Natural Protein Sequences Research Articles

Conformation biases of amino acids based on tripeptide microenvironment from PDB database

We have constructed a bank (FTTP) of tendentious factors of three states of three-peptide units from PDB database based on conformational dihedral angle library and demonstrated that amino acid biases toward protein secondary structure are present in natural protein sequences. Our research results reveal that 20 standard amino acids fall into three groups: nine residues inclined to α-helix with a common character (e.g. direct side chain aliphatic residues or positive/negative charged residues) arrange in three grades, viz EA, QKRLD, and MN, in turn; seven residues are apt to β-strand with 2′-branched side chain aliphatic residues or benzyl-included residues, namely PV, IYTC, and F, in three ranks; and four residues SHWG show a double tendency to both α and β. Noticeably, proline has the strongest ability to form extended conformation, especially the R e value up to 9.5298 at position 3 (Table 3). Thus, biases of codons show an evident tendency in protein folding, where GC-rich codons are mainly in charge of forming contracted conformation, especially the codon's first letter plays a dominant role in translating the genomic GC signature into protein sequences and structures. So, biases of amino acids will play an important role in protein folding, folding codons, refining domain, structure prediction, and structural genomics/proteomics.

An Immunologically Cryptic Epitope of Plasmodium falciparum Circumsporozoite Protein Facilitates Liver Cell Recognition and Induces Protective Antibodies That Block Liver Cell Invasion

Circumsporozoite, a predominant surface protein, is involved in invasion of liver cells by Plasmodium sporozoites, which leads to malaria. We have previously reported that the amino terminus region (amino acids 27-117) of P. falciparum circumsporozoite protein plays a critical role in the invasion of liver cells by the parasite. Here we show that invasion-blocking antibodies are induced by a polypeptide encoding these 91 amino acids, only when it is presented in the absence of the rest of the protein. This suggests that when present in the whole protein, the amino terminus remains immunologically cryptic. A single reactive epitope was identified and mapped to a stretch of 21 amino acids from position 93 to 113. The epitope is configurational in nature, since its recognition was affected by deleting as little as 3 amino acids from either end of the 21-residue peptide. Lysine 104, the only known polymorphic position in the epitope, affected its recognition by the antibodies, and its conversion to leucine in the protein led to a substantial loss of binding activity of the protein to the hepatocytes. This indicated that in the protein, the epitope serves as a binding ligand and facilitates the interaction between sporozoite and hepatic cells. When considered along with the observation that in its native state this motif is immunologically unresponsive, we suggest that hiding functional moieties of the protein from the immune system is an evasion strategy to preserve liver cell binding function and may be of importance in designing anti-sporozoite vaccines.

A glimpse at the organization of the protein universe

The amino acid sequences of most natural proteins result in an ability to fold to specific structures that generate biological activity, and simultaneously to avoid misfolding and aggregation (1). It appears from the data available to us at present that the overall architecture (the “fold”) of these structures is much more highly conserved during evolution than the sequences that encode them. These folds have therefore emerged as ideal candidates for classifying proteins (Fig. 1) and hence to begin to make order of the protein universe (2). The continuing advances in structural biology, and particularly the recent emergence of structural genomics initiatives in which particular emphasis is placed on the discovery of new folds (3), are providing an opportunity to build up a comprehensive map of the protein universe. Of particular significance is the fact that the number of distinct structural archetypes, or folds, is thought to be relatively small, less than ≈10,000 by most estimates, with many different sequences able to encode the same basic fold of the polypeptide chain (4). A key question in the analysis of protein sequences and structures is the way in which they relate to their functions. Clues as to the answer will not only begin to enlighten us as to the fundamental organization of the protein universe, and the location within it of natural proteins, but will also provide a means of predicting the functions of those proteins for which this information is not yet defined by experiment. The ability to predict function will be of tremendous value, for example, in interpreting the output of genome sequencing programs, or in the design of new proteins with specific functional characteristics. In a recent issue of PNAS, Kim and colleagues (5) take a significant step toward this objective by extending their earlier study (6) to show that proteins with similar functions can be found close together in the protein universe—provided that the latter is organized through structural considerations in a suitable way, termed the structure space map (SSM). Fig. 1. The totality of all possible proteins can be looked at in three different ways: (i) the protein universe, which is formed by all possible amino acid sequences; (ii) the protein fold universe, which contains all possible folds associated with these sequences; ...

