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PubMed2006

Analysing the ability to retain sidechain hydrogen-bonds in mutant proteins.

MOTIVATION: Hydrogen bonds are one of the most important inter-atomic interactions in biology. Previous experimental, theoretical and bioinformatics analyses have shown that the hydrogen bonding potential of amino acids is generally satisfied and that buried unsatisfied hydrogen-bond-capable residues are destabilizing. When studying mutant proteins, or introducing mutations to residues involved in hydrogen bonding, one needs to know whether a hydrogen bond can be maintained. Our aim, therefore, was to develop a rapid method to evaluate whether a sidechain can form a hydrogen-bond. RESULTS: A novel knowledge-based approach was developed in which the conformations accessible to the residues involved are taken into account. Residues involved in hydrogen bonds in a set of high resolution crystal structures were analyzed and this analysis is then applied to a given protein. The program was applied to assess mutations in the tumour-suppressor protein, p53. This raised the number of distinct mutations identified as disrupting sidechain-sidechain hydrogen bonding from 181 in our previous analysis to 202 in this analysis.

Algorithms↗
PubMed2002

RPD3 is required for the inactivation of yeast ribosomal DNA genes in stationary phase.

rRNA transcription in Saccharomyces cerevisiae is performed by RNA polymerase I and regulated by changes in growth conditions. During log phase, approximately 50% of the ribosomal DNA (rDNA) genes in each cell are transcribed and maintained in an open, psoralen-accessible conformation. During stationary phase, the percentage of open rDNA genes is greatly reduced. In this study we found that the Rpd3 histone deacetylase was required to inactivate (close) individual rDNA genes as cells entered stationary phase. Even though approximately 50% of the rDNA genes remained open during stationary phase in rpd3Delta mutants, overall rRNA synthesis was still reduced. Using electron microscopy of Miller chromatin spreads, we found that the number of RNA polymerases transcribing each open gene in the rpd3Delta mutant was significantly reduced when cells grew past log phase. Bulk levels of histone H3 and H4 acetylation were reduced during stationary phase in an RPD3-dependent manner. However, histone H3 and H4 acetylation was not significantly altered at the rDNA locus in an rpd3Delta mutant. Rpd3 therefore regulates the number of open rDNA repeats.

Acetylation↗
PubMed2000

The role of hydrophobicity patterns in prion folding as revealed by recurrence quantification analysis of primary structure.

It has been suggested that the number and strength of local contacts are the major factors governing conformation accessibility of model two ground-state polypeptide chains. This phenomenology has been posed as a possible factor influencing prion folding. To test this conjecture, recurrence quantification analysis was applied to two model 36mers, and the Syrian hamster prion protein. A unique divergence of the radius function for the recurrence quantification variable %DET of hydrophobicity patterns was observed for both 36mers, and in a critical region of the hamster prion protein. This divergence suggests a partition between strong short- and long-range hydrophobicity patterns, and may be an important factor in prion phenomenology, along with other global thermodynamic factors.

Algorithms↗
PubMed1975

Self-assembly of biological macromolecules.

The genetic apparatus of the cell is responsible for the accurate biosynthesis of the primary structure of macromolecules which then spontaneously fold up and, in certain circumstances, aggregate to yield the complex tertiary and quaternary structures of the biologically active molecules. Structures capable of self-assembly in this range from simple monomers through oligomers to complex multimeric structures that may contain more than one type of polypeptide chain and components other than protein. It is becoming clear that even with the simpler monomeric enzymes there is becoming clear that even with the simpler monomeric enzymes there is a kinetically determined pathway for the folding process and that a folded protein must now be regarded as the minimum free energy form of the kinetically accessible conformations. It is argued that the denatured subunits of oligomeric enzymes are likely to fold to something like their final structure before aggregating to give the native quaternary structure and the available evidence would suggest that this is so. The importance of nucleation events and stable intermediates in the self-assembly of more complex structures is clear. Many self-assembling structures contain only identical subunits and symmetry arguments are very successful in accounting for the structures formed. Because proteins are themselves complex molecules and not inelastic geometric objects, the rules of strict symmetry can be bent and quasi-equivalent bonding between subunits permitted. This possibility is frequently employed in biological structures. Conversely, symmetry arguments can offer a reliable means of choosing between alternative models for a given structure. It can be seen that proteins gain stability by growing larger and it is argued in evolutionary terms that aggregation of subunits is the preferred way to increase the size of proteins. The possession of quaternary structure by enzymes allows conferral of other biologically important properties, such as cooperativity between active sites, changes of specificity, substrate channelling and sequential reactions within a multi-enzyme complex. Comparison is made of the invariant subunit compositions of the simpler oligomeric enzymes with the variation evidently open to, say, the 2-oxoacid dehydrogenase complexes of E. coli. With viruses, on the other hand, the function of the quaternary structure is to package nucleic acid and, as an example, the assembly and breakdown of tobacco mosaic virus is discussed. Attention is drawn to the possible ways in which the principles of self-assembly can be extended to make structures more complicated than those that can be formed by simple aggregation of the comonent parts.

