Synaptonemal Complex Proteins: Specific Proteins of Meiotic Chromosomes

The review considers proteins of the synaptonemal complex (SC), a specific structure formed between homologous chromosomes in maturing germline cells during meiotic prophase I. The structure and functions are described for proteins that form ultrastructural SC elements in mammals, in yeast, and in higher plants. The roles of cohesins and of the SC proteins in meiotic sister-chromatid cohesion are considered. Though still scarce, data are summarized on the SC self-assembly and dissociation and on the molecular composition of SC-associated recombination nodules, which provide a compartment for meiotic recombination enzymes. The accumulating data on the SC molecular components and on their structure, properties, and interactions improve the understanding of the SC function.     

Synaptonemal complex damage as a measure of genotoxicity at meiosis

Synaptonemal complex aberrations can provide a sensitive measure of chemical-specific alterations to meiotic chromosomes. Mitomycin C, cyclophosphamide, amsacrine, ellipticine, colchicine, vinblastine sulfate, and cis-platin exposures in mice have been shown to cause various patterns of synaptonemal complex structural damage and synaptic irregularity. These effects are suggestive of abnormal homologue pairing/synapsis/recombination effects which, theoretically, could be implicated in mechanisms leading to aneuploidy and other potentially heritable chromosomal disorders.     

Msh4 and Msh5 Function in SC-Independent Chiasma Formation During the Streamlined Meiosis of Tetrahymena

ZMM proteins have been defined in budding yeast as factors that are collectively involved in the formation of interfering crossovers (COs) and synaptonemal complexes (SCs), and they are a hallmark of the predominant meiotic recombination pathway of most organisms. In addition to this so-called class I CO pathway, a minority of crossovers are formed by a class II pathway, which involves the Mus81-Mms4 endonuclease complex. This is the only CO pathway in the SC-less meiosis of the fission yeast. ZMM proteins (including SC components) were always found to be co-occurring and hence have been regarded as functionally linked. Like the fission yeast, the protist Tetrahymena thermophila does not possess a SC, and its COs are dependent on Mus81-Mms4. Here we show that the ZMM proteins Msh4 and Msh5 are required for normal chiasma formation, and we propose that they have a pro-CO function outside a canonical class I pathway in Tetrahymena. Thus, the two-pathway model is not tenable as a general rule.     

PFDB: A Protein Families DataBase for Macintosh Computers. The Effectiveness of Its Organization in Searching for Protein Similarity

A protein sequence database (PFDB) containing about 11,000 entries is available for Macintosh computers. The PFDB can be easily updated by importing sequences from the PIR collection through the internet. The most important feature of the database is its organization in families of closely related sequences, each family being characterized by its average dipeptide composition [Petrilli (1993), Comput. Appl. Biosci. 2, 89–93]. This allows one to perform a rapid and sensitive protein similarity search by comparing the precalculated family dipeptide composition with that of the query sequence by a linear correlation coefficient. An example of an application in which a new protein was classsified by using a sequence of a fragment just 19 residues long is reported.     

Presence of a Fibronectin Type III Domain in a Plant Protein

A hidden Markov model (HMM) approach was used to identify potential candidates in sequence databases for fibronectin type III domains in plants, a kingdom heretofore bereft of these structures. Fortuitously, one of the proteins uncovered had already had a crystal structure published, allowing direct structural confirmation of the existence of this domain in plants. Received: 19 December 1997 / Accepted: 23 December 1997     

融合标签技术在膜蛋白结构研究中的应用

膜蛋白高级结构的研究包括不同的层次,即膜蛋白拓扑学结构的研究、利用核磁共振技术和蛋白质晶体衍射技术对三维结构的研究,以及膜蛋白复合体的研究。在研究过程中,如果能够基于膜蛋白的拓扑学结构预测,选择合适的蛋白质或多肽融合标签,利用基因融合技术在基因水平上对膜蛋白进行改造,可以产生含有融合标签的重组膜蛋自,不仅具有原有膜蛋白的功能活性,还具有融合标签所特有的生理生化特性,将会极大地促进膜蛋白结构和功能的研究。我们就目前膜蛋白结构研究中所涉及的融合标签技术及其应用策略和所取得的进展做一简述。     

Synthetic Peptide Fragments as Probes for Structure Determination of Potassium Ion-Channel Proteins

