Situation determinants of heroin consumption in opiate-dependent subjects: a psychometric approach

BACKGROUND: Chymotrypsin inhibitor 2 (CI2) is a member of the class of fast-folding small proteins, which is very suitable for testing theories of folding. CI2 folds around a diffuse extended nucleus consisting of the single alpha helix and a set of hydrophobic residues. In particular, Ala16 has been predicted and independently found to interact with Leu49 and Ile57, hydrophobic residues that are highly conserved among homologues. We have characterised in detail the interactions between these residues in the folding nucleus of the protein by using double-mutant cycles. RESULTS: Surprisingly, we find that there is some destabilising strain in the transition state for folding of the wild-type protein between Ala16 and Ile57. Further, we find that the strain is larger in the native state of the protein. This is shown directly in the unfolding kinetics, which clearly show a release of strain. The net result of this is that the presence of both residues speeds up folding. Ala16 and Leu49 interact favourably in the transition state, but have no net interaction energy in the native state. CONCLUSIONS: Part of the folding nucleus of the protein fits together more snugly in the transition state than it does in the native state. Interactions between some of the closely packed residues in the folding nucleus of CI2 may perhaps be optimised for the rate of folding and not for stability.     

Identification of kinetically hot residues in proteins

A number of recent studies called attention to the presence of kinetically important residues underlying the formation and stabilization of folding nuclei in proteins, and to the possible existence of a correlation between conserved residues and those participating in the folding nuclei. Here, we use the Gaussian network model (GNM), which recently proved useful in describing the dynamic characteristics of proteins for identifying the kinetically hot residues in folded structures. These are the residues involved in the highest frequency fluctuations near the native state coordinates. Their high frequency is a manifestation of the steepness of the energy landscape near their native state positions. The theory is applied to a series of proteins whose kinetically important residues have been extensively explored: chymotrypsin inhibitor 2, cytochrome c, and related C2 proteins. Most of the residues previously pointed out to underlie the folding process of these proteins, and to be critically important for the stabilization of the tertiary fold, are correctly identified, indicating a correlation between the kinetic hot spots and the early forming structural elements in proteins. Additionally, a strong correlation between kinetically hot residues and loci of conserved residues is observed. Finally, residues that may be important for the stability of the tertiary structure of CheY are proposed.     

Correlating Streptococcus mutans counts in saliva with plaque amount, gingival inflammation and caries experience in school children

The DNA methyltransferases, M.HhaI and M.TaqI, and catechol O-methyl-transferase (COMT) catalyze the transfer of a methyl group from the cofactor S-adenosyl-L-methionine (AdoMet) to carbon-5 of cytosine, to nitrogen-6 of adenine, and to a hydroxyl group of catechol, respectively. The catalytic domains of the bilobal proteins, M.HhaI and M.TaqI, and the entire single domain of COMT have similar folding with an alpha/beta structure containing a mixed central beta-sheet. The functional residues are located in equivalent regions at the carboxyl ends of the parallel beta-strands. The cofactor binding sites are almost identical and the essential catalytic amino acids coincide. The comparable protein folding and the existence of equivalent amino acids in similar secondary and tertiary positions indicate that many (if not all) AdoMet-dependent methyltransferases have a common catalytic domain structure. This permits tertiary structure prediction of other DNA, RNA, protein, and small-molecule AdoMet-dependent methyltransferases from their amino acid sequences.     

The subunit interfaces of oligomeric enzymes are conserved to a similar extent to the overall protein sequences

It is well established that, within families of homologous enzymes, amino acid residues that are involved in the chemistry of the reaction are highly conserved. To determine if residues at the subunit interface of oligomeric enzymes with shared active sites are also conserved, comparative analysis of five enzyme families was undertaken. For the chosen enzyme families, sequence data were available for a large number of proteins and a three-dimensional structure was known for at least two members of each family. The analysis indicates that the subunit interface and the hydrophobic core of proteins from all five families have diverged to a similar extent to the overall protein sequences.     