Binding Sites↗
PubMed2006

Electric dipole moments of water clusters from a beam deflection measurement.

The response of (H2O)(n=3-18) clusters to an electric field is studied by beam deflection. All clusters deflect uniformly, behaving as polarizable particles. The effective polarizabilities exceed the electronic component and increase as the clusters are cooled, revealing a large permanent dipole contribution. The results resolve a discrepancy concerning the polarity of water clusters and show that all species access conformations with moments exceeding 1 D. The data show no evidence for a freezing transition down to approximately 120 K, but suggest a shift in the conformer arrangement at n=8-9.

Journal Article↗
PubMed1999

Actin surface structure revealed by antibody imprints: evaluation of phage-display analysis of anti-actin antibodies.

Phage-display peptide library analysis of an anti-F actin polyclonal antibody identified 12 amino acid residues of actin that appear, in its X-ray crystal structure, to be grouped together in a surface accessible conformational epitope. Phage epitope mapping was carried out by isolating immune complexes containing members of the J404 nonapeptide phage-display library formed in diluted antiserum and isolated on a protein A affinity matrix. Immunoreactive clones were grown as plaques, replica plated onto nitrocellulose, and labeled with anti-actin immune serum. One hundred and forty-four positively staining clones identified in this way were sequenced. Of these, 54 displayed peptides with sequence similarities. When the most abundantly selected sequence, KQTWQQLWD, was produced as a synthetic peptide and derivatized to ovalbumin, the complex was strongly recognized by the antiserum on Western blots and inhibited the binding of the antibody to immobilized F-actin by 60%. A scrambled version of this sequence WQDK WLQTQ, when coupled to ovalbumin, was not recognized by the antiserum and minimally inhibited binding of antiserum to immobilized F-actin by 10%. KQTWQQLWD contained four residues that corresponded, in frame, to a highly conserved six residue region of the chicken beta-actin sequence 351TFQQMW356 (identical residues are shown in bold). Examination of the rabbit skeletal muscle X-ray crystal structure suggested that within a 15 A radius of W356, nine additional residues were arranged on the actin surface in such a way that they could be mimicked by several of the selected phage sequences with root-mean-square deviation fits of 2.1-2.5 A. We conclude that phage-display analysis can provide information about the relative location of amino acids on the surfaces of proteins using antibody imprints of the protein surface structure.

Actins↗
PubMed1996

Protein engineering studies of A-chain loop 47-56 of Escherichia coli heat-labile enterotoxin point to a prominent role of this loop for cytotoxicity.

Heat-labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, is a close relative of cholera toxin (CT). These two toxins share approximately 80% sequence identity, and consists of one 240-residue A chain and five 103-residue B subunits. The B pentamer is responsible for GM1 receptor recognition, whereas the A subunit carries out an ADP-ribosylation of an arginine residue in the G protein, Gs alpha, in the epithelial target cell. This paper explores the importance of specific amino acids in loop 47-56 of the A subunit. This loop was observed to be highly mobile in the inactive R7K mutant of the A subunit. The position of the loop in wild-type protein is such that it might require considerable reorganization during substrate binding and is likely to have a crucial role in substrate binding. Five single-site substitutions have been made in the LT-A subunit 47-56 loop to investigate its possible role in the enzymatic activity and toxicity of LT and CT. The wild-type residues Thr-50 and Val-53 were replaced either by a glycine or by a proline. The glycine substitutions were intended to increase the mobility of this active-site loop, and the proline substitutions were intended to decrease the mobility of this same loop by restricting the accessible conformational space. Under the hypothesis that mobility of the loop is important for catalysis, the glycine-substitution mutants T50G and V53G would be expected to exhibit activity equal to or greater than that of the wild-type A subunit, while the proline substitution mutants T50P and T53P would be less active. Cytotoxicity assays showed, however, that all four of these mutants were considerably less active than wild-type LT. These results lend support for assignment of a prominent role to loop 47-56 in catalysis by LT and CT.

Animals↗
PubMed2004

Neuroserpin Portland (Ser52Arg) is trapped as an inactive intermediate that rapidly forms polymers: implications for the epilepsy seen in the dementia FENIB.

The dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB) is caused by point mutations in the neuroserpin gene. We have shown a correlation between the predicted effect of the mutation and the number of intracerebral inclusions, and an inverse relationship with the age of onset of disease. Our previous work has shown that the intraneuronal inclusions in FENIB result from the sequential interaction between the reactive centre loop of one neuroserpin molecule with beta-sheet A of the next. We show here that neuroserpin Portland (Ser52Arg), which causes a severe form of FENIB, also forms loop-sheet polymers but at a faster rate, in keeping with the more severe clinical phenotype. The Portland mutant has a normal unfolding transition in urea and a normal melting temperature but is inactive as a proteinase inhibitor. This results in part from the reactive loop being in a less accessible conformation to bind to the target enzyme, tissue plasminogen activator. These results, with those of the CD analysis, are in keeping with the reactive centre loop of neuroserpin Portland being partially inserted into beta-sheet A to adopt a conformation similar to an intermediate on the polymerization pathway. Our data provide an explanation for the number of inclusions and the severity of dementia in FENIB associated with neuroserpin Portland. Moreover the inactivity of the mutant may result in uncontrolled activity of tissue plasminogen activator, and so explain the epileptic seizures seen in individuals with more severe forms of the disease.

Amino Acid Substitution↗
PubMed1999

Specificity determinants of proteolytic processing of Aspergillus PacC transcription factor are remote from the processing site, and processing occurs in yeast if pH signalling is bypassed.

The Aspergillus nidulans transcription factor PacC, which mediates pH regulation, is proteolytically processed to a functional form in response to ambient alkaline pH. The full-length PacC form is unstable in the presence of an operational pH signal transduction pathway, due to processing to the relatively stable short functional form. We have characterized and used an extensive collection of pacC mutations, including a novel class of "neutrality-mimicking" pacC mutations having aspects of both acidity- and alkalinity-mimicking phenotypes, to investigate a number of important features of PacC processing. Analysis of mutant proteins lacking the major translation initiation residue or truncated at various distances from the C terminus showed that PacC processing does not remove N-terminal residues, indicated that processing yields slightly heterogeneous products, and delimited the most upstream processing site to residues approximately 252 to 254. Faithful processing of three mutant proteins having deletions of a region including the predicted processing site(s) and of a fourth having 55 frameshifted residues following residue 238 indicated that specificity determinants reside at sequences or structural features located upstream of residue 235. Thus, the PacC protease cuts a peptide bond(s) remote from these determinants, possibly thereby resembling type I endonucleases. Downstream of the cleavage site, residues 407 to 678 are not essential for processing, but truncation at or before residue 333 largely prevents it. Ambient pH apparently regulates the accessibility of PacC to proteolytic processing. Alkalinity-mimicking mutations L259R, L266F, and L340S favor the protease-accessible conformation, whereas a protein with residues 465 to 540 deleted retains a protease-inaccessible conformation, leading to acidity mimicry. Finally, not only does processing constitute a crucial form of modulation for PacC, but there is evidence for its conservation during fungal evolution. Transgenic expression of a truncated PacC protein, which was processed in a pH-independent manner, showed that appropriate processing can occur in Saccharomyces cerevisiae.

Amino Acid Sequence↗
PubMed2001

Ambient pH signaling regulates nuclear localization of the Aspergillus nidulans PacC transcription factor.

The Aspergillus nidulans zinc finger transcription factor PacC is activated by proteolytic processing in response to ambient alkaline pH. The pH-regulated step is the transition of full-length PacC from a closed to an open, protease-accessible conformation. Here we show that in the absence of ambient pH signaling, the C-terminal negative-acting domain prevents the nuclear localization of full-length closed PacC. In contrast, the processed PacC form is almost exclusively nuclear at any ambient pH. In the presence of ambient pH signaling, the fraction of PacC that is in the open conformation but has not yet been processed localizes to the nucleus. Therefore, ambient alkaline pH leads to an increase in nuclear PacC by promoting the proteolytic elimination of the negative-acting domain to yield the processed form and by increasing the proportion of full-length protein that is in the open conformation. These findings explain why mutations resulting in commitment of PacC to processing irrespective of ambient pH lead to permanent PacC activation and alkalinity mimicry. A nuclear import signal that targets Escherichia coli beta-galactosidase to the nucleus has been located to the PacC zinc finger region. A mutation abolishing DNA binding does not prevent nuclear localization of the processed form, showing that PacC processing does not lead to nuclear localization by passive diffusion of the protein made possible by the reduction in size, followed by retention in the nucleus after DNA binding.

