabolite found in plants that is recognized for its nutraceutical value and is an essential constituent of various foods and beverages. It is reported to exhibit a plethora of activities, including antioxidant, antimicrobial, antidiabetic, anticancer, and anti-inflammatory. Alpha-2-macroglobulin (α2M) is a major plasma anti-proteinase that can inhibit proteinases of both human and non-human origin, regardless of their specificity and catalytic mechanism. Here, we explored the interaction of MYR-α2M using various biochemical and biophysical techniques. It was found that the interaction of MYR brings subtle change in its anti-proteolytic potential and thereby alters its structure and function, as can be seen from absorbance and fluorescence spectroscopy. UV spectroscopy of α2M in presence of MYR indicated the occurrence of hyperchromism, suggesting complex formation. Fluorescence spectroscopy reveals that MYR reduces the fluorescence intensity of native α2M with a shift in the wavelength maxima. At 318.15K, MYR binds to α2M with a binding constant of 2.4 × 103M-1, which indicates significant binding. The ΔG value was found to be - 7.56kcalmol-1 at 298.15K, suggesting the interaction to be spontaneous and thermodynamically favorable. The secondary structure of α2M does not involve any major change as was confirmed by CD analysis. The molecular docking indicates that Asp-146, Ser-172, Glu-174, and Tyr-180 were the key residues involved in α2M-MYR complex formation. This study contributes to our understanding of the function and mechanism of protein and flavonoid binding by providing a molecular basis of the interaction between MYR and α2M.

Unnatural helical peptidic foldamers as protein segment mimics.

Unnatural helical peptidic foldamers have attracted considerable attention owing to their unique folding behaviours, diverse artificial protein binding mechanisms, and promising applications in chemical, biological, medical, and material fields. Unlike the conventional α-helix consisting of molecular entities of native α-amino acids, unnatural helical peptidic foldamers are generally comprised of well-defined backbone conformers with unique and unnatural structural parameters. Their folded structures usually arise from unnatural amino acids such as N-substituted glycine, N-substituted-β-alanine, β-amino acid, urea, thiourea, α-aminoxy acid, α-aminoisobutyric acid, aza-amino acid, aromatic amide, γ-amino acid, as well as sulfono-γ-AA amino acid. They can exhibit intriguing and predictable three-dimensional helical structures, generally featuring superior resistance to proteolytic degradation, enhanced bioavailability, and improved chemodiversity, and are promising in mimicking helical segments of various proteins. Although it is impossible to include every piece of research work, we attempt to highlight the research progress in the past 10 years in exploring unnatural peptidic foldamers as protein helical segment mimics, by giving some representative examples and discussing the current challenges and future perspectives. We expect that this review will help elucidate the principles of structural design and applications of existing unnatural helical peptidic foldamers in protein segment mimicry, thereby attracting more researchers to explore and generate novel unnatural peptidic foldamers with unique structural and functional properties, leading to more unprecedented and practical applications.

Crystal structure of transcription factor TGA7 from Arabidopsis

TGA family of transcription factors play important roles in the systemic acquired resistance (SAR) in plants. In SAR, TGA7 binds to the activation sequence-1 (as-1) in the promoter region of SAR related genes and regulates their expressions in an NPR1 dependent manner. Despite its important roles in plant immunity, the molecular mechanism for DNA binding of TGA7 remains unclear. In the present work, we resolved the crystal structure of TGA7 dimers at a resolution of 2.06 Å, in which each monomer binds one molecule of palmitate. Further biochemical studies revealed that TGA7 specifically binds to the TGACG boxes of as-1 DNA in the form of homodimers, and it has specific requirements for the relative spacing and orientation of the two TGACG boxes. Moreover, we built a TGA7-DNA complex model and confirmed by site-directed mutagenesis that amino acid residue R109 in the DNA binding domain (DBD) of TGA7 is a key residue responsible for DNA recognition. Our work offers a good example for structural and functional studies of TGA proteins, and provides key clues to understand the DNA binding mechanism of TGA proteins in the SAR.

13C-NMR Chemical Shifts in 1,3-Benzazoles as a Tautomeric Ratio Criterion.

