Consecutive Residues Research Articles

ACA‐specific RNA sequence recognition is acquired via the loop 2 region of MazF mRNA interferase

MazF is an mRNA interferase that cleaves mRNAs at a specific RNA sequence. MazF from E. coli (MazF-ec) cleaves RNA at A^CA. To date, a large number of MazF homologs that cleave RNA at specific three- to seven-base sequences have been identified from bacteria to archaea. MazF-ec forms a dimer, in which the interface between the two subunits is known to be the RNA substrate-binding site. Here, we investigated the role of the two loops in MazF-ec, which are closely associated with the interface of the MazF-ec dimer. We examined whether exchanging the loop regions of MazF-ec with those from other MazF homologs, such as MazF from Myxococcus xanthus (MazF-mx) and MazF from Mycobacterium tuberculosis (MazF-mt3), affects RNA cleavage specificity. We found that exchanging loop 2 of MazF-ec with loop 2 regions from either MazF-mx or MazF-mt3 created a new cleavage sequence at (A/U)(A/U)AA^C in addition to the original cleavage site, A^CA, whereas exchanging loop 1 did not alter cleavage specificity. Intriguingly, exchange of loop 2 with 8 or 12 consecutive Gly residues also resulted in a new RNA cleavage site at (A/U)(A/U)AA^C. The present study suggests a method for expanding the RNA cleavage repertoire of mRNA interferases, which is crucial for potential use in the regulation of specific gene expression and for biotechnological applications.

Proline: The Distribution, Frequency, Positioning, and Common Functional Roles of Proline and Polyproline Sequences in the Human Proteome

Proline is an anomalous amino acid. Its nitrogen atom is covalently locked within a ring, thus it is the only proteinogenic amino acid with a constrained phi angle. Sequences of three consecutive prolines can fold into polyproline helices, structures that join alpha helices and beta pleats as architectural motifs in protein configuration. Triproline helices are participants in protein-protein signaling interactions. Longer spans of repeat prolines also occur, containing as many as 27 consecutive proline residues. Little is known about the frequency, positioning, and functional significance of these proline sequences. Therefore we have undertaken a systematic bioinformatics study of proline residues in proteins. We analyzed the distribution and frequency of 687,434 proline residues among 18,666 human proteins, identifying single residues, dimers, trimers, and longer repeats. Proline accounts for 6.3% of the 10,882,808 protein amino acids. Of all proline residues, 4.4% are in trimers or longer spans. We detected patterns that influence function based on proline location, spacing, and concentration. We propose a classification based on proline-rich, polyproline-rich, and proline-poor status. Whereas singlet proline residues are often found in proteins that display recurring architectural patterns, trimers or longer proline sequences tend be associated with the absence of repetitive structural motifs. Spans of 6 or more are associated with DNA/RNA processing, actin, and developmental processes. We also suggest a role for proline in Kruppel-type zinc finger protein control of DNA expression, and in the nucleation and translocation of actin by the formin complex.

Skeletal Calsequestrin - Calcium Interaction: Role of Acidic C-Terminus

Skeletal isoform of calsequestrin (CASQ1) is expressed primarily in fast twitch skeletal muscles in all vertebrates and buffers Ca2+ inside the sarcoendoplasmic reticulum (SR), the intracellular Ca2+ store. CASQ1 has a very unique C-terminus composed sole of aspartic acid residues. Presence of more than 10 consecutive aspartic acid residues in a protein sequence [referred as consecutive aspartate stretch (CAS)] is a very rare feature that is found only in about 20 proteins in the human genome. However, the role of CAS in CASQ1 function has not been investigated. Here we applied computational approach to understand the role of CAS in Ca2+-binding. The recent structure of CASQ1 has resolved the structure of its whole protein except for the CAS. We prepared the model by adding the CAS residues and performed molecular dynamics simulations for 50 nanoseconds in the presence of various Ca2+ concentrations. Our study shows that the CAS assumes a compact structure at higher Ca2+ concentrations and indicates that the CAS might work as a metal sensor. We found that the CAS undergoes maximal Ca2+-binding before the rest of the surface is saturated. The study revealed various Ca2+-binding sites with differing affinities and geometry. Interestingly, some sites are Ca2+-concentrations dependent while some others are independent of Ca2+ concentration. The low affinity sites of CASQ1 bind Ca2+ transiently that is mediated by water molecules and can dissociate quickly to support Ca2+-release during contraction. These studies collectively indicate the CAS works as a Ca2+-sensor that may be a novel metal sensing motif. We propose the term “Dn-motif” for CAS.

