Conservation of inter-residue interactions and prediction of folding rates of domain repeats

Domains are the main structural and functional units of larger proteins. They tend to be contiguous in primary structure and can fold and function independently. It has been observed that 10–20% of all encoded proteins contain duplicated domains and the average pairwise sequence identity between them is usually low. In the present study, we have analyzed the structural similarity between domain repeats of proteins with known structures available in the Protein Data Bank using structure-based inter-residue interaction measures such as the number of long-range contacts, surrounding hydrophobicity, and pairwise interaction energy. We used RADAR program for detecting the repeats in a protein sequence which were further validated using Pfam domain assignments. The sequence identity between the repeats in domains ranges from 20 to 40% and their secondary structural elements are well conserved. The number of long-range contacts, surrounding hydrophobicity calculations and pairwise interaction energy of the domain repeats clearly reveal the conservation of 3-D structure environment in the repeats of domains. The proportions of mainchain–mainchain hydrogen bonds and hydrophobic interactions are also highly conserved between the repeats. The present study has suggested that the computation of these structure-based parameters will give better clues about the tertiary environment of the repeats in domains. The folding rates of individual domains in the repeats predicted using the long-range order parameter indicate that the predicted folding rates correlate well with most of the experimentally observed folding rates for the analyzed independently folded domains.     

Mechanical unfolding of a titin Ig domain: structure of transition state revealed by combining atomic force microscopy,protein engineering and molecular dynamics simulations

Titin I27 shows a high resistance to unfolding when subject to external force. To investigate the molecular basis of this mechanical stability, protein engineering Phi-value analysis has been combined with atomic force microscopy to investigate the structure of the barrier to forced unfolding. The results indicate that the transition state for forced unfolding is significantly structured, since highly destabilising mutations in the core do not affect the force required to unfold the protein. As has been shown before, mechanical strength lies in the region of the A' and G-strands but, contrary to previous suggestions, the results indicate clearly that side-chain interactions play a significant role in maintaining mechanical stability. Since Phi-values calculated from molecular dynamics simulations are the same as those determined experimentally, we can, with confidence, use the molecular dynamics simulations to analyse the structure of the transition state in detail, and are able to show loss of interactions between the A' and G-strands with associated A-B and E-F loops in the transition state. The key event is not a simple case of loss of hydrogen bonding interactions between the A' and G-strands alone. Comparison with Phi-values from traditional folding studies shows differences between the force and "no-force" transition states but, nevertheless, the region important for kinetic stability is the same in both cases. This explains the correspondence between hierarchy of kinetic stability (measured in stopped-flow denaturant studies) and mechanical strength in these titin domains.     

Ig-like domains on bacteriophages: a tale of promiscuity and deceit

The immunoglobulin (Ig) fold is one of the most important structures in biology, playing essential roles in the vertebrate immune response, cell adhesion, and many other processes. Through bioinformatic analysis, we have discovered that Ig-like domains are often found in the constituent proteins of tailed double-stranded (ds) DNA bacteriophage particles, and are likely displayed on the surface of these viruses. These phage Ig-like domains fall into three distinct sequence families, which are similar to the classic immunoglobulin domain (I-Set), the fibronectin type 3 repeat (FN3), and the bacterial Ig-like domain (Big2). The phage Ig-like domains are very promiscuous. They are attached to more than ten different functional classes of proteins, and found in all three morphogenetic classes of tailed dsDNA phages. In addition, they reside in phages that infect a diverse set of gram negative and gram positive bacteria. These domains are deceptive because many are added to larger proteins through programmed ribosomal frameshifting, so that they are not always detected by standard protein sequence searching procedures. In addition, the presence of unrecognized Ig-like domains in a variety of phage proteins with different functions has led to gene misannotation. Our results demonstrate that horizontal gene transfer involving Ig-like domain encoding DNA has occurred commonly between diverse classes of both lytic and temperate phages, which otherwise display very limited sequence similarities to one another. We suggest that phage may have been an important vector in the spread of Ig-like domains through diverse species of bacteria. While the function of the phage Ig-like domains is unknown, several lines of evidence suggest that they may play an accessory role in phage infection by weakly interacting with carbohydrates on the bacterial cell surface.     

