Viruses are the most abundant life form and infect practically all organisms. Consequently, these obligate parasites are a major cause of human suffering and economic loss. Rossmann‐like fold is the most populated fold among α/β‐folds in the Protein Data Bank and proteins containing Rossmann‐like fold constitute 22% of all known proteins 3D structures. Thus, analysis of viral proteins containing Rossmann‐like domains could provide an understanding of viral biology and evolution as well as could propose possible targets for antiviral therapy. We provide functional and evolutionary analysis of viral proteins containing a Rossmann‐like fold found in the evolutionary classification of protein domains (ECOD) database developed in our lab. We identified 81 protein families of bacterial, archeal, and eukaryotic viruses in light of their evolution‐based ECOD classification and Pfam taxonomy. We defined their functional significance using enzymatic EC number assignments as well as domain‐level family annotations. 相似文献
Several studies based on the known three-dimensional (3-D) structures of proteins show that two homologous proteins with insignificant
sequence similarity could adopt a common fold and may perform same or similar biochemical functions. Hence, it is appropriate
to use similarities in 3-D structure of proteins rather than the amino acid sequence similarities in modelling evolution of
distantly related proteins. Here we present an assessment of using 3-D structures in modelling evolution of homologous proteins.
Using a dataset of 108 protein domain families of known structures with at least 10 members per family we present a comparison
of extent of structural and sequence dissimilarities among pairs of proteins which are inputs into the construction of phylogenetic
trees. We find that correlation between the structure-based dissimilarity measures and the sequence-based dissimilarity measures
is usually good if the sequence similarity among the homologues is about 30% or more. For protein families with low sequence
similarity among the members, the correlation coefficient between the sequence-based and the structure-based dissimilarities
are poor. In these cases the structure-based dendrogram clusters proteins with most similar biochemical functional properties
better than the sequence-similarity based dendrogram. In multi-domain protein families and disulphide-rich protein families
the correlation coefficient for the match of sequence-based and structure-based dissimilarity (SDM) measures can be poor though
the sequence identity could be higher than 30%. Hence it is suggested that protein evolution is best modelled using 3-D structures
if the sequence similarities (SSM) of the homologues are very low. 相似文献
We compare side chain prediction and packing of core and non‐core regions of soluble proteins, protein‐protein interfaces, and transmembrane proteins. We first identified or created comparable databases of high‐resolution crystal structures of these 3 protein classes. We show that the solvent‐inaccessible cores of the 3 classes of proteins are equally densely packed. As a result, the side chains of core residues at protein‐protein interfaces and in the membrane‐exposed regions of transmembrane proteins can be predicted by the hard‐sphere plus stereochemical constraint model with the same high prediction accuracies (>90%) as core residues in soluble proteins. We also find that for all 3 classes of proteins, as one moves away from the solvent‐inaccessible core, the packing fraction decreases as the solvent accessibility increases. However, the side chain predictability remains high (80% within ) up to a relative solvent accessibility, , for all 3 protein classes. Our results show that % of the interface regions in protein complexes are “core”, that is, densely packed with side chain conformations that can be accurately predicted using the hard‐sphere model. We propose packing fraction as a metric that can be used to distinguish real protein‐protein interactions from designed, non‐binding, decoys. Our results also show that cores of membrane proteins are the same as cores of soluble proteins. Thus, the computational methods we are developing for the analysis of the effect of hydrophobic core mutations in soluble proteins will be equally applicable to analyses of mutations in membrane proteins. 相似文献
Cell‐free protein synthesis (CFPS) is a valuable method for the fast expression of difficult‐to‐express proteins as well as posttranslationally modified proteins. Since cell‐free systems circumvent possible cytotoxic effects caused by protein overexpression in living cells, they significantly enlarge the scale and variety of proteins that can be characterized. We demonstrate the high potential of eukaryotic CFPS to express various types of membrane proteins covering a broad range of structurally and functionally diverse proteins. Our eukaryotic cell‐free translation systems are capable to provide high molecular weight membrane proteins, fluorescent‐labeled membrane proteins, as well as posttranslationally modified proteins for further downstream analysis. 相似文献
We have classified 865 sequences of EF‐hand proteins from five proteomes into 156 subfamilies. These subfamilies were put into six groups. Evolutionary relationships among subfamilies and groups were analyzed from the inferred ancestral sequence for each subfamily. CTER, CPV, and PEF groups arose from a common EF‐lobe (pair of adjacent EF‐hands). They have two or more EF‐lobes; the relative positions of their EF‐lobes differ from each other. Comparisons of the ancestral sequences and the inferred structures of the EF‐lobes of these groups indicate that the mutual positions of EF‐lobes were established soon after divergence of an EF‐lobe for each group and before the duplication and fusion of EF‐lobe gene(s). These ancestral sequences reveal that some subfamilies in low similarity and isolated groups did not evolve from the EF‐lobe precursor, even if their conformations are similar to the canonical EF‐hand. This is an example of convergent evolution. 相似文献