Phosphatidylinositol 4-kinases

Polyphosphoinositides are involved in many signal transduction pathways in eukaryotic cells. The first committed step is catalysed by phosphatidylinositol 4-kinase leading to the formation of phosphatidylinositol 4-phosphate. In the last four years, ten cDNA molecules have been cloned which code isoforms of phosphatidylinositol 4-kinase; some of which are highly related. Characteristically, they contain a C-terminal catalytic domain which is similar to that of (poly)phosphoinositide 3-kinases and to that of more distantly related lipid/protein kinases. Alignment has characterised cDNAs from Chaenorabditis, Dictyostelium and Schizostaphyloccus pombe as those of phosphatidylinositol 4-kinases also. All these lipid kinases are related to the superfamily of protein kinases. Several amino acids are highly conserved in catalytic domains of lipid and protein kinases. Employing the catalytic subunit of the cAMP-dependent protein kinase as template, these residues can be assigned functionally. On the basis of the alignment, a phylogenetic tree of the superfamily of phosphatidylinositol kinases has been constructed. Three families, the phosphatidylinositol 4-kinases, phosphoinositide 3-kinases, and the phosphatidylinositol related lipid/protein kinases, can be recognised. Each family comprises two subfamilies. The involvement of the phosphatidylinositol 4-kinases in signal transduction processes is summarised and a new hypothesis for the function of their isoforms in polyphosphoinositide signalling is presented. The involvement of phosphatidylinositol 4-kinases in formation of lipid-protein interactions with cytoskeleton proteins and the metabolism of polyphosphoinositide in the nucleus is discussed.     

Qualitative and quantitative analysis of the secondary structure of cytochrome C Langmuir-Blodgett films

HP1 integrase promotes site-specific recombination of the HP1 genome into that of Haemophilus influenzae. The isolated C-terminal domain (residues 165-337) of the protein interacts with the recombination site and contains the four catalytic residues conserved in the integrase family. This domain represents a novel fold consisting principally of well-packed alpha helices, a surface beta sheet, and an ordered 17-residue C-terminal tail. The conserved triad of basic residues and the active-site tyrosine are contributed by a single monomer and occupy fixed positions in a defined active-site cleft. Dimers are formed by mutual interactions of the tail of one monomer with an adjacent monomer; this orients active-site clefts antiparallel to each other.     

Insect prothoracicotropic hormone: a new member of the vertebrate growth factor superfamily

Prothoracicotropic hormone (PTTH) is a brain neurosecretory protein that controls insect development. PTTH of the silkmoth Bombyx mori is a homodimeric protein, the subunit of which consists of 109 amino acids. Clear-cut sequence similarity to any other proteins has not been observed. By disulfide-bond pattern analysis and modeling of the PTTH structure based on the known three-dimensional (3D) structures of growth factor family with cystine-knot motif, we propose that the PTTH protomer adopts the fold unique to the structural superfamily of the growth factors, beta-nerve growth factor (beta-NGF), transforming growth factor-beta 2 (TGF-beta 2), and platelet-derived growth factor-BB (PDGF-BB). The insect neurohormone PTTH appears to be a member of the growth factor superfamily, sharing a common ancestral gene with the three vertebrate growth factors, beta-NGF, TGF-beta 2 and PDGF-BB.     

Computer-assisted dissection of rolling circle DNA replication

A comparative analysis of the proteins involved in initiation and termination of rolling circle replication (RCR) was performed using computer-assisted methods of data based screening, motif search and multiple amino acid sequence alignment. Two vast classes of such proteins were delineated, one of these being associated with RCR proper, and the other with mobilization (conjugal transfer) of plasmid DNA. The common denominator of the two classes was found to be a conserved amino acid motif that consists of the sequence HisUHisUUU (U--bulky hydrophobic residue; hereafter HUH motif). Based on analogies with metalloenzymes, it is hypothesized that the two conserved His residues this motif may be involved in metal ion coordination required for the activity of the RCR and mobilization proteins. The proteins of the replication (Rep) class contained two additional conserved motifs, with the motif around the Tyr residue(s) forming the covalent link with nicked DNA being located C-proximally of the HUH motif. This class further split into two large superfamilies and several smaller families, with the proteins belonging to a single but not to different (super)families demonstrating statistically significant similarity to each other. Superfamily I, prototyped by the gene A proteins of small isometric single-stranded (ss) DNA bacteriophages, included also Rep proteins of P2-related double-stranded (ds) DNA bacteriophages, the small phage-plasmid hybrid phasyl, and several cyanobacterial and archaebacterial plasmids. These proteins contained two invariant Tyr residues separated by three partially conserved amino acids, suggesting that they all may share the cleavage-ligation mechanism proposed for phi X174 A protein and involving alternate covalent binding of both tyrosines to DNA (Van Mansfeld, A.D., Van Teeffelen, H.A., Baas, P.D., Jansz, H.S., 1986. Nucl. Acids Res. 14, 4229-4238). Superfamily II included Rep proteins of a number of ssDNA plasmids replicating mainly in gram-positive bacteria that unexpectedly were shown to be related to the Rep proteins of plant geminiviruses. Conservation of the "HUH" motif and a motif around the putative DNA-linking Tyr residue was observed also in the Rep proteins of animal parvoviruses containing linear ssDNA with a terminal hairpin and replicating via the rolling hairpin mechanism. The class of plasmid mobilization (Mob) proteins was characterized by the opposite orientation of the conserved motifs, with the (putative) DNA-linking Tyr being located N-proximally of the "HUH" motif.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protein folding and protein evolution: common folding nucleus in different subfamilies of c-type cytochromes?

