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PubMed2006

The influence of urea on the structure of proteins in reversed micelles.

Static fluorescence measurements from the protein Tryptophan (Trp) residues and circular dichroism (CD) spectroscopy were used to investigate changes on the tertiary and secondary structures of the protein bovine serum albumin (BSA) and the dimeric enzyme hexokinase (HK) type PII from yeast, entrapped in reversed micelles (RMs). The latter were obtained from the amphiphilic AOT (sodium bis-2-ethylhexyl sulfosuccinate) in n-hexane, at several water to surfactant ratios, W. BSA and HK were found to be anchored at the RM interface in close contact with the surfactant layer, regardless of the size of the waterpool. For BSA, such interaction promotes partial protein unfolding, according to CD data that showed a decrease in the content of helical structure from 66% in a buffer solution to 48% in the micellar moiety. When urea was present in the micelle, further loss in helical structure occurred, thus indicating that the combined effect (micellar environment and urea) altered the BSA conformation to a greater extent than did RM or urea alone. Interestingly, Trps probed the same environment in the micelle, regardless of the presence of urea, but the fluorescence was quenched to a higher extent with urea. Thus, the fluorofore emission must have been affected either by the direct interaction of urea or by indirect exchange of water structure caused by urea interacting with water and the micellar interface. Both mechanisms might be of relevance in the solvation properties nearby the Trps. For HK, an association between the enzyme and the micelle interface was indicated in the CD spectra, which exhibited a randomized structure upon interaction, whatever the RM droplet size. The urea addition to the micelle water pool did not cause further impact on the HK conformation. In addition, the influence of urea at the RM interface was not sensed by the exposed tryptophans of the enzyme, unlike the results for BSA.

Animals↗

PubMed2007

Spectroscopic study of conformational changes accompanying self-assembly of HCV core protein.

Electron microscopy and infrared and Raman spectroscopy have been used here to study the morphology, size distribution, secondary and tertiary structures of protein particles assembled from a truncated hepatitis C virus (HCV) core protein covering the first 120 aa. Particles of pure protein, having similar morphology and size distribution of those of nucleocapsids found in sera from HCV-infected patients, have been visualized for the first time. The secondary structure of these protein particles involve beta-sheet enrichment in relation to its protein monomer. Tertiary/quaternary structure has also been studied using the dynamics of H/D exchange. With this aim infrared spectra were measured as a function of H/D exchange time and subsequently analyzed by principal component analysis and two-dimensional correlation spectroscopy. Temporal dynamics of exchange for these protein particles were as follows: arginine residues exchanged first, followed by turn and unordered structures, followed by beta-sheets which may act as linkers of protein monomers.

Arginine↗

PubMed2006

Protemot: prediction of protein binding sites with automatically extracted geometrical templates.

UNLABELLED: Geometrical analysis of protein tertiary substructures has been an effective approach employed to predict protein binding sites. This article presents the Protemot web server that carries out prediction of protein binding sites based on the structural templates automatically extracted from the crystal structures of protein-ligand complexes in the PDB (Protein Data Bank). The automatic extraction mechanism is essential for creating and maintaining a comprehensive template library that timely accommodates to the new release of PDB as the number of entries continues to grow rapidly. The design of Protemot is also distinctive by the mechanism employed to expedite the analysis process that matches the tertiary substructures on the contour of the query protein with the templates in the library. This expediting mechanism is essential for providing reasonable response time to the user as the number of entries in the template library continues to grow rapidly due to rapid growth of the number of entries in PDB. This article also reports the experiments conducted to evaluate the prediction power delivered by the Protemot web server. Experimental results show that Protemot can deliver a superior prediction power than a web server based on a manually curated template library with insufficient quantity of entries. AVAILABILITY: http://protemot.csie.ntu.edu.tw/step1.cgi http://bioinfo.mc.ntu.edu.tw/protemot/step1.cgi.

Binding Sites↗

PubMed2005

Control of protein functional dynamics by peptide linkers.

