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We study the effect of the peptide QAKTFLDKFNHEAEDLFYQ on the kinetics of the SARS-CoV-2 spike protein S1 binding to angiotensin-converting enzyme 2 (ACE2), with the aim to characterize the interaction mechanism of the SARS-CoV2 virus with its host cell. This peptide corresponds to the sequence 24-42 of the ACE2 α1 domain, which marks the binding site for the S1 protein. The kinetics of S1-ACE2 complex formation was measured in the presence of various concentrations of the peptide using bio-layer interferometry. Formation of the S1-ACE2 complex was inhibited by the peptide in cases where it was preincubated with S1 protein before the binding experiment. The kinetic analysis of S1-ACE2 complex dissociation revealed that preincubation stabilized this complex, and this effect was dependent on the peptide concentration as well as the preincubation time. The results point to the formation of the ternary complex of S1 with ACE2 and the peptide. This is possible in the presence of another binding site for the S1 protein beside the receptor-binding domain for ACE2, which binds the peptide QAKTFLDKFNHEAEDLFYQ. Therefore, we conducted computational mapping of the S1 protein surface, revealing two additional binding sites located at some distance from the main receptor-binding domain on S1. We suggest the possibility to predict and test the short protein derived peptides for development of novel strategies in inhibiting virus infections., Supplementary Information: The online version contains supplementary material available at 10.1007/s10989-021-10324-7., Competing Interests: Conflict of interestThe authors declare no competing interests., (© The Author(s), under exclusive licence to Springer Nature B.V. 2021.)

Two of the most popular positron emission tomography (PET) tracers, [ 11 C]PE2I and [ 18 F]FE-PE2I, used to quantify dopamine transporters (DAT), display dissimilar kinetic behavior in in vivo assays. This difference can be explained by comparing values of kinetic rate constants, which characterize interaction of these tracers with DAT sites in vitro . At the same time, this kinetic analysis showed that the overall binding mechanism is similar for these two tracers and includes a fast step of complex formation followed by a slow isomerization step of this complex. Comparison with previous PE2I data revealed that isomerization of the DAT complex with PE2I occurs three times faster than in the case of FE-PE2I, which leads to the slower onset of peak specific binding of the former tracer in the DAT-rich regions. Therefore, ligands with slower isomerization on-rate, including [ 18 F]FE-PE2I, seem to be better tracers in vivo , and their properties can be predicted in vitro ., Competing Interests: The authors declare no competing financial interest.

The allosteric influence of adenosine triphosphate (ATP) on the binding effectiveness of a series of peptide inhibitors with the catalytic subunit of 3'5'-cyclic adenosine monophosphate dependent protein kinase was investigated, and the dependence of this effect on peptide structure was analyzed. The allosteric effect was calculated as ratio of peptide binding effectiveness with the enzyme-ATP complex and with the free enzyme, quantified by the competitive inhibition of the enzyme in the presence of ATP excess, and by the enzyme-peptide complex denaturation assay, respectively It was found that the principle "better binding-stronger allostery" holds for interactions of the studied peptides with the enzyme, indicating that allostery and peptide binding with the free enzyme are governed by the same specificity pattern. This means that the allosteric regulation does not include new ligand-protein interactions, but changes the intensity (strength) of the interatomic forces that govern the complex formation in the case of each individual ligand. We propose that the allosteric regulation can be explained by the alteration of the intrinsic dynamics of the protein by ligand binding, and that this phenomenon, in turn, modulates the ligand off-rate from its binding site as well as the binding affinity. The positive allostery could therefore be induced by a reduction in the enzyme's overall intrinsic dynamics.

Fluorescence spectroscopy was used to differentiate between different states of acrylodan-labeled cAMP-dependent protein kinase catalytic subunits in urea, guanidine hydrochloride and 3-(N-morpholino)propanesulfonic acid solutions, by measuring changes in the emission spectrum of the protein-coupled dye, which is very sensitive to its microenvironment. Decomposition of the observed fluorescence spectra by a parameterized log-normal distribution function allowed the resolution of overlapping spectral bands and revealed the formation of three distinct protein states, denominated as native, denatured and unfolded structures. At low denaturant concentrations the formation of the denatured form from the native protein was observed, and this process was characterized by a blue-shift of the fluorescence spectrum of acrylodan, indicating that the dye was transferred into some water-deficit hydrophobic environment inside the protein molecule. Therefore, formation of a "dry molten globule" structure could be suggested in state. At high denaturant concentrations a red-shift of the emission spectrum of the protein-coupled probe was observed indicating significant extrusion of the dye molecule into water environment as a result of the unfolding of the protein structure.