1P078 天然蛋白質のアミノ酸配列に見るフォールディング中間体の不安定化機構(蛋白質 C) 物生(安定性、折れたたみなど)))

1P078 天然蛋白質のアミノ酸配列に見るフォールディング中間体の不安定化機構(蛋白質 C) 物生(安定性、折れたたみなど)))

A small molecule transcriptional activation domain.

Artificial transcriptional activators are excellent tools for studying the mechanistic details of transcriptional regulation. Furthermore, as the correlation between a wide range of human diseases and misregulated transcription becomes increasingly evident, such molecules may in the long run serve as the basis for transcription-based therapeutic agents. The greatest challenge in this arena has been the discovery of organic molecules that are functional mimics of transcriptional activation domains, sequences of natural proteins that participate in a variety of protein-protein interactions to control transcriptional levels. We describe the first examples of small molecules that function in this capacity, isoxazolidines containing an array of polar and hydrophobic functional groups. Despite their small size, the most potent of the structures functions nearly as well as a natural activation domain.

Mitochondrial malate dehydrogenase from the thermophilic, filamentous fungus Talaromyces emersonii.

Mitochondrial malate dehydrogenase (m-MDH; EC 1.1.1.37), from mycelial extracts of the thermophilic, aerobic fungus Talaromyces emersonii, was purified to homogeneity by sequential hydrophobic interaction and biospecific affinity chromatography steps. Native m-MDH was a dimer with an apparent monomer mass of 35 kDa and was most active at pH 7.5 and 52 degrees C in the oxaloacetate reductase direction. Substrate specificity and kinetic studies demonstrated the strict specificity of this enzyme, and its closer similarity to vertebrate m-MDHs than homologs from invertebrate or mesophilic fungal sources. The full-length m-MDH gene and its corresponding cDNA were cloned using degenerate primers derived from the N-terminal amino acid sequence of the native protein and multiple sequence alignments from conserved regions of other m-MDH genes. The m-MDH gene is the first oxidoreductase gene cloned from T. emersonii and is the first full-length m-MDH gene isolated from a filamentous fungal species and a thermophilic eukaryote. Recombinant m-MDH was expressed in Escherichia coli, as a His-tagged protein and was purified to apparent homogeneity by metal chelate chromatography on an Ni2+-nitrilotriacetic acid matrix, at a yield of 250 mg pure protein per liter of culture. The recombinant enzyme behaved as a dimer under nondenaturing conditions. Expression of the recombinant protein was confirmed by Western blot analysis using an antibody against the His-tag. Thermal stability studies were performed with the recombinant protein to investigate if results were consistent with those obtained for the native enzyme.

Cotranslational incorporation of a structurally diverse series of proline analogues in an Escherichia coli expression system.

A set of Escherichia coli expression strains have been defined that are competent for the incorporation of a structurally diverse series of proline analogues under culture conditions that are compatible with high levels of analogue substitution within a proline-rich protein substrate. These bacterial strains have been employed to assay the efficacy of incorporation of noncanonical amino acids into a recombinant-protein test substrate and to create variant polypeptides in which native protein sequences have been globally substituted with imino acid analogues in response to proline codons. We envision that these methods may be used to interrogate the effect of imino acid substitution on protein structure and function and may be particularly informative in the context of structural comparison of a series of modified proteins with respect to the stereoelectronic differences between the incorporated proline analogues.

Short amino acid stretches can mediate amyloid formation in globular proteins: the Src homology 3 (SH3) case.

Protein misfolding and deposition underlie an increasing number of debilitating human disorders. We have shown that model proteins unrelated to disease, such as the Src homology 3 (SH3) domain of the p58alpha subunit of bovine phosphatidyl-inositol-3'-kinase (PI3-SH3), can be converted in vitro into assemblies with structural and cytotoxic properties similar to those of pathological aggregates. By contrast, homologous proteins, such as alpha-spectrin-SH3, lack the capability of forming amyloid fibrils at a measurable rate under any of the conditions we have so far examined. However, transplanting a small sequence stretch (6 aa) from PI3-SH3 to alpha-spectrin-SH3, comprising residues of the diverging turn and adjacent RT loop, creates an amyloidogenic protein closely similar in its behavior to the original PI3-SH3. Analysis of specific PI3-SH3 mutants further confirms the involvement of this region in conferring amyloidogenic properties to this domain. Moreover, the inclusion in this stretch of two consensus residues favored in SH3 sequences substantially inhibits aggregation. These findings show that short specific amino acid stretches can act as mediators or facilitators in the incorporation of globular proteins into amyloid structures, and they support the suggestion that natural protein sequences have evolved in part to code for structural characteristics other than those included in the native fold, such as avoidance of aggregation.