Control of structural flexibility is essential for the proper functioning of a large number of proteins and multiprotein complexes. At the residue level, such flexibility occurs due to local relaxation of peptide bond angles whose cumulative effect may result in large changes in the secondary, tertiary or quaternary structures of protein molecules. Such flexibility, and its absence, most often depends on the nature of interdomain linkages formed by oligopeptides. Both flexible and relatively rigid peptide linkers are found in many multidomain proteins. Linkers are thought to control favorable and unfavorable interactions between adjacent domains by means of variable softness furnished by their primary sequence. Large-scale structural heterogeneity of multidomain proteins and their complexes, facilitated by soft peptide linkers, is now seen as the norm rather than the exception. Biophysical discoveries as well as computational algorithms and databases have reshaped our understanding of the often spectacular biomolecular dynamics enabled by soft linkers. Absence of such motion, as in so-called molecular rulers, also has desirable functional effects in protein architecture. We review here the historic discovery and current understanding of the nature of domains and their linkers from a structural, computational, and biophysical point of view. A number of emerging applications, based on the current understanding of the structural properties of peptides, are presented in the context of domain fusion of synthetic multifunctional chimeric proteins.

Amino Acid Sequence↗

PubMed1992

Poly(L-alanine) as a universal reference material for understanding protein energies and structures.

We present a proposition, the "poly(L-alanine) hypothesis," which asserts that the native backbone geometry for any polypeptide or protein of M residues has a closely mimicking, mechanically stable, image in poly(L-alanine) of the same number of residues. Using a molecular mechanics force field to represent the relevant potential energy hypersurfaces, we have carried out calculations over a wide range of M values to show that poly(L-alanine) possesses the structural versatility necessary to satisfy the proposition. These include poly(L-alanine) representatives of minima corresponding to secondary and supersecondary structures, as well as poly(L-alanine) images for tertiary structures of the naturally occurring proteins bovine pancreatic trypsin inhibitor, crambin, ribonuclease A, and superoxide dismutase. The successful validation of the hypothesis presented in this paper indicates that poly(L-alanine) will serve as a good reference material in thermodynamic perturbation theory and calculations aimed at evaluating relative free energies for competing candidate tertiary structures in real polypeptides and proteins.

Algorithms↗

PubMedDate not supplied

[Various views on the formation of the spatial structure of proteins].

Basing on the protein tertiary structure data analysis, the peculiarities of enzymatic catalysis, as well as on the results of ab initio conformational energy map calculations of dipeptides, the conclusion is drawn, that the synthesis of polypeptide chains on the ribosome occurs on the right hand conformation of amino acid residues. For the number of amino acid residues to transfer to left hand conformation, local and electoral conditions are necessary. Some possible errors in the X-ray crystal structure data of proteins are pointed out.

Amino Acids↗

PubMed1996

Destabilisation of native tertiary structural interactions is linked to helix-induction by 2,2,2-trifluoroethanol in proteins.

The effect of 2,2,2-trifluoroethanol (TFE) on the structure of an all beta-sheet protein, cardiotoxin analogue 111 (CTX III) from the Taiwan cobra (Naja naja atra) is studied. It is found that high concentrations (> 80% v/v) of TFE induced a beta-sheet to alpha-helix structural transition. It is found that in denatured and reduced CTX III (rCTX III) helical conformation is induced even upon addition of low concentrations (> 10% v/v) of TFE. Using three other proteins, namely, ribonuclease A (RNase A), lysozyme and alpha-lactalbumin, it is been observed that helix-induction by TFE is intricately linked to drastic destabilization of native tertiary structural interactions in the proteins.

Circular Dichroism↗

PubMed2001

Structure prediction of protein complexes by an NMR-based protein docking algorithm.