Amino acid sequences of seven subfamilies of cytochromes c (mitochondrial cytochromes c, c1; chloroplast cytochromes c6, cf; bacterial cytochromes c2, c550, c551; in total 164 sequences) have been compared. Despite extensive homology within eukaryotic subfamilies, homology between different subfamilies is very weak. Other than the three heme-binding residues (Cys13, Cys14, His18, in numeration of horse cytochrome c) there are only four positions which are conserved in all subfamilies: Gly/Ala6, Phe/Tyr10, Leu/Val/Phe94 and Tyr/Trp/Phe97. In all 17 cytochromes c with known 3D-structures, these residues form a network of conserved contacts (6-94, 6-97, 10-94, 10-97 and 94-97). Especially strong is the contact between aromatic groups in positions 10 and 97, which corresponds to 13 interatomic contacts. As residues 6, 10 and residues 94, 97 are in (i, i+4) and (i, i+3) positions in the N and C-terminal helices, respectively, the above mentioned system of conserved contacts consists mainly of contacts between one turn of N-terminal helix and one turn of C-terminal helix. The importance of the contacts between interfaces of these helices has been confirmed by the existence of these contacts in both equilibrium and kinetic molten globule-like folding intermediates, as well as by mutational evidence that these contacts are involved in tight packing between the N and C-helices. Since these four residues are not involved in heme binding and have no other apparent functional role, their conservation in highly diverged cytochromes c suggests that they are of a critical importance for protein folding. The author assumes that they are involved in a common folding nucleus of all subfamilies of c-type cytochromes.     

Therapeutic opportunities from the pharmacological manipulation of the Fas system

The major facilitator superfamily (MFS) is one of the two largest families of membrane transporters found on Earth. It is present ubiquitously in bacteria, archaea, and eukarya and includes members that can function by solute uniport, solute/cation symport, solute/cation antiport and/or solute/solute antiport with inwardly and/or outwardly directed polarity. All homologous MFS protein sequences in the public databases as of January 1997 were identified on the basis of sequence similarity and shown to be homologous. Phylogenetic analyses revealed the occurrence of 17 distinct families within the MFS, each of which generally transports a single class of compounds. Compounds transported by MFS permeases include simple sugars, oligosaccharides, inositols, drugs, amino acids, nucleosides, organophosphate esters, Krebs cycle metabolites, and a large variety of organic and inorganic anions and cations. Protein members of some MFS families are found exclusively in bacteria or in eukaryotes, but others are found in bacteria, archaea, and eukaryotes. All permeases of the MFS possess either 12 or 14 putative or established transmembrane alpha-helical spanners, and evidence is presented substantiating the proposal that an internal tandem gene duplication event gave rise to a primordial MFS protein prior to divergence of the family members. All 17 families are shown to exhibit the common feature of a well-conserved motif present between transmembrane spanners 2 and 3. The analyses reported serve to characterize one of the largest and most diverse families of transport proteins found in living organisms.     

Chaos game representation of proteins

The present report proposes a new method for the chaos game representation (CGR) of different families of proteins. Using concatenated amino acid sequences of proteins belonging to a particular family and a 12-sided regular polygon, each vertex of which represents a group of amino acid residues leading to conservative substitutions, the method can generate the CGR of the family and allows pictorial representation of the pattern characterizing the family. An estimation of the percentages of points plotted in different segments of the CGR (grid points) allows quantification of the nonrandomness of the CGR patterns generated. The CGRs of different protein families exhibited distinct visually identifiable patterns. This implies that different functional classes of proteins follow specific statistical biases in the distribution of different mono-, di-, tri-, or higher order peptides along their primary sequences. The potential of grid counts as the discriminative and diagnostic signature of a family of proteins is discussed.     