Potassium channels are a diverse class of transmembrane proteins that are responsible for diffusion of potassium ion across cell membranes. The lack of large quantities of these proteins from natural sources, is a major hindrance in their structural characterization using biophysical techniques. Synthetic peptide fragments corresponding to functionally important domains of these proteins provide an attractive approach towards characterizing the structural organization of these ion-channels. Conformational properties of peptides from three different potassium channels (Shaker, ROMK1 and minK) have been characterized in aqueous media, organic solvents and in phospholipid membranes. Techniques used for these studies include FTIR, CD and 2D-NMR spectroscopy. FTIR spectroscopy has been a particularly valuable tool for characterizing the folding of the ion-channel peptides in phospholipid membranes; the three different types of potassium channels all share a common transmembrane folding pattern that is composed of a predominantly -helical structure. There is no evidence to suggest the presence of any significant -sheet structure. These results are in excellent agreement with the crystal structure of a bacterial potassium channel (Doyle, D. A. et al. (1998) Science 280:69–77), and suggest that all potassium channel proteins may share a common folding motif where the ion-channel structure is constructed entirely from -helices.     

Design of Chimeric Proteins on the Basis of a Pentameric Superhelical Fragment of Human Cartilage Oligomeric Matrix Protein: I. The Properties of a Hybrid Containing the Immunodominant Domain of the Circumsporozoite Protein of Plasmodium falciparum

The oligomeric recombinant protein DB-1 containing the immunodominant sites of the circumsporozoite protein of Plasmodium falciparum and tetanus toxin was constructed to optimize the schemes of presentation of B-cell epitopes during vaccination with chimeric proteins without the use of adjuvants. A fragment of the pentameric coiled-coil human cartilage oligomeric matrix protein was used as an oligomerization matrix. The expression of the protein in Escherichia coli cells was studied, a method for its purification was developed, and it was biochemically characterized.     

Structural relationships of homologous proteins as a fundamental principle in homology modeling

Protein structure prediction is based mainly on the modeling of proteins by homology to known structures; this knowledgebased approach is the most promising method to date. Although it is used in the whole area of protein research, no general rules concerning the quality and applicability of concepts and procedures used in homology modeling have been put forward yet. Therefore, the main goal of the present work is to provide tools for the assessment of accuracy of modeling at a given level of sequence homology. A large set of known structures from different conformational and functional classes, but various degrees of homology was selected. Pairwise structure superpositions were performed. Starting with the definition of the structurally conserved regions and determination of topologically correct sequence alignments, we correlated geometrical properties with sequence homology (defined by the 250 PAM Dayhoff Matrix) and identity. It is shown that both the topological differences of the protein backbones and the relative positions of corresponding side chains diverge with decreasing sequence identity. Below 50% identity, the deviation in regions that are structurally not conserved continually increases, thus implying that with decreasing sequence identity modeling has to take into account more and more structurally diverging loop regions that are difficult to predict. © 1993 Wiley-Liss, Inc.     

Proteins as a potential nitrogen storage compound in bark and leaves of several softwoods

Summary The occurrence of vegetative storage proteins in the leaf and bark tissues of several softwood species during overwintering was investigated by sodium dodecyl sulphate polyacrylamide electrophoresis. Monthly protein profiles from leaves and bark of six evergreen softwood species (Pinus strobus, P. sylvestris, Picea abies, P. glauca, Abies balsamea, and Thuja occidentalis) and the bark of one deciduous softwood species (Larix decidua) suggest that storage proteins are present in bark tissues of L. decidua, Pinus sylvestris, and P. strobus. The remaining species did not show similar specific proteins. However, the total soluble protein content which was determined during active growth and during overwintering in the same tissues indicated that protein levels were higher in the winter compared to the summer in the bark of all species and in the leaves of Pinus spp. and T. occidentalis. While vegetative storage proteins do not appear prevalent in all softwood species, proteins may constitute a major form of overwintering nitrogen storage for many species.     

Recombinant immunoglobulin variable domains generated from synthetic genes provide a system for in vitro characterization of light-chain amyloid proteins.

