Oblique orientated alpha-helices and their prediction

Oblique orientated alpha-helices possess hydrophobicity gradients, which allow the parent alpha-helices to penetrate the membrane at a shallow angle, thereby destabilising membrane lipid organisation and promoting a range of biological processes. These alpha-helices occur in a variety of membrane interactive proteins and a number of techniques have been developed to guide their identification using sequence data alone. Hydrophobicity profiling, which provides a one-dimensional analysis of sequence data, identified only 30% of known tilted peptides in a control dataset and was thus of limited predictive use. In contrast, extended hydrophobic moment plot methodology and amphipilic profiling which take residue distribution into account and provide two-dimensional analysis of primary structural data, were found to be good indicators of tilted peptide structure. Amphiphilic profiling identified 67% of tilted peptides in the control dataset and showed that potentially, approximately 40% of transmembrane alpha-helices possess tilted peptide structure. However, it has been shown that extending these simple methods to take into account the three-dimensional spatial distribution of residues gives no clear additional benefit to identifying tilted peptides.     

A novel class of small functional peptides that bind and inhibit human alpha-thrombin isolated by mRNA display

Here we report the in vitro selection of novel small peptide motifs that bind to human alpha-thrombin. We have applied mRNA display to select for thrombin binding peptides from an unbiased library of 1.2 x 10(11) different 35-mer peptides, each containing a random sequence of 15 amino acids. Two clones showed binding affinities ranging from 166 to 520 nM. A conserved motif of four amino acids, DPGR, was identified. Clot formation of human plasma is inhibited by the selected clones, and they downregulate the thrombin-mediated activation of protein C. The identified peptide motifs do not share primary sequence similarities to any of the known natural thrombin binding motifs. As new inhibitors for human thrombin open interesting possibilities in thrombosis research, our newly identified peptides may provide further insights into this field of investigation and may be possible candidates for the development of new anti-thrombotic agents.     

Heterotypic Supramolecular Hydrogels Formed by Noncovalent Interactions in Inflammasomes

The advance of structural biology has revealed numerous noncovalent interactions between peptide sequences in protein structures, but such information is less explored for developing peptide materials. Here we report the formation of heterotypic peptide hydrogels by the two binding motifs revealed by the structures of an inflammasome. Specifically, conjugating a self-assembling motif to the positively or negatively charged peptide sequence from the ASCPYD filaments of inflammasome produces the solutions of the peptides. The addition of the peptides of the oppositely charged and complementary peptides to the corresponding peptide solution produces the heterotypic hydrogels. Rheology measurement shows that ratios of the complementary peptides affect the viscoelasticity of the resulted hydrogel. Circular dichroism indicates that the addition of the complementary peptides results in electrostatic interactions that modulate self-assembly. Transmission electron microscopy reveals that the ratio of the complementary peptides controls the morphology of the heterotypic peptide assemblies. This work illustrates a rational, biomimetic approach that uses the structural information from the protein data base (PDB) for developing heterotypic peptide materials via self-assembly.     

Design of a protein kinase-inducible domain

Protein phosphorylation is a critical regulatory strategy. New tools are necessary which may be used to interrogate and are responsive to the activities of protein kinases and phosphatases. We have used protein design to develop a protein motif, termed a protein kinase-inducible domain, whose structure is dependent on its phosphorylation state. Based on an EF hand calcium-binding loop, the key design element is the replacement of a structurally critical Glu residue, which binds metal in a bidentate manner, with a serine residue, which is expected to bind metal tightly when phosphorylated but poorly when not phosphorylated. The design comprises an EF hand consensus sequence, a tryptophan at residue 7 to sensitize lanthanide luminescence, and the recognition sequence of a serine/threonine kinase. Designed peptides, which contain minimal substrate recognition motifs of the protein kinases PKA, PKC, or the MAP kinase Erk, form complexes with Tb3+ when phosphorylated, showing strong Tb3+ luminescence emission at 544 nm, but show weak luminescence when not phosphorylated. The change in fluorescence on phosphorylation is comparable to or greater than that observed in described kinase sensors. Site-specific lanthanide binding was confirmed by NMR with diamagnetic and paramagnetic metals. The kinase-inducible domain peptides comprise an expressible sequence, potentially enabling their use as genetically encoded tags of protein kinase activity. The motif is general and potentially applicable to the majority of serine/threonine kinases.     