Identification of spike protein residues of murine coronavirus responsible for receptor-binding activity by use of soluble receptor-resistant mutants

We previously demonstrated by site-directed mutagenesis analysis that the amino acid residues at positions 62 and 214 to 216 in the N-terminal region of mouse hepatitis virus (MHV) spike (S) protein are important for receptor-binding activity (H. Suzuki and F. Taguchi, J. Virol. 70:2632-2636, 1996). To further identify the residues responsible for the activity, we isolated the mutant viruses that were not neutralized with the soluble form of MHV receptor proteins, since such mutants were expected to have mutations in amino acids responsible for receptor-binding activity. Five soluble-receptor-resistant (srr) mutants isolated had mutations in a single amino acid at three different positions: one was at position 65 (Leu to His) (srr11) in the S1 subunit and three were at position 1114 (Leu to Phe) (srr3, srr4, and srr7) and one was at position 1163 (Cys to Phe) (srr18) in the S2 subunit. The receptor-binding activity examined by a virus overlay protein blot assay and by a coimmunoprecipitation assay showed that srr11 S protein had extremely reduced binding activity, while the srr7 and srr18 proteins had binding activity similar to that of wild-type cl-2 protein. However, when cell surface receptors were used for the binding assay, all srr mutants showed activity similar to that of the wild type or only slightly reduced activity. These results, together with our previous observations, suggest that amino acids located at positions 62 to 65 of S1, a region conserved among the MHV strains examined, are important for receptor-binding activity. We also discuss the mechanism by which srr mutants with a mutation in S2 showed high resistance to neutralization by a soluble receptor, despite their sufficient level of binding to soluble receptors.     

Scanning mutagenesis of transmembrane domain 3 of the M1 muscarinic acetylcholine receptor

Scanning mutagenesis of transmembrane domain 3 of the M1 muscarinic acetylcholine receptor has revealed a highly-differentiated alpha-helical structure. Lipid-facing residues are distinguished from a patch of residues which selectively stabilise the ground state of the receptor, and from a band of amino acids extending the full length of the helix, which contribute to the active agonist-receptor-G protein complex. The most important residues are strongly conserved in the GPCR superfamily.     

Probing the structure and mechanism of Ras protein with an expanded genetic code

Mutations in Ras protein at positions Gly12 and Gly13 (phosphate-binding loop L1) and at positions Ala59, Gly60, and Gln61 (loop L4) are commonly associated with oncogenic activation. The structural and catalytic roles of these residues were probed with a series of unnatural amino acids that have unusual main chain conformations, hydrogen bonding abilities, and steric features. The properties of wild-type and transforming Ras proteins previously thought to be uniquely associated with the structure of a single amino acid at these positions were retained by mutants that contained a variety of unnatural amino acids. This expanded set of functional mutants provides new insight into the role of loop L4 residues in switch function and suggests that loop L1 may participate in the activation of Ras protein by effector molecules.     

Fast and one-step folding of closely and distantly related homologous proteins of a four-helix bundle family

Bovine acyl-coenzyme A binding protein is a four-helix bundle protein belonging to a group of homologous eukaryote proteins that binds medium and long-chain acyl-coenzyme A esters with a very high affinity. The three-dimensional structure of both the free and the ligated protein together with the folding kinetics have been described in detail for the bovine protein and with four new sequences reported here, a total of 16 closely related sequences ranging from yeasts and plants to human are known. The kinetics of folding and unfolding in different concentrations of guanidine hydrochloride together with equilibrium unfolding have been measured for bovine, rat and yeast acyl-coenzyme A binding protein. The bovine and rat sequences are closely related whereas the yeast is more distantly related to these. In addition to the three natural variants, kinetics of a bovine mutant protein, Tyr31 --> Asn, have been studied. Both the folding and unfolding rates in water of the yeast protein are 15 times faster than those of bovine. The folding rates in water of the two mammalian forms, rat and bovine, are similar, though still significantly different. A faster unfolding rate both for rat and the bovine mutant protein results from a lower stability of the native states of these. These hydrophobic regions, mini cores, have been identified in the three-dimensional structure of the bovine protein and found to be formed primarily by residues that have been conserved throughout the entire eukaryote evolution from yeasts to both plants and mammals as seen in the sample of 16 sequences. The conserved residues are found to stabilize helix-helix interactions and serve specific functional purposes for ligand binding. The fast one-step folding mechanism of ACBP has been shown to be a feature that seems to be maintained throughout evolution despite numerous differences in sequence and even dramatic differences in folding kinetics and protein stability. The protein study raises the question to what extent does the conserved hydrophobic residues provide a scaffold for an efficient one-step folding mechanism.     