Capping Protein Binding to S100B: IMPLICATIONS FOR THE “TENTACLE” MODEL FOR CAPPING THE ACTIN FILAMENT BARBED END

S100B binds tightly to a 12-amino acid peptide derived from heterodimeric capping protein. In native intact capping protein, this sequence is in the C terminus of the alpha-subunit, which is important for capping the actin filament. This C-terminal region is proposed to act as a flexible "tentacle," extending away from the body of capping protein in order to bind actin. To this hypothesis, we analyzed the interaction between S100B and capping protein in solution. The C-terminal 28 amino acids of the alpha-subunit, the proposed tentacle, bound to S100B as a free synthetic peptide or a glutathione S-transferase fusion (K(d) approximately 0.4-1 microm). In contrast, S100B did not bind to whole native capping protein or functionally affect its capping activity. S100B does not bind, with any significant affinity, to the proposed alpha-tentacle sequence of whole native capping protein in solution. In the NMR structure of S100B complexed with the alpha-subunit-derived 12-amino acid peptide, the hydrophobic side of a short alpha-helix in the peptide, containing an important tryptophan residue, contacts S100B. In the x-ray structure of native capping protein, the corresponding sequence of the alpha-subunit C terminus, including Trp(271), interacts closely with the body of the protein. Therefore, our results suggest the alpha-subunit C terminus is not mobile as predicted by the tentacle model. Addition of non-ionic detergent allowed whole capping protein to bind weakly to S100B, indicating that the alpha-subunit C terminus can be mobilized from the surface of the capping protein molecule, presumably by weakening the hydrophobic binding at the contact site.

Lower limit to the size of the primeval amino acid alphabet.

Here I systematically examine the information complexity of all primary sequences of natural proteins deposited in the Swiss-Prot database. The sequence complexity is assessed by determining the frequency of occurrence of each amino acid type on sequence windows of fixed length, calculating the Shannon entropy of the window and then averaging over all windows covering the sequence. The minimum value in information content obtained from the present-day record imposes a lower limit in the number of letters that a primeval amino acid alphabet must have had.

Consensus-Based Engineering of Protein Stability: From Intrabodies to Thermostable Enzymes

Publisher Summary This chapter elaborates the consensus-based engineering of protein stability. Protein stability is an experimental metric of the information encoded in a protein sequence. This approach has allowed to engineer hyperstable immunoglobulin domains that can be expressed as intrabodies. Its application by a number of groups to a diverse set of proteins has demonstrated this procedure to be the most reliable strategy for the rational generation of stabilizing mutations. The ability to generate synthetic enzymatically active proteins from consensus sequences emphasizes the statistical nature of natural protein sequences as products of neutral drift and selective pressure. Stabilization is not the only objective for which the approach is useful, and others could include the selection of residues for nondisruptive destabilizing mutations to reduce the lifetime of proteins, or to generate proteins that can be used to screen for second-site revertants in evolutionary engineering strategies. It is found that the final steps of mutagenesis, characterization, and combination of successful residues are specific for each experimental system at hand and should be straightforward.

Facile synthesis of site-specifically acetylated and methylated histone proteins: reagents for evaluation of the histone code hypothesis.

The functional capacity of genetically encoded histone proteins can be powerfully expanded by posttranslational modification. A growing body of biochemical and genetic evidence clearly links the unique combinatorial patterning of side chain acetylation, methylation, and phosphorylation mainly within the highly conserved N termini of histones H2A, H2B, H3, and H4 with the regulation of gene expression and chromatin assembly and remodeling, in effect constituting a "histone code" for epigenetic signaling. Deconvoluting this code has proved challenging given the inherent posttranslational heterogeneity of histone proteins isolated from biological sources. Here we describe the application of native chemical ligation to the preparation of full-length histone proteins containing site-specific acetylation and methylation modifications. Peptide thioesters corresponding to histone N termini were prepared by solid phase peptide synthesis using an acid labile Boc/HF assembly strategy, then subsequently ligated to recombinantly produced histone C-terminal globular domains containing an engineered N-terminal cysteine residue. The ligation site is then rendered traceless by hydrogenolytic desulfurization, generating a native histone protein sequence. Synthetic histones generated by this method are fully functional, as evidenced by their self-assembly into a higher order H3/H4 heterotetramer, their deposition into nucleosomes by human ISWI-containing (Imitation of Switch) factor RSF (Remodeling and Spacing Factor), and by enzymatic modification by human Sirt1 deacetylase and G9a methyltransferase. Site-specifically modified histone proteins generated by this method will prove invaluable as novel reagents for the evaluation of the histone code hypothesis and analysis of epigenetic signaling mechanisms.