Fusogenic tilted peptides induce nanoscale holes in supported phosphatidylcholine bilayers

Tilted peptides are known to insert in lipid bilayers with an oblique orientation, thereby destabilizing membranes and facilitating membrane fusion processes. Here, we report the first direct visualization of the interaction of tilted peptides with lipid membranes using in situ atomic force microscopy (AFM) imaging. Phase-separated supported dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers were prepared by fusion of small unilamellar vesicles and imaged in buffer solution, in the absence and in the presence of the simian immunodeficiency virus (SIV) peptide. The SIV peptide was shown to induce the rapid appearance of nanometer scale bilayer holes within the DPPC gel domains, while keeping the domain shape unaltered. We attribute this behavior to a local weakening and destabilization of the DPPC domains due to the oblique insertion of the peptide molecules. These results were directly correlated with the fusogenic activity of the peptide as determined using fluorescently labeled DOPC/DPPC liposomes. By contrast, the nontilted ApoE peptide did not promote liposome fusion and did not induce bilayer holes but caused slight erosion of the DPPC domains. In conclusion, this work provides the first direct evidence for the production of stable, well-defined nanoholes in lipid bilayer domains by the SIV peptide, a behavior that we have shown to be specifically related to the tilted character of the peptide. A molecular mechanism underlying spontaneous insertion of the SIV peptide within lipid bilayers and the subsequent removal of bilayer patches is proposed, and its relevance to membrane fusion processes is discussed.     

Individual b2 ion fragmentation profiles combined with AspN digestion improve N-terminal peptide sequencing

The N terminus of peptides generated by AspN is restricted to about 40 dipeptide motifs starting with D or E. These motifs are visible upon collision-induced dissociation (CID) as b₂ ions, which are often the most abundant low-mass fragment ions. It was observed that b₂ ions are accompanied by a set of sequence-specific neutral losses of CO, H₂O, NH₃, and some other small units. To test the utility of these profiles as additional parameters for reliable assignment of the b₂ ion motif besides its m/z value, the CID spectra of 221 different AspN-generated peptides covering all N-terminal D-X and E-X motifs were recorded. Qualitatively, the b₂ ion fragmentation profiles of individual motifs were found to exhibit little dependency on the rest of the peptide sequence. Thus, it is concluded that the set of b₂ ion fragmentation profiles recorded in this study can be used as reference set. Knowledge of these profiles provides an increased specificity for b₂ ion annotation of AspN-generated peptides compared to the use of only a solitary b₂ ion m/z value. Recognition of the b₂ ion motif provides a two-amino-acid sequence including its direction; it provides the location of this motif at the N terminus, and it sets a starting point for further extension of the b ion series.     

Rapid determination of multiple linear kinase substrate motifs by mass spectrometry

Kinase-substrate recognition depends on the chemical properties of the phosphorylatable residue as well as the surrounding linear sequence motif. Detailed knowledge of these characteristics increases the confidence of linking identified phosphorylation sites to kinases, predicting phosphorylation sites, and designing optimal peptide substrates. Here, we present a mass spectrometry-based approach for determining linear kinase substrate motifs by elaborating the positional and chemical preference of the kinase for a phosphorylatable residue using libraries of naturally-occurring peptides that are amenable to peptide identification by commonly used proteomics platforms. We applied this approach to a structurally and functionally diverse set of purified kinases, which recapitulated their previously described substrate motifs and discovered additional ones, including preferences of certain kinases for phosphorylatable residues adjacent to peptide termini. Furthermore, we identify specific and distinguishable motif elements for the four members of the polo-like kinase (Plk) family and verify members of these motif elements for Plk1 in vivo.     

Recognition of multiple substrate motifs by the c-ABL protein tyrosine kinase

Using a combinatorial peptide library that is based on the one-bead one-peptide approach we identified 14 peptide substrates for the c-ABL protein tyrosine kinase, which define three distinct consensus sequence groups. This is distinct from many serine/threonine kinases, which often phosphorylate only one major consensus sequence. The three consensus sequences accurately predict phosphorylation sites in cellular ABL substrates proven to play a role in cell signaling. Our data suggest that protein tyrosine kinases have evolved to recognize multiple substrate motifs.     