Protein docking algorithms can be used to study the driving forces and reaction mechanisms of docking processes. They are also able to speed up the lengthy process of experimental structure elucidation of protein complexes by proposing potential structures. In this paper, we are discussing a variant of the protein-protein docking problem, where the input consists of the tertiary structures of proteins A and B plus an unassigned one-dimensional 1H-NMR spectrum of the complex AB. We present a new scoring function for evaluating and ranking potential complex structures produced by a docking algorithm. The scoring function computes a 'theoretical' 1H-NMR spectrum for each tentative complex structure and subtracts the calculated spectrum from the experimental one. The absolute areas of the difference spectra are then used to rank the potential complex structures. In contrast to formerly published approaches (e.g. [Morelli et al. (2000) Biochemistry, 39, 2530-2537]) we do not use distance constraints (intermolecular NOE constraints). We have tested the approach with four protein complexes whose three-dimensional structures are stored in the PDB data bank (Bernstein et al., 1977) and whose 1H-NMR shift assignments are available from the BMRB database. The best result was obtained for an example, where all standard scoring functions failed completely. Here, our new scoring function achieved an almost perfect separation between good approximations of the true complex structure and false positives.

Algorithms↗

PubMed1998

Crystal structure of the catalytic domain of protein-tyrosine phosphatase SHP-1.

The crystal structures of the protein-tyrosine phosphatase SHP-1 catalytic domain and the complex it forms with the substrate analogue tungstate have been determined and refined to crystallographic R values of 0.209 at 2.5 A resolution and 0.207 at 2.8 A resolution, respectively. Despite low sequence similarity, the catalytic domain of SHP-1 shows high similarity in secondary and tertiary structures with other protein-tyrosine phosphatases (PTPs). In contrast to the conformational changes observed in the crystal structures of PTP1B and Yersinia PTP, the WPD loop (Trp419-Pro428) in the catalytic domain of SHP-1 moves away from the substrate binding pocket after binding the tungstate ion. Sequence alignment and structural analysis suggest that the residues in the WPD loop, especially the amino acid following Asp421, are critical for the movement of WPD loop on binding substrates and the specific activity of protein-tyrosine phosphatases. Our mutagenesis and kinetic measurements have supported this hypothesis.

Amino Acid Sequence↗

PubMed2002

Three-dimensional domain swapping in homooligomeric proteins and its functional significance.

In the process of oligomeric structure formation through a mechanism of three-dimensional domain swapping, one domain of a monomeric protein is replaced by the same domain from an identical monomer. The swapped "domain" can represent an entire tertiary globular domain or an element of secondary protein structure, such as an alpha-helix or a beta-strand. Different examples of three-dimensional domain swapping are reviewed; the functional importance of this phenomenon and its role in the development of new properties by some proteins in the process of evolution are considered. The contribution of three-dimensional domain swapping to the formation of linear protein polymers and amyloids is discussed.

Amyloid↗

PubMed2005

Evolutionary information for specifying a protein fold.

Classical studies show that for many proteins, the information required for specifying the tertiary structure is contained in the amino acid sequence. Here, we attempt to define the sequence rules for specifying a protein fold by computationally creating artificial protein sequences using only statistical information encoded in a multiple sequence alignment and no tertiary structure information. Experimental testing of libraries of artificial WW domain sequences shows that a simple statistical energy function capturing coevolution between amino acid residues is necessary and sufficient to specify sequences that fold into native structures. The artificial proteins show thermodynamic stabilities similar to natural WW domains, and structure determination of one artificial protein shows excellent agreement with the WW fold at atomic resolution. The relative simplicity of the information used for creating sequences suggests a marked reduction to the potential complexity of the protein-folding problem.

Algorithms↗

PubMed1994

Investigating the role of conserved residue Asp134 in Escherichia coli ribonuclease HI by site-directed random mutagenesis.

