Alignment of helical membrane protein sequences using AlignMe

Few sequence alignment methods have been designed specifically for integral membrane proteins, even though these important proteins have distinct evolutionary and structural properties that might affect their alignments. Existing approaches typically consider membrane-related information either by using membrane-specific substitution matrices or by assigning distinct penalties for gap creation in transmembrane and non-transmembrane regions. Here, we ask whether favoring matching of predicted transmembrane segments within a standard dynamic programming algorithm can improve the accuracy of pairwise membrane protein sequence alignments. We tested various strategies using a specifically designed program called AlignMe. An updated set of homologous membrane protein structures, called HOMEP2, was used as a reference for optimizing the gap penalties. The best of the membrane-protein optimized approaches were then tested on an independent reference set of membrane protein sequence alignments from the BAliBASE collection. When secondary structure (S) matching was combined with evolutionary information (using a position-specific substitution matrix (P)), in an approach we called AlignMePS, the resultant pairwise alignments were typically among the most accurate over a broad range of sequence similarities when compared to available methods. Matching transmembrane predictions (T), in addition to evolutionary information, and secondary-structure predictions, in an approach called AlignMePST, generally reduces the accuracy of the alignments of closely-related proteins in the BAliBASE set relative to AlignMePS, but may be useful in cases of extremely distantly related proteins for which sequence information is less informative. The open source AlignMe code is available at https://sourceforge.net/projects/alignme​/, and at http://www.forrestlab.org, along with an online server and the HOMEP2 data set

Computational and Experimental Studies of Substrate Binding, Conformational Change and Importance of the Trimeric State in the Glycine Betaine Transporter BetP

The glycine betaine/sodium symporter BetP responds to changes in external osmolality by regulation of its transport activity. A recent X-ray structure of BetP confirms that it is a homotrimer and in this structure each protomer adopts an identical conformation, in which the pathway is occluded from both sides. Despite the availability of a wealth of experimental data for BetP, the structures of the alternate states (e.g., open to the outside of the cell), molecular mechanisms of substrate and Na⁺ binding and transport, as well as the functional implications of the trimeric state remain poorly understood. To address these questions, we carried out computational studies using a range of techniques to derive hypotheses that were then tested experimentally. First, to identify structural features of the alternate states, we developed a procedure for flexible fitting of the X-ray structure of BetP into a lower-resolution cryo-EM map of BetP in a more native lipid environment, in which the three protomers have different conformations. These results suggest that: (i) the protomers adopt distinct conformational states relevant to the transport cycle; and (ii) there is conformational coupling between the protomers. Second, we performed all-atom molecular dynamics simulations and in silico alanine scanning of BetP trimers in order to identify interface residues crucial for maintaining the trimeric state. Mutations of these residues to alanine were introduced experimentally revealing that the isolated monomers are functional, and that the trimeric state is important for the regulation and higher activity of the protein. Finally, using molecular modeling and biochemical experiments we identified two Na⁺ binding sites in BetP that could not be resolved in the 3.35 Å resolution X-ray structure

Bastroviruses (<i>Astroviridae</i>): genetic diversity and potential impact on human and animal health

Introduction. Bastroviruses were discovered in the Netherlands in 2016 in human stool samples and show partial genetic similarities to astroviruses and hepatitis E viruses. Their association with disease onset has not yet been established. Materials and methods. Metagenomic sequencing of fecal samples of Nyctalus noctula bats collected in the Russian Federation in 2023 was performed. Two almost complete genomes of bastroviruses were assembled. The zoonotic potential of these viruses was assessed using machine learning methods, their recombination was studied, and phylogenetic trees were constructed. Results. A nearly complete bastrovirus genome was de novo assembled in one of the samples, and it was used to assemble another genome in another sample. The zoonotic potential of the virus from one of these samples was estimated as high. The existence of recombination between structural and non-structural polyproteins was demonstrated. Conclusion. Two bastrovirus genomes were assembled, phylogenetic and recombination analyses were performed, and the zoonotic potential was evaluated

GoLoco motif proteins binding to Gαi1: insights from molecular simulations

Molecular dynamics simulations, computational alanine scanning and sequence analysis were used to investigate the structural properties of the Gαi1/GoLoco peptide complex. Using these methodologies, binding of the GoLoco motif peptide to the Gαi1 subunit was found to restrict the relative movement of the helical and catalytic domains in the Gαi1 subunit, which is in agreement with a proposed mechanism of GDP dissociation inhibition by GoLoco motif proteins. In addition, the results provide further insights into the role of the “Switch IV” region located within the helical domain of Gα, the conformation of which might be important for interactions with various Gα partners

Rapid diagnostics of novel coronavirus infection by loop-mediated isothermal amplification

This review presents the basic principles of application of the loop-mediated isothermal amplification (LAMP) reaction for the rapid diagnosis of coronavirus infection caused by SARS-CoV-2. The basic technical details of the method, and the most popular approaches of specific and non-specific detection of amplification products are briefly described. We also discuss the first published works on the use of the method for the detection of the nucleic acid of the SARS-CoV-2 virus, including those being developed in the Russian Federation. For commercially available and published LAMP-based assays, the main analytical characteristics of the tests are listed, which are often comparable to those based on the method of reverse transcription polymerase chain reaction (RT-PCR), and in some cases are even superior. The advantages and limitations of this promising methodology in comparison to other methods of molecular diagnostics, primarily RT-PCR, are discussed, as well as the prospects for the development of technology for the detection of other infectious agents

G protein inactive and active forms investigated by simulation methods

Molecular dynamics and computational alanine scanning techniques have been used to investigate G proteins in their inactive state (the Gαi1β1γ2 heterotrimer) as well as in their empty and monomeric active states (Gαi1 subunit). We find that: (i) the residue Q204 of Gαi1 plays a key role for binding Gβ1γ2 and is classified among the most relevant in the interaction with a key cellular partner, the so- called regulator of G protein signaling protein. The mutation of this residue to L, which is observed in a variety of diseases, provides still fair stability to the inactive state because of the formation of van der Waals interactions. (ii) The empty state turns out to adopt some structural features of the active one, including a previously unrecognized rearrangement of a key residue (K46). (iii) The so-called Switch IV region increases its mobility on passing from the empty to the active state, and, even more, to the inactive state. Such change in mobility could be important for its several structural and functional roles. (iv) A large scale motion of the helical domain in the inactive state might be important for GDP release upon activation by GPCR, consistently with experimental data

The role of trimerization in the osmoregulated betaine transporter BetP

The osmoregulated betaine transporter BetP is a stable trimer. Structural studies have shown that individual protomers can adopt distinct transport conformations, implying a functional role for the trimeric state in transport, although the role of trimerization in regulation is not yet understood. We designed putative monomeric mutants by molecular-dynamics simulations and in silico alanine-scanning mutagenesis. Several mutants including BetP-W101A/T351A were monomeric in detergent as well as in the membrane, as shown by blue native gel electrophoresis, crosslinking and electron microscopy. This monomeric form retains the ability to accumulate betaine, but is no longer regulated by hyperosmotic shock