Structural and thermodynamic insight into phenylalanine hydroxylase from the human pathogen Legionella pneumophila

Phenylalanine hydroxylase from Legionella pneumophila (lpPAH) has a major functional role in the synthesis of the pigment pyomelanin, which is a potential virulence factor. We present here the crystal structure of lpPAH, which is a dimeric enzyme that shows high thermostability, with a midpoint denaturation temperature of 79 °C, and low substrate affinity. The structure revealed a dimerization motif that includes ionic interactions and a hydrophobic core, composed of both β-structure and a C-terminal region, with the specific residues (P255, P256, Y257 and F258) interacting with the same residues from the adjacent subunit within the dimer. This unique dimerization interface, together with a number of aromatic clusters, appears to contribute to the high thermal stability of lpPAH. The crystal structure also explains the increased aggregation of the enzyme in the presence of salt. Moreover, the low affinity for substrate l-Phe could be explained from three consecutive glycine residues (G181, 182, 183) located at the substrate-binding site. This is the first structure of a dimeric bacterial PAH and provides a framework for interpreting the molecular and kinetic properties of lpPAH and for further investigating the regulation of the enzyme.

The intrinsically disordered C-terminal region of Arabidopsis thaliana TCP8 transcription factor acts both as a transactivation and self-assembly domain

TCPs are plant specific transcription factors with non-canonical basic helix-loop-helix domains. While Arabidopsis thaliana has 24 TCPs involved in cell proliferation and differentiation, their mode of action has not been fully elucidated. Using bioinformatic tools, we demonstrate that TCP transcription factors belong to the intrinsically disordered proteins (IDP) family and that disorder is higher in class I TCPs than in class II TCPs. In particular, using bioinformatic and biochemical approaches, we have characterized TCP8, a class I TCP. TCP8 exhibits three intrinsically disordered regions (IDR) made of more than 50 consecutive residues, in which phosphorylable Ser residues are mainly clustered. Phosphorylation of Ser-211 that belongs to the central IDR was confirmed by mass spectrometry. Yeast two-hybrid assays also showed that the C-terminal IDR corresponds to a transactivation domain. Moreover, biochemical experiments demonstrated that TCP8 tends to oligomerize in dimers, trimers and higher-order multimers. Bimolecular fluorescence complementation (BiFC) experiments carried out on a truncated form of TCP8 lacking the C-terminal IDR indicated that it is effectively required for the pronounced self-assembly of TCP8. These data were reinforced by the prediction of a coiled coil domain in this IDR. The C-terminal IDR acts thus as an oligomerization domain and also a transactivation domain. Moreover, many Molecular Recognition Features (MoRFs) were predicted, indicating that TCP8 could interact with several partners to fulfill a fine regulation of transcription in response to various stimuli.