RUNS OF AMINO ACIDS ARE LONGER THAN EXPECTED IN PROTEINS BASED ON A GRAPH THEORY REPRESENTATION OF THE GENETIC CODE

An in silico study of mRNA secondary structure has found a bias within the coding sequences of genes that favors "in-frame" pairing of nucleotides. This pairing of codons, each with its reverse-complement, partitions the 20 amino acids into three subsets. The genetic code can therefore be represented by a three-component graph. The composition of proteins in terms of amino acid membership in the three subgroups has been measured, and sequence runs of members within the same subgroup have been analyzed using a runs statistic based on Z-scores. In a GENBANK database of over 416,000 protein sequences, the distribution of this runs-test statistic is negatively skewed. To assess whether this statistical bias was due to a chance grouping of the amino acids in the real genetic code, several alternate partitions of the genetic code were examined by permuting the assignment of amino acids to groups. A metric was constructed to define the difference, or "distance", between any two such partitions, and an exhaustive search was conducted among alternate partitions maximally distant from the natural partition of the genetic code, to select sets of partitions that were also maximally distant from one another. The statistical skewness of the runs statistic distribution for native protein sequences were significantly more negative under the natural partition than they were under all of the maximally different partition of codons, although for all partitions, including the natural one, the randomized sequences had quite similar skewness. Hence under the natural graph theory partition of the genetic code there is a preference for more protein sequences to contain fewer runs of amino acids, than they do under the other partitions, meaning that the average run must be longer under the natural partition. This suggests that a corresponding bias may exist in the coding sequences of the actual genes that code for these proteins.

Intein-mediated affinity-fusion purification of the Escherichia coli RecA protein

The RecA protein of Escherichia coli plays important roles in homologous recombination, recombinational DNA repair, and SOS induction. Because its functions are conserved among the phylogenetic kingdoms, RecA investigations have provided a paradigm for understanding these biological processes. The RecA protein has been overproduced in E. coli and purified using a variety of purification schemes requiring multiple, time-intensive steps. The purification schemes share a dependence on appropriate RecA structure and/or function at one or more steps. In this report, we used a modified protein splicing element (intein) and a chitin-binding domain, fused to the C-terminus of RecA, to facilitate a one-step affinity purification of RecA protein without modification of the native protein sequence. Following the single chromatographic step, RecA protein that is greater than 95% physical purity at a concentration of greater than 65 μM was obtained. The protein displays in vitro activities that are identical to those of protein isolated using classical procedures. The purification strategy described here promises to yield mutant RecA proteins in sufficient quantity for rigorous biophysical characterization without dependence on intrinsic RecA function.

Folding free energy function selects native-like protein sequences in the core but not on the surface.

An automatic protein design procedure is used to select amino acid sequences that optimize the folding free energy function for a given protein. The only information used in designing the sequences is a set of known backbone structures for each protein, a rotamer library, and a well established classical empirical force field, which relies on basic physical chemical principles that underlie molecular interactions and protein stability, and has not been adjusted to yield native-like sequences. Applying the procedure to 7 different known protein folds, representing a total of 45 different native protein structures, yields ensembles of designed sequences displaying remarkable similarity to their natural counterparts in the protein core, but which are distinctly non-native on the protein surface. We show that natural and designed sequences for a given fold score significantly higher than random sequences against profiles derived from both, designed and natural sequence ensembles. Furthermore, we find that designed sequence profiles can be used to retrieve the native sequences for many of the analyzed proteins using standard PSI-BLAST searches in sequence databases. These findings may have important implications for our understanding the selection pressures operating on natural protein sequences and hold promise for improving fold recognition.

Dictionary-driven prokaryotic gene finding.

Gene identification, also known as gene finding or gene recognition, is among the important problems of molecular biology that have been receiving increasing attention with the advent of large scale sequencing projects. Previous strategies for solving this problem can be categorized into essentially two schools of thought: one school employs sequence composition statistics, whereas the other relies on database similarity searches. In this paper, we propose a new gene identification scheme that combines the best characteristics from each of these two schools. In particular, our method determines gene candidates among the ORFs that can be identified in a given DNA strand through the use of the Bio-Dictionary, a database of patterns that covers essentially all of the currently available sample of the natural protein sequence space. Our approach relies entirely on the use of redundant patterns as the agents on which the presence or absence of genes is predicated and does not employ any additional evidence, e.g. ribosome-binding site signals. The Bio-Dictionary Gene Finder (BDGF), the algorithm's implementation, is a single computational engine able to handle the gene identification task across distinct archaeal and bacterial genomes. The engine exhibits performance that is characterized by simultaneous very high values of sensitivity and specificity, and a high percentage of correctly predicted start sites. Using a collection of patterns derived from an old (June 2000) release of the Swiss-Prot/TrEMBL database that contained 451 602 proteins and fragments, we demonstrate our method's generality and capabilities through an extensive analysis of 17 complete archaeal and bacterial genomes. Examples of previously unreported genes are also shown and discussed in detail.