Amino Acids↗
PubMed1992

Rabbit lung surfactant protein A gene: identification of a lung-specific DNase I hypersensitive site.

Expression of the gene encoding pulmonary surfactant protein A, SP-A, is lung specific and developmentally and hormonally regulated. Previously, we observed that SP-A gene transcription is initiated in fetal rabbit lung after day 21 of gestation and reaches maximal levels by day 28. In the present study, a cDNA specific for rabbit SP-A was used to isolate the SP-A gene from a rabbit genomic library. A 7.6-kb fragment containing the entire structural gene and approximately 380 bp of 5'-flanking DNA was isolated and characterized. The transcription initiation site, mapped by primer extension analysis, was localized 23 bp downstream of a putative TATA element. The structural gene is composed of five exons and four introns. The first exon encodes the 5'-untranslated region of the mRNA; the translation initiation site is in exon II, and exon V contains the two polyadenylation sites that give rise to the 2.0- and 3.0-kb species of SP-A mRNA. A potential adenosine 3',5'-cyclic monophosphate (cAMP)-regulatory element (CRE) was identified at -261 bp, and sequences with homology to glucocorticoid-regulatory element (GRE) half-sites were found at -150 and -190 bp upstream of the transcription initiation site and within the first intron. A DNase I hypersensitive site was identified in genomic DNA isolated from 21- and 28-day fetal and adult rabbit lung tissues. This site was mapped within the 5'-flanking region of the SP-A gene, at approximately -80 to -180 bp upstream of the transcription initiation site. The absence of this hypersensitive site in genomic DNA of liver, kidney, and heart tissues suggests that altered chromatin structure may serve a role in lung-specific SP-A gene expression. The presence of this tissue-specific DNase I hypersensitive site in lung nuclei from 21-day gestational age fetal rabbits suggests that the SP-A gene may exist in an accessible conformation prior to the time of transcription initiation.

Amino Acid Sequence↗
PubMed2006

Cytochrome C in a dry trehalose matrix: structural and dynamical effects probed by x-ray absorption spectroscopy.

We report on the structure and dynamics of the Fe ligand cluster of reduced horse heart cytochrome c in solution, in a dried polyvinyl alcohol (PVA) film, and in two trehalose matrices characterized by different contents of residual water. The effect of the solvent/matrix environment was studied at room temperature using Fe K-edge x-ray absorption fine structure (XAFS) spectroscopy. XAFS data were analyzed by combining ab initio simulations and multi-parameter fitting in an attempt to disentangle structural from disorder parameters. Essentially the same structural and disorder parameters account adequately for the XAFS spectra measured in solution, both in the absence and in the presence of glycerol, and in the PVA film, showing that this polymer interacts weakly with the embedded protein. Instead, incorporation in trehalose leads to severe structural changes, more prominent in the more dried matrix, consisting of 1), an increase up to 0.2 A of the distance between Fe and the imidazole N atom of the coordinating histidine residue and 2), an elongation up to 0.16 A of the distance between Fe and the fourth-shell C atoms of the heme pyrrolic units. These structural distortions are accompanied by a substantial decrease of the relative mean-square displacements of the first ligands. In the extensively dried trehalose matrix, extremely low values of the Debye Waller factors are obtained for the pyrrolic and for the imidazole N atoms. This finding is interpreted as reflecting a drastic hindering in the relative motions of the Fe ligand cluster atoms and an impressive decrease in the static disorder of the local Fe structure. It appears, therefore, that the dried trehalose matrix dramatically perturbs the energy landscape of cytochrome c, giving rise, at the level of local structure, to well-resolved structural distortions and restricting the ensemble of accessible conformational substates.

Animals↗
PubMed1975

Interaction of L-alpha-palmitoyl lysophosphatidylcholine with the AI polypeptide of high density lipoprotein.

The AI polypeptide chain from human high density serum lipoprotein has two accessible conformational states in aqueous solution. L-alpha-Palmitoyl lysophosphatidylcholine induces the transition between these two states at an equilibrium concentration of ligand of 2 X 10(-5)M, and the protein has a maximum binding capacity of 95 to 100 mol of lipid/mol of protein. The present study, together with previous investigations in this laboratory, suggests that the conformational state of AI in the presence of high levels of bound amphiphiles is similar to the in vivo state, and further, that this complex does not result from the insertion of AI into amphiphilic micelles. The mode of interaction of AI with amphiphilic ligands is shown to be significantly different from that of membrane proteins thus far investigated.

Binding Sites↗
PubMed1991

Actions of three clostridial IgA proteases on distinct forms of immunoglobulin A molecules.