The challenge of increasing Pfam coverage of the human proteome

It is a worthy goal to completely characterize all human proteins in terms of their domains. Here, using the Pfam database, we asked how far we have progressed in this endeavour. Ninety per cent of proteins in the human proteome matched at least one of 5494 manually curated Pfam-A families. In contrast, human residue coverage by Pfam-A families was <45%, with 9418 automatically generated Pfam-B families adding a further 10%. Even after excluding predicted signal peptide regions and short regions (<50 consecutive residues) unlikely to harbour new families, for ∼38% of the human protein residues, there was no information in Pfam about conservation and evolutionary relationship with other protein regions. This uncovered portion of the human proteome was found to be distributed over almost 25 000 distinct protein regions. Comparison with proteins in the UniProtKB database suggested that the human regions that exhibited similarity to thousands of other sequences were often either divergent elements or N- or C-terminal extensions of existing families. Thirty-four per cent of regions, on the other hand, matched fewer than 100 sequences in UniProtKB. Most of these did not appear to share any relationship with existing Pfam-A families, suggesting that thousands of new families would need to be generated to cover them. Also, these latter regions were particularly rich in amino acid compositional bias such as the one associated with intrinsic disorder. This could represent a significant obstacle toward their inclusion into new Pfam families. Based on these observations, a major focus for increasing Pfam coverage of the human proteome will be to improve the definition of existing families. New families will also be built, prioritizing those that have been experimentally functionally characterized.Database URL: http://pfam.sanger.ac.uk/

Characterization and mutational analysis of the UDP-Glc(NAc) 4-epimerase from Marinithermus hydrothermalis

UDP-hexose 4-epimerases are important enzymes that play key roles in various biological pathways, including lipopolysaccharide biosynthesis, galactose metabolism through the Leloir pathway, and biofilm formation. Unfortunately, the determinants of their substrate specificity are not yet fully understood. They can be classified into three groups, with groups 1 and 3 preferring non-acetylated and acetylated UDP-hexoses, respectively, whereas members of group 2 are equally active on both types of substrates. In this study, the UDP-Glc(NAc) 4-epimerase from Marinithermus hydrothermalis (mGalE) was functionally expressed in Escherichia coli and thoroughly characterized. The enzyme was found to be thermostable, displaying its highest activity at 70°C and having a half-life of 23min at 60°C. Activity could be detected on both acetylated and non-acetylated UDP-hexoses, meaning that this epimerase belongs to group 2. This observation correlates well with the identity of the so-called "gatekeeper" residue (Ser279), which has previously been suggested to influence substrate specificity (Schulz et al., J Biol Chem 279:32796-32803, 2004). Furthermore, substituting this serine to a tyrosine brings about a significant preference for non-acetylated sugars, thereby demonstrating that a single residue can determine substrate specificity among type 1 and type 2 epimerases. In addition, two consecutive glycine residues (Gly118 and Gly119) were identified as a unique feature of GalE enzymes from Thermus species, and their importance for activity as well as affinity was confirmed by mutagenesis. Finally, homology modeling and mutational analysis has revealed that the enzyme's catalytic triad contains a threonine residue (Thr117) instead of the usual serine.

EF-P Is Essential for Rapid Synthesis of Proteins Containing Consecutive Proline Residues

Elongation factor P (EF-P) is a translation factor of unknown function that has been implicated in a great variety of cellular processes. Here, we show that EF-P prevents ribosome from stalling during synthesis of proteins containing consecutive prolines, such as PPG, PPP, or longer proline strings, in natural and engineered model proteins. EF-P promotes peptide-bond formation and stabilizes the peptidyl-transfer RNA in the catalytic center of the ribosome. EF-P is posttranslationally modified by a hydroxylated β-lysine attached to a lysine residue. The modification enhances the catalytic proficiency of the factor mainly by increasing its affinity to the ribosome. We propose that EF-P and its eukaryotic homolog, eIF5A, are essential for the synthesis of a subset of proteins containing proline stretches in all cells.

A novel approach to the analysis of SUMOylation with the independent use of trypsin and elastase digestion followed by database searching utilising consecutive residue addition to lysine