A protein sequence that can encode native structure by disfavoring alternate conformations.

The linear sequence of amino acids contains all the necessary information for a protein to fold into its unique three-dimensional structure. Native protein sequences are known to accomplish this by promoting the formation of stable, kinetically accessible structures. Here we describe a Pro residue in the center of the third transmembrane helix of the cystic fibrosis transmembrane conductance regulator that promotes folding by a distinct mechanism: disfavoring the formation of a misfolded structure. The generality of this mechanism is supported by genome-wide transmembrane sequence analyses. Furthermore, the results provide an explanation for the increased frequency of Pro residues in transmembrane alpha-helices. Incorporation by nature of such 'negative folding determinants', aimed at preventing the formation of off-pathway structures, represents an additional mechanism by which folding information is encoded within the evolved sequences of proteins.

Protein folding and its links with human disease

The ability of proteins to fold to their functional states following synthesis in the intracellular environment is one of the most remarkable features of biology. Substantial progress has recently been made towards understanding the fundamental nature of the mechanism of the folding process. This understanding has been achieved through the development and concerted application of a variety of novel experimental and theoretical approaches to this complex problem. The emerging view of folding is that it is a stochastic process, but one biased by the fact that native-like interactions between residues are, on average, more stable than non-native ones. The sequences of natural proteins have emerged through evolutionary processes such that their unique native states can be found very efficiently even in the complex environment inside a living cell. But under some conditions proteins fail to fold correctly, or to remain correctly folded, in living systems, and this failure can result in a wide range of diseases. One group of diseases, known as amyloidoses, which includes Alzheimer's disease and the transmissible spongiform encephalopathies, involves deposition of aggregated proteins in a variety of tissues. These diseases are particularly intriguing because evidence is accumulating that the formation of the highly organized amyloid aggregates is a generic property of polypeptides, and not simply a feature of the few proteins associated with recognized pathological conditions. That such aggregates are not normally found in properly functional biological systems is again a testament to evolution, in this case of a variety of mechanisms inhibiting their formation. Understanding the nature of such protective mechanisms is a crucial step in the development of strategies to prevent and treat these debilitating diseases.

Disulfide bond formation in peptides.

The formation of disulfide bridges is often a crucial final stage in peptide synthesis. There is compelling evidence that the disulfide pattern can be critical in the folding and structural stabilization of many natural peptide and protein sequences, while the artificial introduction of disulfide bridges into natural or designed peptides may often improve biological activities/specificities and stabilities. This unit provides a highly selective, albeit state-of-the-art, menu of procedures that can be performed to establish intramolecular or intermolecular disulfide bridges in targets of varying complexities.

The structural basis of protein folding and its links with human disease.

The ability of proteins to fold to their functional states following synthesis in the intracellular environment is one of the most remarkable features of biology. Substantial progress has recently been made towards understanding the fundamental nature of the mechanism of the folding process. This understanding has been achieved through the development and concerted application of a variety of novel experimental and theoretical approaches to this complex problem. The emerging view of folding is that it is a stochastic process, but one biased by the fact that native-like interactions between residues are on average more stable than non-native ones. The sequences of natural proteins have emerged through evolutionary processes such that their unique native states can be found very efficiently even in the complex environment inside a living cell. But under some conditions proteins fail to fold correctly, or to remain correctly folded, in living systems, and this failure can result in a wide range of diseases. One group of diseases, known as amyloidoses, which includes Alzheimer's and the transmissible spongiform encephalopathies, involves deposition of aggregated proteins in a variety of tissues. These diseases are particularly intriguing because evidence is accumulating that the formation of the highly organized amyloid aggregates is a generic property of polypeptides, and not simply a feature of the few proteins associated with recognized pathological conditions. That such aggregates are not normally found in properly functional biological systems is again a testament to evolution, in this case of a variety of mechanisms inhibiting their formation. Understanding the nature of such protective mechanisms is a crucial step in the development of strategies to prevent and treat these debilitating diseases.