The primary structural features that render human monoclonal light chains amyloidogenic are presently unknown. To gain further insight into the physical and biochemical factors that result in the pathologic deposition of these proteins as amyloid fibrils, we have selected for detailed study three closely homologous protein products of the light-chain variable-region single-gene family VkIV. Two of these proteins, REC and SMA, formed amyloid fibrils in vivo. The third protein, LEN, was excreted by the patient at levels of 50 g/day with no indication of amyloid deposits. Sequences of amyloidogenic proteins REC and SMA differed from the sequence of the nonpathogenic protein LEN at 14 and 8 amino acid positions, respectively, and these amino acid differences have been analyzed in terms of the three-dimensional structure of the LEN dimer. To provide a replenishable source of these human proteins, we constructed synthetic genes coding for the REC, SMA, and LEN variable domains and expressed these genes in Escherichia coli. Immunochemical and biophysical comparisons demonstrated that the recombinant VkIV products have tertiary structural features comparable to those of the patient-derived proteins. This well-defined set of three clinically characterized human kIV light chains, together with the capability to produce these kIV proteins recombinantly, provide a system for biophysical and structural comparisons of two different amyloidogenic light-chain proteins and a nonamyloidogenic protein of the same subgroup. This work lays the foundation for future investigations of the structural basis of light-chain amyloidogenicity.     

Prediction of possible sites for posttranslational modifications in human gamma crystallins: effect of glycation on the structure of human gamma-B-crystallin as analyzed by molecular modeling

Crystallins are recognized as one of the long-lived proteins of lens tissue that might serve as the target for several posttranslational modifications leading to cataract development. We have studied several such sites present in the human gamma-crystallins based either on PROSITE pattern search results or earlier experimental evidences. Their probabilities were examined on the basis of the database analysis of the gamma-crystallin sequences and on their specific locations in the constructed homology models. An N-glycosylation site in human gammaD-crystallin and several phosphorylation sites in all four human gamma-crystallins were predicted by the PROSITE search. Some of these sites were found to be strongly conserved in the gamma-crystallin sequences from different sources. An extensive analysis of these sites was performed to predict their probabilities as potential sites for protein modifications. Glycation studies were performed separately by attaching sugars to the human gammaB-crystallin model, and the effect of binding was analyzed. The studies showed that the major effect of alphaD-glucose (alphaD-G) and alphaD-glucose-6-phosphate (alphaD-G6P) binding was the disruption of charges not only at the surface but also within the molecule. Only a minor alteration in the distances of sulfhydryl groups of cysteines and on their positions in the three-dimensional models were observed, leading us to assume that glycation alone is not responsible for intra- and intermolecular disulfide bond formation.     

Loss of protein structure stability as a major causative factor in monogenic disease

The most common cause of monogenic disease is a single base DNA variant resulting in an amino acid substitution. In a previous study, we observed that a high fraction of these substitutions appear to result in reduction of stability of the corresponding protein structure. We have now investigated this phenomenon more fully. A set of structural effects, such as reduction in hydrophobic area, overpacking, backbone strain, and loss of electrostatic interactions, is used to represent the impact of single residue mutations on protein stability. A support vector machine (SVM) was trained on a set of mutations causative of disease, and a control set of non-disease causing mutations. In jack-knifed testing, the method identifies 74% of disease mutations, with a false positive rate of 15%. Evaluation of a set of in vitro mutagenesis data with the SVM established that the majority of disease mutations affect protein stability by 1 to 3 kcal/mol. The method's effective distinction between disease and non-disease variants, strongly supports the hypothesis that loss of protein stability is a major factor contributing to monogenic disease.     

Small-angle X-ray scattering based structure,modeling and molecular dynamics analyses of a family 5 glycoside hydrolase first endo-mannanase named as RfGH5_7 from Ruminococcus flavefaciens

Abstract

Endo-β-1,4-mannanase named as RfGH5_7 from Ruminococcus flavefaciens cloned, expressed and purified earlier was structurally characterized in present study. The RaptorX modeled structure of RfGH5_7 showed a (β/α)₈ Triose-phosphate Isomerase (TIM) barrel fold. The Ramachandran plot assessment of RfGH5_7 showed that all amino acids fall in allowed region except one, Asn22 in the disallowed region. The superposition of RfGH5_7 modeled structure with its nearest homologues revealed that Glu154 acts as proton donor while Glu249 acts as nucleophile. Secondary structure of RfGH5_7 through Circular Dichroism (CD) analysis revealed 33.5% α-helices, 17% β-strands and 49.5% random coils. Molecular Dynamic (MD) simulation showed Root Mean Square Deviation (RMSD), 0.67?nm and radius of gyration (Rg) between 1.9?nm and 1.85?nm. The binding interaction of mannotetraose on the surface of RfGH5_7 structure displayed polar interactions with His219, Tyr221, Trp278, Ser279 and Gly282 residues. Small-angle X-ray scattering (SAXS) analysis displayed the intact and monodispersed nature of the enzyme RfGH5_7. The radius of gyration (Rg) by Guinier analysis for globular shape was found to be 2.29?±?0.09?nm and for rod-shape it was 0.95?±?0.02?nm. Kratky plot confirmed that RfGH5_7 structure is compact and folded in solution. The ab initio derived dummy model of RfGH5_7 displayed single domain structure of yellow humped fish like shape. The RfGH5_7 modeled structure was well fitted with ab initio derived model from SAXS data.