Identification of sites of protein SUMOylation is of great importance due its functional diversity within the cell. To date, most approaches to this problem rely on site-directed mutagenesis and/or highly specialised mass spectrometry approaches. We present a novel alternative approach to the site mapping of SUMOylation using trypsin and elastase digestion, routine mass spectrometry and an unbiased isotag database searching strategy. SUMOylated protein samples were digested with a number of enzymes and the resulting peptides separated using liquid chromatography. Analysis was carried out on both linear ion trap Orbitrap and quadrupole-time-of-flight (Q-TOF)-based mass spectrometers equipped with electrospray ionisation. The data files were subsequently searched using the Mascot algorithm with multiple variable tag modifications corresponding to SUMO-derived fragments. The utility of this approach was demonstrated with di-SUMO 2, di-SUMO 3, SUMO 1-RanGap(418-587) 1 and an enriched population of SUMOylated proteins. Unbiased database searches led to the identification of a number of analytically useful isotags ranging in length from two to four residues. Isopeptide fragments were generated including QTGG (di-SUMO-2/3), TGG (di-SUMO-2/3) and GG (SUMO-1). The method was validated by successfully mapping a number of sites of SUMO modification on SUMO-modified proteins enriched from a cell lysate. This combination of relaxed enzyme specificity, shortened isotag generation and unbiased database searching enabled confident identification of novel analytically useful SUMOylated isopeptides without a requirement for mutagenesis.

The Size and Conservation of a Coiled-coil Structure in the Ectodomain of Human BST-2/Tetherin Is Dispensable for Inhibition of HIV-1 Virion Release

BST-2/CD317/tetherin is a host factor that inhibits HIV-1 release and is counteracted by HIV-1 Vpu. Structural studies indicate that the BST-2 ectodomain assumes a coiled-coil conformation. Here we studied the role of the BST-2 ectodomain for tethering function. First, we addressed the importance of the length and structure of the ectodomain by adding or substituting heterologous coiled-coil or non-coiled-coil sequences. We found that extending or replacing the BST-2 ectodomain using non-coiled-coil sequences resulted in loss of BST-2 function. Doubling the size of the BST-2 ectodomain by insertion of a heterologous coiled-coil motif or substituting the BST-2 coiled-coil domain with a heterologous coiled-coil motif maintained tethering function. Reductions in the size of the BST-2 coiled-coil domain were tolerated as well. In fact, deletion of the C-terminal half of the BST-2 ectodomain, including a series of seven consecutive heptad motifs did not abolish tethering function. However, slight changes in the positioning of deletions affecting the relative placing of charged or hydrophobic residues on the helix severely impacted the functional properties of BST-2. Overall, we conclude that the size of the BST-2 ectodomain is highly flexible and can be reduced or extended as long as the positioning of residues important for the stability of the dimer interface is maintained.

The proline‐rich tetramerization peptides in equine serum butyrylcholinesterase

Soluble, tetrameric, plasma butyrylcholinesterase from horse has previously been shown to include a non-covalently attached polyproline peptide in its structure. The polyproline peptide matched the polyproline-rich region of human lamellipodin. Our goal was to examine the tetramer-organizing peptides of horse butyrylcholinesterase in more detail. Horse butyrylcholinesterase was denatured by boiling, thus releasing a set of polyproline peptides ranging in mass from 1173 to 2098 Da. The peptide sequences were determined by fragmentation in MALDI-TOF-TOF and linear ion trap quadrupole Orbitrap mass spectrometers. Twenty-seven polyproline peptides grouped into 13 families were identified. Peptides contained a minimum of 11 consecutive proline residues and as many as 21. Many of the peptides had a non-proline amino acid at the N-terminus. A search of the protein databanks matched peptides to nine proteins, although not all peptides matched a known protein. It is concluded that polyproline peptides of various lengths and sequences are included in the tetramer structure of horse butyrylcholinesterase. The function of these polyproline peptides is to serve as tetramer-organizing peptides.

The Coordination of NiII and CuII Ions to the Polyhistidyl Motif of Hpn Protein: Is It as Strong as We Think?