Although the lifestyles and infection strategies of plant pathogens are diverse, a prevailing feature is the use of an arsenal of secreted proteins, known as effectors, which aid in microbial infection. In the case of eukaryotic filamentous pathogens, such as fungi and oomycetes, effector proteins are typically dissimilar, at the protein sequence level, to known protein families and functional domains. Consequently, we currently have a limited understanding of how fungal and oomycete effectors promote disease. Protein biochemistry and structural biology are two methods that can contribute greatly to the understanding of protein function. Both techniques are dependent on obtaining proteins that are pure and functional, and generally require the use of heterologous recombinant protein expression systems. Here, we present a general scheme and methodology for the production and characterization of small cysteine‐rich (SCR) effectors utilizing Escherichia coli expression systems. Using this approach, we successfully produced cysteine‐rich effectors derived from the biotrophic fungal pathogen Melampsora lini and the necrotrophic fungal pathogen Parastagonospora nodorum. Access to functional recombinant proteins facilitated crystallization and functional experiments. These results are discussed in the context of a general workflow that may serve as a template for others interested in understanding the function of SCR effector(s) from their plant pathogen(s) of interest. 相似文献
The interactions of Met and Cys with other amino acid side chains have received little attention, in contrast to aromatic–aromatic, aromatic–aliphatic or/and aliphatic–aliphatic interactions. Precisely, these are the only amino acids that contain a sulfur atom, which is highly polarizable and, thus, likely to participate in strong Van der Waals interactions. Analysis of the interactions present in membrane protein crystal structures, together with the characterization of their strength in small‐molecule model systems at the ab‐initio level, predicts that Met–Met interactions are stronger than Met–Cys ≈ Met–Phe ≈ Cys–Phe interactions, stronger than Phe–Phe ≈ Phe–Leu interactions, stronger than the Met–Leu interaction, and stronger than Leu–Leu ≈ Cys–Leu interactions. These results show that sulfur‐containing amino acids form stronger interactions than aromatic or aliphatic amino acids. Thus, these amino acids may provide additional driving forces for maintaining the 3D structure of membrane proteins and may provide functional specificity. 相似文献
β‐Sheets are quite frequent in protein structures and are stabilized by regular main‐chain hydrogen bond patterns. Irregularities in β‐sheets, named β‐bulges, are distorted regions between two consecutive hydrogen bonds. They disrupt the classical alternation of side chain direction and can alter the directionality of β‐strands. They are implicated in protein‐protein interactions and are introduced to avoid β‐strand aggregation. Five different types of β‐bulges are defined. Previous studies on β‐bulges were performed on a limited number of protein structures or one specific family. These studies evoked a potential conservation during evolution. In this work, we analyze the β‐bulge distribution and conservation in terms of local backbone conformations and amino acid composition. Our dataset consists of 66 times more β‐bulges than the last systematic study (Chan et al. Protein Science 1993, 2:1574–1590). Novel amino acid preferences are underlined and local structure conformations are highlighted by the use of a structural alphabet. We observed that β‐bulges are preferably localized at the N‐ and C‐termini of β‐strands, but contrary to the earlier studies, no significant conservation of β‐bulges was observed among structural homologues. Displacement of β‐bulges along the sequence was also investigated by Molecular Dynamics simulations. 相似文献
The centre of the human lens, which is composed of proteins that were synthesized prior to birth, is an ideal model for the evaluation of long‐term protein stability and processes responsible for the degradation of macromolecules. By analysing the sequences of peptides present in human lens nuclei, characteristic features of intrinsic protein instability were determined. Prominent was the cleavage on the N‐terminal side of serine residues. Despite accounting for just 9% of the amino acid composition of crystallins, peptides with N‐terminal Ser represented one‐quarter of all peptides. Nonenzymatic cleavage at Ser could be reproduced by incubating peptides at elevated temperatures. Serine residues may thus represent susceptible sites for autolysis in polypeptides exposed to physiological conditions over a period of years. Once these sites are cleaved, other chemical processes result in progressive removal or ‘laddering’ of amino acid residues from newly exposed N‐ and C‐termini. As N‐terminal Ser peptides originated from several crystallins with unrelated sequences, this may represent a general feature of long‐lived proteins. 相似文献
Drug delivery to the brain for the treatment of pathologies with a CNS component is a significant clinical challenge. P‐glycoprotein (PgP), a drug efflux pump in the endothelial cell membrane, is a major factor in preventing therapeutics from crossing the blood‐brain barrier (BBB). Identifying PgP regulatory mechanisms is key to developing agents to modulate PgP activity. Previously, we found that PgP trafficking was altered concomitant with increased PgP activity and disassembly of high molecular weight PgP‐containing complexes during acute peripheral inflammatory pain. These data suggest that PgP activity is post‐translationally regulated at the BBB. The goal of the current study was to identify proteins that co‐localize with PgP in rat brain microvessel endothelial cell membrane microdomains and use the data to suggest potential regulatory mechanisms. Using new density gradients of microvessel homogenates, we identified two unique pools (1,2) of PgP in membrane fractions. Caveolar constituents, caveolin1, cavin1, and cavin2, co‐localized with PgP in these fractions indicating the two pools contained caveolae. A chaperone (Hsc71), protein disulfide isomerase and endosomal/lysosomal sorting proteins (Rab5, Rab11a) also co‐fractionated with PgP in the gradients. These data suggest signaling pathways with a potential role in post‐translational regulation of PgP activity at the BBB.
The circular dichroism spectra of membrane suspensions are distorted by differential light scattering and absorption flattening effects, which arise as a consequence of the large size of the membrane particles relative to the wavelength of light and the high concentration of proteins in the membranes. In this paper, the consequences of these phenomena on the protein spectra of large membrane particles are discussed, and methods for eliminating them are examined. The distortions due to differential light scattering are relatively small in membrane systems, and can be compensated for by use of a large detector acceptance angle geometry. Several methods for correcting for differential flattening, which introduces a substantial distortion, have been evaluated, and a new method, the flattening quotient approach, which produces by far the best results, is described. Since the secondary structures calculated from circular dichroism spectra are highly dependent on accurate spectral shape and magnitude, this method for correcting the spectra may find general application in circular dichroism studies of membrane proteins. 相似文献