The role of the conserved Asp134 residue in Escherichia coli ribonuclease HI, which is located at the center of the alpha V helix and lies close to the active site, was analyzed by means of site-directed random mutagenesis. Mutant rnhA genes encoding proteins with ribonuclease H activities were screened by their ability to suppress the ribonuclease-H-dependent, temperature-sensitive growth phenotype of E. coli strain MIC3001. Based on the DNA sequences, nine mutant proteins were predicted to have ribonuclease H activity in vivo. All of these mutant proteins were purified to homogeneity and examined for enzymic activity and protein stability. Among them, only the mutant proteins [D134H]RNase H and [D134N]RNase H were shown to have considerable ribonuclease H activities. Determination of the kinetic parameters revealed that replacement of Asp134 by amino acid residues other than asparagine and histidine dramatically decreased the enzymic activity without seriously affecting the substrate binding. Determination of the CD spectra indicated that none of the mutations seriously affected secondary and tertiary structure. The protein stability was determined from the thermal denaturation curves. All mutant proteins were more stable than the wild-type protein. Such stabilization effects would be a result of a reduction in the negative charge repulsion between Asp134 and the active-site residues, and/or an enhancement of the stability of the alpha V helix. These results strongly suggest that Asp134 does not contribute to the maintenance of the molecular architecture but the carboxyl oxygen at its delta 1 position impacts catalysis.

Amino Acid Sequence↗

PubMed2005

Local stability identification and the role of key acidic amino acid residues in staphylococcal nuclease unfolding.

Staphylococcal nuclease is a single domain protein with 149 amino acids. It has no disulfide bonds, which makes it a simple model for the study of protein folding. In this study, 20 mutants of this protein were generated each with a single base substitution of glycine for negatively charged glutamic acid or aspartic acid. Using differential scanning microcalorimetry in thermal denaturation experiments, we identified two mutants, E75G and E129G, having approximately 43% and 44%, respectively, lower DeltaH(cal) values than the wild-type protein. Furthermore, two mutants, E75Q and E129Q, were created and the results imply that substitution of the Gly residue has little influence on destabilization of the secondary structure that leads to the large perturbation of the tertiary protein structure stability. Two local stable areas formed by the charge-charge interactions around E75 and E129 with particular positively charged amino acids are thus identified as being significant in maintenance of the three-dimensional structure of the protein.

Aspartic Acid↗

PubMed1993

Characterization of a human chorionic gonadotropin-like protein from Candida albicans.

Studies from our laboratory have demonstrated that human CG (hCG), human LH, (hLH), and an hCG-like protein extracted from Xanthomonas maltophilia were able to induce Candida albicans transition from the blastospore to the germ tube stage. In the present study, we describe the characterization of an hCG-like material extracted from Candida albicans blastospores (CaCGLP), which is potent in inducing transition and presumably represents the endogenous transition-inducing substance. This material was extracted from Candida albicans blastospores with glacial acetic acid and purified by affinity chromatography using a polyclonal rabbit anti-hCG antibody. The product obtained is a 68-kilodalton single band protein, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Under reduced conditions a protein smear is seen. Amino acid analysis showed a predominance of glycine (22%), followed by serine (12%), and glutamate (12%). This protein reacted in the following hCG immunoassays: 1) a polyclonal rabbit anti-hCG equilibrium assay, 2) a carboxyl-tail hCG equilibrium assay, 3) two hCG equilibrium assays using monoclonal antibodies (CG no. 4 and CG no. 9), 4) a free alpha-subunit equilibrium assay using a monoclonal antibody, and 5) an ultrasensitive immunoradiometric assay for hCG which does not cross-react with hLH, nor the free beta-subunit of hCG. The CaCGLP showed no reaction in a specific hLH immunoradiometric assay. When CaCGLP was tested in the transition assay, in the presence of 4% rat serum, it was found that this protein was 100 times more potent than hCG in producing Candida albicans transition. We conclude that Candida albicans produces a protein that has certain tertiary structure similarities to hCG and that this material is able to induce germ tube formation. We postulate that CaCGLP has an autocrine/paracrine effect in Candida albicans as a transition factor to control its own pathogenicity.

Amino Acids↗

PubMed1995

Legumin-like and vicilin-like seed storage proteins: evidence for a common single-domain ancestral gene.