Hpn, one of Helicobacter pylori's nickel-accessory proteins, is an amazingly peculiar protein: Almost half of its sequence consists of polyhistidyl (poly-His) residues. Herein, we try to understand the origin of this naturally occurring sequence, thereby shedding some light on the bioinorganic chemistry of Hpn's numerous poly-His repeats. By using potentiometric, mass spectrometric, and various spectroscopic techniques, we studied the Ni(II) - and Cu(II) complexes of the wild-type Ac-THHHHYHGG-NH(2) fragment of Hpn and of its six analogues, in which consecutive residues (His or Tyr) were replaced by Ala (Ala-substitution or Ala-scan approaches), thereby resulting in Ac-TAHHHYHGG-NH(2), Ac-THAHHYHGG-NH(2), Ac-THHAHYHGG-NH(2), Ac-THHHAYHGG-NH(2), Ac-THHHHAHGG-NH(2), and Ac-THHHHYAGG-NH(2) peptides. We found that the His4 residue is critical for both Ni(II) - and Cu(II) -ion binding and the effectiveness of binding varies even if the substituted amino acid does not take part in the direct binding interactions.

Protein loops, solitons, and side-chain visualization with applications to the left-handed helix region

Folded proteins have a modular assembly. They are constructed from regular secondary structures like α helices and β strands that are joined together by loops. Here we develop a visualization technique that is adapted to describe this modular structure. In complement to the widely employed Ramachandran plot that is based on toroidal geometry, our approach utilizes the geometry of a two sphere. Unlike the more conventional approaches that describe only a given peptide unit, ours is capable of describing the entire backbone environment including the neighboring peptide units. It maps the positions of each atom to the surface of the two-sphere exactly how these atoms are seen by an observer who is located at the position of the central C_{α} atom. At each level of side-chain atoms we observe a strong correlation between the positioning of the atom and the underlying local secondary structure with very little if any variation between the different amino acids. As a concrete example we analyze the left-handed helix region of nonglycyl amino acids. This region corresponds to an isolated and highly localized residue independent sector in the direction of the C_{β} carbons on the two-sphere. We show that the residue independent localization extends to C_{γ} and C_{δ} carbons and to side-chain oxygen and nitrogen atoms in the case of asparagine and aspartic acid. When we extend the analysis to the side-chain atoms of the neighboring residues, we observe that left-handed β turns display a regular and largely amino acid independent structure that can extend to seven consecutive residues. This collective pattern is due to the presence of a backbone soliton. We show how one can use our visualization techniques to analyze and classify the different solitons in terms of selection rules that we describe in detail.

Abstract 3850: Production of recombinant lunasin peptides with enhanced anticancer activity using transient expression in tobacco

Abstract Epidemiological observations suggest a correlation between high levels of soybean product consumption and lowered incidence and mortality due to prostate, breast and colon cancer. Lunasin, a 43-44 amino acid peptide derived from soybean, has been implicated as a significant source of this anticancer activity. Lunasin contains nine consecutive aspartic acid residues at the C-terminus that bind to hypoacetlyated core histones, a minimal internal RGD cell adhesion motif and a helical region exhibiting structural homology to conserved sequences of chromatin binding proteins. Initial studies demonstrated that lunasin can prevent the transformation of mammalian cells by chemical carcinogens or viral oncogenes, and inhibits chemically-induced tumors in a mouse skin cancer model. These results suggest that lunasin may be useful as a cancer chemoprevention agent. More recent studies have demonstrated that lunasin can inhibit the proliferation of several established human cancer cell lines in vitro and in vivo, suggesting that lunasin may also be useful as a cancer therapeutic. Lunasin's anticancer effects are currently limited to specific cancer cell types and require 10-100 µM concentrations of peptide. We have now established a transient expression system based on the Tobacco Mosaic Virus vector, Geneware®, for large-scale production of modified forms of recombinant lunasin in tobacco. Our initial studies revealed that we could not detect any significant protein accumulation using constructs expressing lunasin peptides alone. We modified our strategy by expressing lunasin peptides as C-terminal fusions to green fluorescent protein (GFP) that includes a linker sequence containing a thrombin cleavage site. We have demonstrated that this system can produce GFP-lunasin at levels &amp;gt;100 mg/kg fresh weight tissue and demonstrated that the lunasin peptide can be recovered by proteolytic cleavage with thrombin. We have used this system to produce a modified form of lunasin that contains an N-terminal RGD domain sequence that preferentially binds to the αvβ6 integrin that is highly expressed in a number of epithelial-derived carcinomas. In vitro studies using cancer cell lines expressing the αvβ6 integrin demonstrate that the RGD-lunasin peptide is &amp;gt;10-fold more active in inhibiting cancer cell proliferation than the natural form of lunasin. Moreover, RGD-lunasin inhibits the proliferation of some cancer cell lines that are insensitive to 100 µM lunasin. These results demonstrate the feasibility of utilizing plant-based expression to produce more efficacious forms of lunasin that are targeted specifically to cancer cells and indicate that RGD lunasin may be useful as a cancer therapeutic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3850. doi:1538-7445.AM2012-3850