Directed evolution of protease beacons that enable sensitive detection of endogenous MT1-MMP activity in tumor cell lines

Directed evolution was applied to identify peptide substrates with enhanced hydrolysis rates by MT1-MMP suitable for protease beacon development. Screening of a random pentapeptide library, using two-color CLiPS, yielded several substrates identical to motifs in distinct collagens that shared the consensus sequence P-x-G↓L. To identify substrates with enhanced cleavage rates, a second-generation decapeptide library incorporating the consensus was screened under stringent conditions, which resulted in a MxPLG↓(M)/(L)M(G)/(A)R consensus motif. These substrates are hydrolyzed by human-MT1-MMP up to six times faster than reported peptide substrates and are stable in plasma. Finally, incubation of soluble protease beacons incorporating the optimized substrates, but not previous substrates, enabled direct detection of endogenous MT1-MMP activity of human-fibrosarcoma (HT-1080) cells. Extended substrate libraries coupled with CLiPS should be useful to generate more effective activity probes for a variety of proteolytic enzymes.     

Investigations of cyclophilin interactions with oligopeptides containing proline by affinity capillary electrophoresis

Affinity capillary electrophoresis using mobility-shift analysis was utilized to characterize the binding of peptide ligands to cyclophilins, which are members of the enzyme family of peptidyl-prolyl cis/trans isomerases. Peptides derived from the human immunodeficiency virus capsid protein p24 exhibited different affinities to the isoenzymes cyclophilin18 and cyclophilin20. For the interaction of the peptide hormone bradykinin with cyclophilin18, a dissociation constant of 1.4 +/- 0.1 mM was determined. Finally, the affinity of cyclophilin20 to peptides from a cellulose-bound peptide library scanning the sequence of Drosophila melanogaster protein cappuccino was investigated. The affinities of selected peptides to cyclophilin20 and a green fluorescent fusion protein with cyclophilin20 were compared.     

Gas‐phase behavior of noncovalent transmembrane segment complexes

Specific helix oligomerization between transmembrane segments (TMSs) is often promoted by motifs like GxxxG. Disruption of this motif in the transmembrane segments of vesicular stomatitis virus G‐protein and of glycophorin A results in a reduced dimerization level studied by in vivo systems like ToxR. This paper reports the influence of sequence motifs like GxxxG in solution and the gas phase. The transmembrane segments may behave differently in the gas and liquid phase, because of the absence of surrounding solvent molecules in the gas phase. Comparison of experiments depending on peptide properties performed in the gas and liquid phase discloses that the peptides retain ‘some memory’ of their liquid‐phase structure in the gas phase. A direct correlation has been found between helicity in solution as determined by circular dichroism and dimerization in the gas phase monitored by electrospray mass spectrometry. These results show that a proper folding in solution is required for oligomerization. On the other hand, sequence‐specific oligomerization depending on the GxxxG motif was not observed with the mass spectrometric detection. Further on, neither concentration‐dependent complex studies nor studies regarding complex stability in the gas phase – via collision‐induced dissociation (CID) – led to sequence‐specific differences. Finally, the findings show that in mass spectrometric measurements noncovalent interactions of studied TMSs is rather more dependent on the secondary structure and proper folding than on their primary structure. Copyright © 2008 John Wiley & Sons, Ltd.     

Segregated Protein–Cucurbit[7]uril Crystalline Architectures via Modulatory Peptide Tectons

One approach to protein assembly involves water-soluble supramolecular receptors that act like glues. Bionanoarchitectures directed by these scaffolds are often system-specific, with few studies investigating their customization. Herein, the modulation of cucurbituril-mediated protein assemblies through the inclusion of peptide tectons is described. Three peptides of varying length and structural order were N-terminally appended to RSL, a β-propeller building block. Each fusion protein was incorporated into crystalline architectures mediated by cucurbit[7]uril ( Q7 ). A trimeric coiled-coil served as a spacer within a Q7 -directed sheet assembly of RSL, giving rise to a layered material of varying porosity. Within the spacer layers, the coiled-coils were dynamic. This result prompted consideration of intrinsically disordered peptides (IDPs) as modulatory tectons. Similar to the coiled-coil, a mussel adhesion peptide (Mefp) also acted as a spacer between protein– Q7 sheets. In contrast, the fusion of a nucleoporin peptide (Nup) to RSL did not recapitulate the sheet assembly. Instead, a Q7 -directed cage was adopted, within which disordered Nup peptides were partially “captured” by Q7 receptors. IDP capture occurred by macrocycle recognition of an intrapeptide Phe-Gly motif in which the benzyl group was encapsulated by Q7 . The modularity of these protein–cucurbituril architectures adds a new dimension to macrocycle-mediated protein assembly. Segregated protein crystals, with alternating layers of high and low porosity, could provide a basis for new types of materials.     