Mechanical unfolding of a titin Ig domain: structure of unfolding intermediate revealed by combining AFM,molecular dynamics simulations,NMR and protein engineering

The mechanical unfolding of an immunoglobulin domain from the human muscle protein titin (TI I27) has been shown to proceed via a metastable intermediate in which the A-strand is detached. The structure and properties of this intermediate are characterised in this study. A conservative destabilising mutation in the A-strand has no effect on the unfolding force, nor the dependence of the unfolding force on the pulling speed, indicating that the unfolding forces measured in an AFM experiment are those required for the unfolding of the intermediate and not the native state. A mutant of TI I27 with the A-strand deleted (TI I27-A) is studied by NMR and standard biophysical techniques, combined with protein engineering. Molecular dynamics simulations show TI I27-A to be a good model for the intermediate. It has a structure very similar to the native state, and is surprisingly stable. Comparison with a Phi-value analysis of the unfolding pathway clearly shows that the protein unfolds by a different pathway under an applied force than on addition of denaturant.     

Thermodynamic characterisation of two transition states along parallel protein folding pathways

Recent work has shown that a β-sandwich domain from the human muscle protein titin (TI I27) unfolds via more than one pathway, providing experimental evidence for a long-standing theoretical prediction in protein folding. Here we present a thermodynamic analysis of two transition states along different folding pathways for this protein. The unusual upwards curvature previously observed in the denaturant-dependent unfolding kinetics is increased at both high and low temperatures, indicating that the high denaturant pathway is becoming more accessible. The transition states in each pathway are structurally distinct and have very different heat capacities. Interestingly the nucleation-condensation pathway is dominant at all physiologically relevant temperatures, supporting the suggestion that pathways with diffuse rather than localised transition states have been selected for by evolution to prevent misfolding.     

Discrimination of folding types of globular proteins based on average distance maps constructed from their sequences

It has been shown that probable portions which form contacts in a protein can be predicted by means of an average distance map (ADM) as well as regular structures (-helices and -turns) defined as short-range compact regions (Kikuchiet al., 1988a,c). In this paper, we analyze the occurrence of those portions and short-range compact regions on ADMs for various proteins regarding their folding types. We have found out that each folding type of proteins shows characteristic distribution of such parts on ADMS. We also discuss the possibility of the prediction of folding types of proteins by ADMs.     

Transition-states in protein folding kinetics: the structural interpretation of Phi values

Phi values are experimental measures of the effects of mutations on the folding kinetics of a protein. A central question is what structural information Phi values give about the transition-state of folding. Traditionally, a Phi value is interpreted as representing the "nativeness" of a mutated residue in the transition-state. However, this interpretation is often problematic. We present here a better structural interpretation of Phi values for mutations within a given helix. Our interpretation is based on a simple physical model that distinguishes between secondary and tertiary free energy contributions of helical residues. From a linear fit of the model to experimental data, we obtain two structural parameters: the extent of helix formation in the transition-state, and the nativeness of tertiary interactions in the transition-state. We apply the model to all proteins with well-characterized helices for which more than 10 Phi values are available: protein A, CI2, and protein L. The model is simple to apply to experimental data, captures nonclassical Phi values <0 or >1 in these helices, and explains how different mutations at a given site can lead to different Phi values.     

模式匹配分析方法检索免疫球蛋白样功能区

运用计算机进行核酸和蛋白质的序列分析是分子生物学研究的一个较新发展,这项技术已越来越多地用于研究大量积累的序列数据。蛋白质功能区是蛋白质分子中能独立折叠成具有一定结构并执行特定功能的结构域,所有具有同一类功能区的分子统称为一个蛋白质的超族(protein superfamily)。本文通过对免疫球蛋白(Ig)超族及其功能区序列所进行的分析,建立了一种根据功能区之保守片段残基组成的模式匹配分析检索蛋白质功能区的方法,它先根据多序列的对准比较确定某一类功能区之保守片段,再对已知的保守片段各位置上氨基酸残基组成进行统计分析,然后根据与统计数值相匹配的方法,计算待检序列残基组成的统计学意义,由此确定功能区的存在。该方法的优点在于它不仅可以检出已知的具有某一类功能区的分子,而且还可能发现新的具有该功能区的分子,从而推测后者的功能。     