Structure of a dominant-negative helix-loop-helix transcriptional regulator suggests mechanisms of autoinhibition

Helix-loop-helix (HLH) family transcription factors regulate numerous developmental and homeostatic processes. Dominant-negative HLH (dnHLH) proteins lack DNA-binding ability and capture basic HLH (bHLH) transcription factors to inhibit cellular differentiation and enhance cell proliferation and motility, thus participating in patho-physiological processes. We report the first structure of a free-standing human dnHLH protein, HHM (Human homologue of murine maternal Id-like molecule). HHM adopts a V-shaped conformation, with N-terminal and C-terminal five-helix bundles connected by the HLH region. In striking contrast to the common HLH, the HLH region in HHM is extended, with its hydrophobic dimerization interfaces embedded in the N- and C-terminal helix bundles. Biochemical and physicochemical analyses revealed that HHM exists in slow equilibrium between this V-shaped form and the partially unfolded, relaxed form. The latter form is readily available for interactions with its target bHLH transcription factors. Mutations disrupting the interactions in the V-shaped form compromised the target transcription factor specificity and accelerated myogenic cell differentiation. Therefore, the V-shaped form of HHM may represent an autoinhibited state, and the dynamic conformational equilibrium may control the target specificity.

On the interaction between konjac glucomannan and xanthan in mixed gels: An analysis based on the cascade model

The rheological properties and critical behavior of konjac glucomannan (KGM)/xanthan (XG) mixed gels were investigated and analyzed using a two-component cascade model. The fitting results show that the optimal functionality value for KGM (fKGM) is 3, whereas the possible functionality value for XG (fXG) is 100–1000 obtained from the modulus data, or 25 obtained from the critical data. The van’t Hoff analysis of the critical data shows that the binding of KGM with XG has a high enthalpy/entropy ratio (ΔH/300ΔS=5.52), which can be explained by the gain in the hydrational entropy due to the release of water molecules during the binding reaction. From these results, we proposed that the binding of KGM with XG takes place on the consecutive glucose residues of KGM with six or more units.

Mapping Structural Determinants within Third Intracellular Loop That Direct Signaling Specificity of Type 1 Corticotropin-releasing Hormone Receptor

The type 1 corticotropin-releasing hormone receptor (CRH-R1) influences biological responses important for adaptation to stressful stimuli, through activation of multiple downstream effectors. The structural motifs within CRH-R1 that mediate G protein activation and signaling selectivity are unknown. The aim of this study was to gain insights about important structural determinants within the third intracellular loop (IC3) of the human CRH-R1α important for cAMP and ERK1/2 pathways activation and selectivity. We investigated the role of the juxtamembrane regions of IC3 by mutating amino acid cassettes or specific residues to alanine. Although simultaneous tandem alanine mutations of both juxtamembrane regions Arg(292)-Met(295) and Lys(311)-Lys(314) reduced ligand binding and impaired signaling, all other mutant receptors retained high affinity binding, indistinguishable from wild-type receptor. Agonist-activated receptors with tandem mutations at the proximal or distal terminal segments enhanced activation of adenylyl cyclase by 50-75% and diminished activation of inositol trisphosphate and ERK1/2 by 60-80%. Single Ala mutations identified Arg(292), Lys(297), Arg(310), Lys(311), and Lys(314) as important residues for the enhanced activation of adenylyl cyclase, partly due to reduced inhibition of adenylyl cyclase activity by pertussis toxin-sensitive G proteins. In contrast, mutation of Arg(299) reduced receptor signaling activity and cAMP response. Basic as well as aliphatic amino acids within both juxtamembrane regions were identified as important for ERK1/2 phosphorylation through activation of pertussis toxin-sensitive G proteins as well as G(q) proteins. These data uncovered unexpected roles for key amino acids within the highly conserved hydrophobic N- and C-terminal microdomains of IC3 in the coordination of CRH-R1 signaling activity.