Communicated by Ramaswamy H. Sarma     

Secondary structure length as a determinant of folding rate of proteins with two- and three-state kinetics

We present a simple method for determining the folding rates of two- and three-state proteins from the number of residues in their secondary structures (secondary structure length). The method is based on the hypothesis that two- and three-state foldings share a common pattern. Three-state proteins first condense into metastable intermediates, subsequent forming of alpha-helices, turns, and beta-sheets at slow rate-limiting step. The folding rate of such proteins anticorrelate with the length of these beta-secondary structures. It is also assumed that in two-state folding, rapidly folded alpha-helices and turns may facilitate formation of fleeting unobservable intermediates and thus show two-state behavior. There is an inverse relationship between the folding rate and the length of beta-sheets and loops. Our study achieves 94.0 and 88.1% correlations with folding rates determined experimentally for 21 three- and 38 two-state proteins, respectively, suggesting that protein-folding rates are determined by the secondary structure length. The kinetic kinds are selected on the basis of a competitive formation of hydrophobic collapse and alpha-structure in early intermediates.     

Problems Associated with the Identification of Proteins in Homologous Families: The Wool Keratin Family as a Case Study

The keratin proteins from wool can be divided into two classes: the intermediate filament proteins (IFPs) and the matrix proteins. Using peptide mass spectral fingerprinting it was possible to match spots to the known theoretical sequences of some IFPs in web-based databases, as enzyme digestion generated sufficient numbers of peptides from each spot to achieve this. In contrast, it was more difficult to obtain good matches for some of the lower molecular weight matrix proteins. Relatively few peaks were generated from tryptic digests of high-sulfur proteins because of their lower molecular weight and the absence of basic residues in the first two-thirds of the sequence. Their high sequence homology also means that generally only a few of these peptides could be considered to be unique identifiers for each protein. Nevertheless, it was still possible to uniquely identify some of these proteins, while the presence of two peptides in the matrix-assisted laser desorption/ionization time-of-flight mass spectrum allowed classification of other protein spots as being members of this family. Only one major peptide peak was generated by the high-glycine tyrosine proteins (HGTPs) and there were relatively few sequences available in web-based databases, limiting their identification to one HGTP family.     

The Brain 68-Kilodalton Microtubule-Associated Protein Is a Cognate Form of the 70-Kilodalton Mammalian Heat-Shock Protein and Is Present as a Specific Isoform in Synaptosomal Membranes

The relationship between the 68-kilodalton microtubule-associated protein (68KMAP) and the major heat-induced protein (HSP70) in rat and human cells was investigated by comparison of their heat induction properties and by tryptic and Cleveland peptide mapping procedures. HSP70 synthesis was induced by heat shock of rat and human cells, whereas 68KMAP was a major synthesised protein in the absence of heat shock, with its synthesis being only slightly increased on heat shock. Tryptic peptide mapping, however, indicated strong peptide homology between the two proteins. These data, therefore, confirm that 68KMAP represents a constitutively expressed, heat-shock cognate gene. Two-dimensional gel electrophoretic analysis of subcellular fractions of rat brain, combined with peptide mapping procedures, indicated that 68KMAP exists as at least two isoforms separable by isofocussing, the more acidic of which (alpha 68KMAP) is present in fractions enriched in microtubules, cytosol, microsomes, synaptosomal plasma membranes, and synaptic vesicles, and the more basic of which (beta 68KMAP) is present predominantly in fractions enriched in synaptic vesicles and synaptosomal plasma membranes. These two forms are distinguishable in terms of changes in Cleveland peptide maps, and we conclude that alpha- and beta 68KMAP, therefore, represent distinct forms. The significance of these findings to the molecular pathogenesis of Down's syndrome in the human brain is discussed.