A predicted consensus structure for the protein kinase C2 homology (C2H) domain,the repeating unit of synaptotagmin

A secondary structure has been predicted for the protein kinase C2 regulatory domain found in homologous form in synaptotagmin, some phospholipases, and some GTP activated proteins. The proposed structure is built from seven consecutive beta strands followed by a terminal alpha helix. Considerations of overall surface exposure of individual secondary structural elements suggest that these are packed into a 2-sheet beta sandwich structure, with one of only three of the many possible folds being preferred. © 1995 Wiley-Liss, Inc.     

Structural homology between Drosophila melanogaster and Escherichia coli acidic ribosomal proteins

Antibodies raised against Drosophila melanogaster ribosomal proteins (r-proteins) were used to examine possible structural relationships between eukaryotic and prokaryotic r-proteins. The antisera were raised against either groups of r-proteins or individually purified r-proteins. Two antisera showed a cross-reaction with total Escherichia coli r-proteins in Ouchterlony double immunodiffusion assays: an antiserum against the D. melanogaster small subunit protein S14 (anti-S14) and an antiserum against a group of D. melanogaster r-proteins (anti-TP80). The specificity of the antisera and the identity of the homologous E. coli r-proteins were characterized by using immunooverlay and immunoblot assays. These assays indicated that anti-S14 was highly specific for protein S14 and anti-TP80 was a multispecific serum that recognized several of the D. melanogaster ribosomal proteins. The E. coli protein homologous to D. melanogaster protein S14 was identified as E. coli protein S6. By adsorption of the anti-TP80 serum, we determined that D. melanogaster protein 7/8 is homologous to the acidic E. coli protein L7/L12. D. melanogaster acidic protein 13 was also shown to be immunologically related to D. melanogaster protein 7/8.This research was supported by National Institutes of Health Grant GM23410 awarded to WYC. LMS was the recipient of a predoctoral fellowship from Molecular, Cellular, and Developmental Biology Training Grant PHS T32 CM07227. We are very grateful to Dr. Anthony Mahowald for providing us with embryos.     

MURF-1 and MURF-2 target a specific subset of myofibrillar proteins redundantly: towards understanding MURF-dependent muscle ubiquitination

MURF-1, MURF-2 and MURF-3 are a specific class of RING finger proteins that are expressed in striated muscle tissues. MURF-1 has been suggested to act as an ubiquitin ligase, thereby controlling proteasome-dependent degradation of muscle proteins. Here, we performed yeast two-hybrid (YTH) screens of skeletal muscle cDNA libraries with MURF-1 baits to identify potential myocellular targets of MURF-1-dependent ubiquitination. This identified eight myofibrillar proteins as binding partners of MURF-1: titin, nebulin, the nebulin-related protein NRAP, troponin-I (TnI), troponin-T (TnT), myosin light chain 2 (MLC-2), myotilin and T-cap. YTH mating studies with MURF-1,2,3 baits indicated that these eight myofibrillar proteins are all targeted redundantly by both MURF-1 and MURF-2. Western blot studies on cardiac tissues from wild-type and MURF-1-deficient mice suggested that titin and nebulin were ubiquitinated at similar levels, and MLC-2 and TnI at reduced levels in MURF-1 KO mice. Mapping of the TnI and titin binding sites on MURF-1 peptide scans demonstrated their binding to motifs highly conserved between MURF-1 and MURF-2. Our data are consistent with a model in which MURF-1 and MURF-2 together target a specific set of myofibrillar proteins redundantly, most likely to control their ubiquitination-dependent degradation. Finally, our YTH screens identified the interaction of MURF-1 with 11 enzymes required for ATP/energy production in muscle including the mitochondrial ATP synthase and cytoplasmic creatine kinase. These data raise the possibility that MURF-1 may coordinately regulate the energy metabolism of mitochondrial and cytoplasmic compartments.