Legumin-like 11S and vicilin-like 7S globulins are the main storage proteins of most angiosperms and gymnosperms. The subunits of the hexameric legumin are synthesized as a precursor comprising a N-terminal acidic alpha- and a C-terminal basic beta-chain. The trimeric vicilin molecule consists of subunits composed of two symmetrical N- and C-terminal structural domains. In a multiple alignment we have compared the N-terminal and C-terminal domains of 11 legumins and seven vicilins of several dicot, monocot, and gymnosperm species. The comparisons using all six possible pairwise combinations reveal that the N-terminal and C-terminal domains of both protein families are similar to each other. These results together with data on the distribution of variable and conserved regions, on the positions of susceptible sites for proteolytic attack, as well as on the published 7S protein tertiary structure suggest that both protein families share a common single-domain ancestor molecule and lead to the hypothesis that a triplication event has occurred during the evolution of a putative legumin/vicilin ancestor gene. Moreover, the comparison of the intron/exon pattern reveals that at least three out of five intron positions are precisely conserved between the genes of both protein families, further supporting the idea of a common evolutionary origin of recent legumin and vicilin encoding genes.

Amino Acid Sequence↗

PubMedDate not supplied

[Are synthetic proteins relevant to the problem of protein folding?].

The possibility to derive the analogs of native proteins by the chemical synthesis is considered to be a serious argument for the concept of posttranslational protein folding. The present paper analyzes for the first time chemically synthesized proteins to reveal whether they are relevant to the problem of protein folding. The results enable the following conclusions to be drawn. The acquisition of the peculiar conformations by the chemically synthesized proteins to exhibit the specific functions is conditioned by the highly marked features of the secondary and tertiary structures of the corresponding native proteins. These features will make themselves evident only if favorable conditions are carefully chosen during the experiments for each individual protein. Thus, in our opinion, the possibility to derive a synthetic protein is hardly evidence for the posttranslational folding of proteins.

Protein Biosynthesis↗

PubMed2002

Solution structure of a cathelicidin-derived antimicrobial peptide, CRAMP as determined by NMR spectroscopy.

CRAMP was identified from a cDNA clone derived from mouse femoral marrow cells as a member of cathelicidin-derived antimicrobial peptides. This peptide shows potent antimicrobial activity against gram-positive and gram-negative bacteria but no hemolytic activity against human erythrocytes. CRAMP was known to cause rapid permeabilization of the inner membrane of Escherichia coli. In this study, the structure of CRAMP in TFE/H2O (1 : 1, v/v) solution was determined by CD and NMR spectroscopy. CD spectra showed that CRAMP adopts a mainly alpha-helical conformation in TFE/H2O solution, DPC micelles, SDS micelles and liposomes, whereas it has a random structure in aqueous solution. The tertiary structure of CRAMP in TFE/H2O (1 : 1, v/v), as determined by NMR spectroscopy, consists of two amphipathic alpha-helices from Leu4 to Lys10 and from Gly16 to Leu33. These two helices are connected by a flexible region from Gly11 to Gly16. Previous analysis of series of fragments composed of various portion of CRAMP revealed that an 18-residue fragment with the sequence from Gly16 to Leu33 was found to retain antibacterial activity. Therefore, the amphipathic alpha-helical region from Gly16 to Leu33 of CRAMP plays important roles in spanning the lipid bilayers as well as its antibiotic activity. Based on this structure, novel antibiotic peptides having strong antibiotic activity, with no hemolytic effect will be developed.

Amino Acid Sequence↗

PubMed2005

Orthogonal multipolar interactions in structural chemistry and biology.

The past few decades of molecular recognition studies have greatly enhanced our knowledge on apolar, ion-dipole, and hydrogen-bonding interactions. However, much less attention has been given to the role that multipolar interactions, in particular those with orthogonal dipolar alignment, play in organizing a crystal lattice or stabilizing complexes involving biological receptors. By using results from database mining, this review attempts to give an overview of types and structural features of these previously rather overlooked interactions. A number of illustrative examples of these interactions found in X-ray crystal structures of small molecules and protein-ligand complexes demonstrate their propensity and thus potential importance for both, chemical and biological molecular recognition processes.

Biology↗