Structure of the cAMP-dependent protein kinase catalytic subunit, where the asparagine residue 326 was replaced with acrylodan-cystein conjugate to implement this fluorescence reporter group into the enzyme, was modeled by molecular dynamics (MD) method and the positioning of the dye molecule in protein structure was characterized at temperatures 300K, 500K and 700K. It was found that the acrylodan moiety, which fluorescence is very sensitive to solvating properties of its microenvironment, was located on the surface of the native protein at 300K that enabled its partial solvation with water. At high temperatures the protein structure significantly changed, as the secondary and tertiary structure elements were unfolded and these changes were sensitively reflected in positioning of the dye molecule. At 700K complete unfolding of the protein occurred and the reporter group was entirely expelled into water. However, at 500K an intermediate of the protein unfolding process was formed, where the fluorescence reporter group was directed towards the protein interior and buried in the core of the formed molten globule state. This different positioning of the reporter group was in agreement with the two different shifts of emission spectrum of the covalently bound acrylodan, observed in the unfolding process of the protein., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Mood disorders are associated with alterations in serotonergic system, deficient BDNF (brain-derived neurotrophic factor) signaling and abnormal synaptic plasticity. Increased degradation and reduced functions of NCAM (neural cell adhesion molecule) have recently been associated with depression and NCAM deficient mice show depression-related behavior and impaired learning. The aim of the present study was to investigate potential changes in serotonergic and BDNF systems in NCAM knock-out mice. Serotonergic nerve fiber density and SERT (serotonin transporter) protein levels were robustly reduced in the hippocampus, prefrontal cortex and basolateral amygdala of adult NCAM(-)(/-) mice. This SERT reduction was already evident during early postnatal development. [(3)H]MADAM binding experiments further demonstrated reduced availability of SERT in cell membranes of NCAM(-)(/-) mice. Moreover, the levels of serotonin and its major metabolite 5-HIAA were down regulated in the brains of NCAM(-)(/-) mice. NCAM(-)(/-) mice also showed a dramatic reduction in the BDNF protein levels in the hippocampus and prefrontal cortex. This BDNF deficiency was associated with reduced phosphorylation of its receptor TrkB. Importantly, chronic administration of antidepressant amitriptyline partially or completely restored these changes in serotonergic and BDNF systems, respectively. In conclusion, NCAM deficiency lead to prominent and persistent abnormalities in brain serotonergic and BDNF systems, which likely contributes to the behavioral and neurobiological phenotype of NCAM(-/-) mice., (Copyright © 2015 Elsevier B.V. and ECNP. All rights reserved.)

The thermal stabilities of the rat and mouse dopamine transporter (DAT) proteins were studied within the temperature range of 0-37°C. The inactivation of the protein was followed by monitoring changes in radioligand-specific binding. We found that the process followed a rate equation with first-order kinetics and was characterized by having a single rate constant k inact. The activation energies (E a) that were calculated from the Arrhenius plots (ln k inact vs. 1/T) were 43 ± 5 and 45 ± 6 kJ/mol for the rat (rDAT) and mouse (mDAT) transporters, respectively, and 44 ± 7 kJ/mol for rDAT from PC-6.3 cell line. These E a values were similar to the E a values of thermal inactivation of the muscarinic receptor from rat brain cortex and to the thermal inactivation of other transmembrane proteins. However, all of these activation energy values were significantly lower than the E a values for soluble single-subunit proteins of similar size. These results therefore suggest that the thermal stability of transmembrane proteins may be governed to a significant extent by cell membrane properties and by interactions between the membrane components and the protein. In contrast, the stability of soluble proteins seems to be mostly governed by protein structure and size, which determine the sum of the stabilizing intramolecular interactions within the protein molecule. It is therefore not surprising that cell membrane properties and composition may have significant effects on the functional properties of transmembrane proteins.