Exploring the folding funnel of a polypeptide chain by biophysical studies on protein fragments

We are examining possible roles of native and non-native interactions in early events in protein folding by a systematic analysis of the structures of fragments of proteins whose folding pathways are well characterised. Seven fragments of the 110-residue protein barnase, corresponding to the progressive elongation from its N terminus, have been characterised by a battery of biophysical and spectroscopic methods. Barnase is a multi-modular protein that folds via an intermediate in which the C-terminal region of its major alpha-helix (alpha-helix1, residues Thr6-His18) is substantially formed as is also its anti-parallel beta-sheet, centred around a beta-hairpin (residues Ser92-Leu95). Fragments up to, and including, residues 1-95 (fragment B95), appeared to be mainly disordered, although a small amount of helical secondary structure in each was inferred from far-UV CD experiments, and fluorescence studies indicated some native-like tertiary interactions in B95. The largest fragment (residues 1-105, B105) is compactly folded. The secondary structure in alpha-helix1 in the seven fragments was found by NMR to increase with increasing chain length faster than the build-up of tertiary interactions, indicating that alpha-helix1 is being stabilised by non-native interactions. This behaviour contrasts with that in fragments of the 64-residue chymotrypsin inhibitor 2 (CI2), in which tertiary and secondary structures build up in parallel with increasing length. CI2 consists of a single module of structure that folds without a detectable intermediate. The largest fragment of barnase, B105, has interactions that resemble its folding intermediate, whereas one of the largest fragments of CI2 (residues 1-60) resembles the folding transition state. The folding pathways of both proteins are consistent with a scheme in which there are low levels of native-like secondary structure in the denatured state that become stabilised by long-range interactions as folding proceeds. Neither protein forms a stable fold when lacking the last ten residues at the C terminus. Since at least 20 amino acid residues are bound to the ribosome during protein biosynthesis, these small proteins do not fold until they have left the ribosome, and so the studies of the folding of such proteins in vitro may be relevant to their folding in vivo, especially as the molecular chaperone GroEL binds only weakly to denatured CI2 and does not discernibly alter the folding mechanism of barnase.     

Structure of the zinc endoprotease from Streptomyces caespitosus

A zinc endoprotease produced by Streptomyces caespitosus (ScNP) specifically hydrolyzes the peptide bond at the imino side of aromatic residues and is the smallest protease found to date. Although ScNP carries the zinc-binding sequence HEXXH, its primary structure of 132 amino acid residues differs from those of other known zinc metalloendoproteases. X-ray structural analysis of ScNP at 1.6 A resolution revealed that despite a lack of sequence homology, the common topological feature of main-chain folding and a beta-turn containing methionine, which is a feature of the zinc metalloendoprotease superfamily of metzincins, is conserved in ScNP. The zinc atom of ScNP is tetrahedrally ligated by the two histidines in the HEXXH sequence, an aspartate residue and a water molecule. Thus, ScNP represents a novel subfamily of metzincins with a HEXXHXXGXXD zinc-binding sequence. A plausible substrate recognition pocket to which aromatic residues bind is located near the catalytic zinc ion.     

Identification of a new member of transforming growth factor-beta superfamily in Drosophila: the first invertebrate activin gene

Activins, a subgroup of the transforming growth factor-beta (TGF-beta) superfamily, have been extensively studied in vertebrates for their roles in growth and development. However, activins are not thought to be expressed in invertebrates. The identification of the first invertebrate activin gene is reported here. A genomic clone representing 102 F region of the Drosophila chromosome 4 is found to encode a putative activin beta. The predicted protein sequence has a multibasic protease site that would generate a mature C-terminal peptide containing 113 amino acids showing > 60% similarity to the vertebrate activin beta B (inhibin beta B) sequences. A TGF-beta family signature as well as all 9 cysteine residues conserved in the vertebrate activins are also present in this mature peptide sequence. Northern blot and RT-PCR analyses indicated that the activin beta gene is expressed in embryo, larva and adult stages of Drosophila.