Polyglutamine Repeats Are Associated to Specific Sequence Biases That Are Conserved among Eukaryotes

Nine human neurodegenerative diseases, including Huntington's disease and several spinocerebellar ataxia, are associated to the aggregation of proteins comprising an extended tract of consecutive glutamine residues (polyQs) once it exceeds a certain length threshold. This event is believed to be the consequence of the expansion of polyCAG codons during the replication process. This is in apparent contradiction with the fact that many polyQs-containing proteins remain soluble and are encoded by invariant genes in a number of eukaryotes. The latter suggests that polyQs expansion and/or aggregation might be counter-selected through a genetic and/or protein context. To identify this context, we designed a software that scrutinize entire proteomes in search for imperfect polyQs. The nature of residues flanking the polyQs and that of residues other than Gln within polyQs (insertions) were assessed. We discovered strong amino acid residue biases robustly associated to polyQs in the 15 eukaryotic proteomes we examined, with an over-representation of Pro, Leu and His and an under-representation of Asp, Cys and Gly amino acid residues. These biases are conserved amongst unrelated proteins and are independent of specific functional classes. Our findings suggest that specific residues have been co-selected with polyQs during evolution. We discuss the possible selective pressures responsible of the observed biases.

Predict impact of single amino acid change upon protein structure

BackgroundAmino acid point mutations (nsSNPs) may change protein structure and function. However, no method directly predicts the impact of mutations on structure. Here, we compare pairs of pentamers (five consecutive residues) that locally change protein three-dimensional structure (3D, RMSD>0.4Å) to those that do not alter structure (RMSD<0.2Å). Mutations that alter structure locally can be distinguished from those that do not through a machine-learning (logistic regression) method.ResultsThe method achieved a rather high overall performance (AUC>0.79, two-state accuracy >72%). This discriminative power was particularly unexpected given the enormous structural variability of pentamers. Mutants for which our method predicted a change of structure were also enriched in terms of disrupting stability and function. Although distinguishing change and no change in structure, the new method overall failed to distinguish between mutants with and without effect on stability or function.ConclusionsLocal structural change can be predicted. Future work will have to establish how useful this new perspective on predicting the effect of nsSNPs will be in combination with other methods.

Interfacial adsorption of cationic peptideamphiphiles: a combined study of in situspectroscopic ellipsometry and liquid AFM

The adsorption of cationic amphiphilic peptides, composed of six consecutive hydrophobic amino acid residues (G, A, and V) at the N-terminus and one cationic charge residue (K) at the C-terminus, at the hydrophilic silica/water interface has been investigated at pH 6.0 by in situspectroscopic ellipsometry (SE) and liquid atomic force microscopy (AFM). In the order of G6K, A6K, and V6K, the peptides showed decreasing critical aggregation concentration (CAC) and increasing interfacial aggregation. No solution or surface aggregated structure was observed for G6K due to its weak hydrophobic character. A6K showed a swift increase of interfacial adsorption from 0.2 to 0.5 mM (CAC = 0.5 mM) within which interfacial aggregation also started. Above this range, the adsorption attained a saturated amount of about 3.3 mg m−2, and peptide stacks, fibrillar fragments and long fibrils coexisted at the interface, in contrast to the dominant solution aggregates of long peptide fibrils formed. For V6K, interfacial aggregation occurred far below the CAC of 0.15 mM and the adsorption isotherm was characterized by two plateaus below and above the CAC, with a characteristic structural transition from fibrillar dominant aggregates to lamellar bilayer sheets accompanying increasing interfacial adsorption, reflecting the rebalance of different interfacial interactions.