Benzimidazole is an important heterocyclic fragment, present in many biologically active compounds with a great variety of therapeutic purposes. Most of the benzimidazole activities are explained through the existence of 1,3-tautomeric equilibrium. As the binding affinity of each tautomer to a protein target depends on an established bioactive conformation, the effect of tautomers on the ligand protein binding mechanism is determinant. In this work, we searched and analyzed a series of reported 13C-NMR spectra of benzazoles and benzazolidine-2-thiones with the purpose of estimating their tautomeric equilibrium. Herein, several approaches to determine this problem are presented, which makes it a good initial introduction to the non-expert reader. This chemical shift difference and C4/C7 signals of benzimidazolidine-2-thione and 1-methyl-2-thiomethylbenzimidazole as references were used in this work to quantitatively calculate, in solution, the pyrrole–pyridine tautomeric ratio in equilibrium. The analysis will help researchers to correctly assign the chemical shifts of benzimidazoles and to calculate their intracyclic or exocyclic tautomeric ratio as well as mesomeric proportion in benzimidazoles.

"In Litero" Screening: Retrospective Evaluation of Clinical Evidence to Establish a Reference List of Human Chemical Respiratory Sensitizers.

Despite decades of investigation, test methods to identify respiratory sensitizers remain an unmet regulatory need. In order to support the evaluation of New Approach Methodologies in development, we sought to establish a reference set of low molecular weight respiratory sensitizers based on case reports of occupational asthma. In this context, we have developed an “in litero” approach to identify cases of low molecular weight chemical exposures leading to respiratory sensitization in clinical literature. We utilized the EPA-developed Sifter literature review tool to maximize the retrieval of publications relevant to respiratory effects in humans for each chemical in a list of chemicals suspected of inducing respiratory sensitization. The literature retrieved for each of these candidate chemicals was sifted to identify relevant case reports and studies, and then evaluated by applying defined selection criteria. Clinical diagnostic criteria were defined around exposure history, respiratory effects, and specific immune response to conclusively demonstrate occupational asthma as a result of sensitization, rather than irritation. This approach successfully identified 28 chemicals that can be considered as human respiratory sensitizers and used to evaluate the performance of NAMs as part of a weight of evidence approach to identify novel respiratory sensitizers. Further, these results have immediate implications for the development and refinement of predictive tools to distinguish between skin and respiratory sensitizers. A comparison of the protein binding mechanisms of our identified “in litero” clinical respiratory sensitizers shows that acylation is a prevalent protein binding mechanism, in contrast to Michael addition and Schiff base formation common to skin sensitizers. Overall, this approach provides an exemplary method to evaluate and apply human data as part of the weight of evidence when establishing reference chemical lists.

Interaction and binding mechanism of lipid oxidation products to sturgeon myofibrillar protein in low temperature vacuum heating conditions: Multispectroscopic and molecular docking approaches.

In this work, the binding mechanism of myofibrillar protein (MP) with malondialdehyde and 4-hydroxy-2-nonenal under low temperature vacuum heating was investigated via multispectroscopic and molecular docking. The results showed that binding interaction and increasing temperature caused significant changes in the conformations as well as a decrease in the value of protein intrinsic fluorescence, surface hydrophobicity, and fluorescence excitation-emission matrix spectra. Furthermore, the decrease in α-helix and β-turn, increase in β-sheet and a random coil of MP, imply the MP molecules to be more unfolded. Isothermal titration calorimetry and molecular docking results showed that main driving force for binding with MP was hydrogen bond, and the binding ability of malondialdehyde was superior to that of 4-hydroxy-2-nonenal. Moreover, increasing the heating temperature was beneficial to the binding reaction and intensified the conformational transition of MP. These results will provide a reference for further studies on the lipid and protein interaction of sturgeon.

A litmus test for classifying recognition mechanisms of transiently binding proteins

Partner recognition in protein binding is critical for all biological functions, and yet, delineating its mechanism is challenging, especially when recognition happens within microseconds. We present a theoretical and experimental framework based on straight-forward nuclear magnetic resonance relaxation dispersion measurements to investigate protein binding mechanisms on sub-millisecond timescales, which are beyond the reach of standard rapid-mixing experiments. This framework predicts that conformational selection prevails on ubiquitin’s paradigmatic interaction with an SH3 (Src-homology 3) domain. By contrast, the SH3 domain recognizes ubiquitin in a two-state binding process. Subsequent molecular dynamics simulations and Markov state modeling reveal that the ubiquitin conformation selected for binding exhibits a characteristically extended C-terminus. Our framework is robust and expandable for implementation in other binding scenarios with the potential to show that conformational selection might be the design principle of the hubs in protein interaction networks.