Three bacterial species of Clostridium (septicum, tertium and sporogenes) were identified to produce extracellular proteases cleaving IgA to Fab and Fc fragments, as demonstrated by SDS-PAGE and immunoelectrophoretic procedures. These enzymes acted on monometric IgA1 paraproteins and normal serum IgA1 but had no activity on IgA2 paraproteins and intact secretory IgA1 from human colostrum. Their action on polyclonal serum IgA1 suggested the absence of neutralizing anti-clostridial IgA protease activity. Although the enzymes were shown not to act on secretory IgA1, they were, however, able to digest free alpha-heavy chains of the dimeric IgA molecules. Susceptibility of the alpha-heavy chain to the proteases was more likely due to the change to a more accessible conformation than because of the absence of neutralizing anti-enzymic activity.

Clostridium↗
PubMed1984

Naturally occurring autoantibodies to exoplasmic and cryptic regions of band 3 protein, the major integral membrane protein of human red blood cells.

Immunoglobulin G (IgG) of healthy human blood donors and IgG from pooled sera (Sandoglobulin) contain natural (auto)antibodies to band 3 protein, the major integral membrane protein of human red blood cells. Affinity-purified and 125I-iodinated anti-band 3 antibodies bound specifically to band 3 protein on immunoblots from membrane proteins in the presence of unlabeled, absorbed IgG. Purified (auto)antibodies also bound nonspecifically to band 4.2 and weakly to band 5 and 6, when assayed with second antibody and 125I-iodinated protein A. The antibodies were directed to regions of band 3 protein that were cryptic and in part exoplasmic but with a low accessibility to surface modifications. The antigenic sites were located within the 65K, but not the 38K-dalton chymotryptic fragment of band 3 protein. Antigenic band 3 protein was equally present in membranes of young and senescent red cells. Hence, if these antibodies were involved in recognizing a few exoplasmic sites of band 3 protein on senescent red cells, antigen exposure would require alterations in band 3 accessibility (conformation, topology) rather than an enzymatic generation of antigenic sites.

Adult↗
PubMed1982

Accessibility of newly synthesized chromatin to histone acetylase.

Pulse-chase experiments with [3H]lysine-labeled tissue culture cells reveal that newly synthesized nucleosomal histones H2B, H3, H4 (and possibly H2A) in chromatin are more accessible to histone acetylase in vivo than are older, pre-existing histones. Thus, when rat hepatoma cells are first pulse-labeled and then incubated in medium containing n-butyrate which blocks histone deacetylation, these newly synthesized histones become acetylated to a far greater extent than do their older homologues. As judged by its increased susceptibility to acetylation, the new chromatin matures at a surprisingly slow rate, the estimated half-time for maturation being about 35 min. Based on this data, we suggest that newly synthesized chromatin is in a relatively extended, accessible conformation, and that it slowly returns to a more compact conformation as it matures.

Acetyltransferases↗
PubMedDate not supplied

[Characteristics of isolating influenza virus hemagglutinin using purified bromelin].

Gel filtration and ion-exchange chromatography were used to isolate a fraction with the highest proteolytic activity and virtually lacking glycosidases from a commercial bromeline preparation ("Serva", grade B) showing, in addition to proteolytic, also glycosidase activity. The utilization of purified bromeline allows isolation of hemagglutinin without residual neuraminidase activity from influenza virus of various serotypes (H1, H1, and H3). The results are discussed on the basis of an assumption of analogous localization but different conformation accessibility of bromeline-sensitive parts of the light chains of influenza viruses of different subtypes, and a possible role of the carbohydrate component of surface glycoproteins, hemagglutinin and neuraminidase of influenza virus virions.

Bromelains↗
PubMed1997

Theoretical and algorithmical optimization of the dead-end elimination theorem.

The dead-end elimination theorem has proved to be a powerful method to reduce the theoretically accessible conformational space when modeling protein side chains by using a rotameric representation of possible conformations. In this work, theoretical details about variants to the original criterion are discussed. We also provide information on how the equations can be algorithmically implemented in such a way that both computational performance and structural accuracy are optimized. In addition, we discuss the theoretical and practical aspects of three new methods called the "bottom line theorem", dead-end elimination assisted by local modeling and a combinatorial search combined with conventional dead-end elimination. It is shown that the algorithm in its current from enables the determination of the global minimum energy side chain conformation of large proteins on a time scale of hours while for small proteins of up to 30 residues the calculations are done on a time scale of seconds. The latter opens a way to combine a main chain sampling algorithm with the dead-end elimination method to locally model entire fragments of a protein chain.

Algorithms↗