Application and Structural Analysis of Triazole-Bridged Disulfide Mimetics in Cyclic Peptides

Ruthenium-catalysed azide–alkyne cycloaddition (RuAAC) provides access to 1,5-disubstituted 1,2,3-triazole motifs in peptide engineering applications. However, investigation of this motif as a disulfide mimetic in cyclic peptides has been limited, and the structural consequences remain to be studied. We report synthetic strategies to install various triazole linkages into cyclic peptides through backbone cyclisation and RuAAC cross-linking reactions. These linkages were evaluated in four serine protease inhibitors based on sunflower trypsin inhibitor-1. NMR and X-ray crystallography revealed exceptional consensus of bridging distance and backbone conformations (RMSD<0.5 Å) of the triazole linkages compared to the parent disulfide molecules. The triazole-bridged peptides also displayed superior half-lives in liver S9 stability assays compared to disulfide-bridged peptides. This work establishes a foundation for the application of 1,5-disubstituted 1,2,3-triazoles as disulfide mimetics.     

Biomolecular engineering by combinatorial design and high-throughput screening: small, soluble peptides that permeabilize membranes

Rational design and engineering of membrane-active peptides remains a largely unsatisfied goal. We have hypothesized that this is due, in part, to the fact that some membrane activities, such as permeabilization, are not dependent on specific amino acid sequences or specific three-dimensional peptide structures. Instead they depend on interfacial activity: the ability of a molecule to partition into the membrane-water interface and to alter the packing and organization of lipids. Here we test that idea by taking a nonclassical approach to biomolecular engineering and design of membrane-active peptides. A 16,384-member rational combinatorial peptide library, containing peptides of 9-15 amino acids in length, was screened for soluble members that permeabilize phospholipid membranes. A stringent, two-phase, high-throughput screen was used to identify 10 unique peptides that had potent membrane-permeabilizing activity but were also water soluble. These rare and uniquely active peptides do not share any particular sequence motif, peptide length, or net charge, but instead they share common compositional features, secondary structure, and core hydrophobicity. We show that they function by a common mechanism that depends mostly on interfacial activity and leads to transient pore formation. We demonstrate here that composition-space peptide libraries coupled with function-based high-throughput screens can lead to the discovery of diverse, soluble, and highly potent membrane-permeabilizing peptides.     

The prediction of amphiphilic alpha-helices

A number of sequence-based analyses have been developed to identify protein segments, which are able to form membrane interactive amphiphilic alpha-helices. Earlier techniques attempted to detect the characteristic periodicity in hydrophobic amino acid residues shown by these structure and included the Molecular Hydrophobic Potential (MHP), which represents the hydrophobicity of amino acid residues as lines of isopotential around the alpha-helix and analyses based on Fourier transforms. These latter analyses compare the periodicity of hydrophobic residues in a putative alpha-helical sequence with that of a test mathematical function to provide a measure of amphiphilicity using either the Amphipathic Index or the Hydrophobic Moment. More recently, the introduction of computational procedures based on techniques such as hydropathy analysis, homology modelling, multiple sequence alignments and neural networks has led to the prediction of transmembrane alpha-helices with accuracies of the order of 95% and transmembrane protein topology with accuracies greater than 75%. Statistical approaches to transmembrane protein modeling such as hidden Markov models have increased these prediction levels to an even higher level. Here, we review a number of these predictive techniques and consider problems associated with their use in the prediction of structure / function relationships, using alpha-helices from G-coupled protein receptors, penicillin binding proteins, apolipoproteins, peptide hormones, lytic peptides and tilted peptides as examples.     

An algorithm for interpretation of low-energy collision-induced dissociation product ion spectra for de novo sequencing of peptides

An algorithm for interpretation of product ion spectra of peptides generated from ion trap mass spectrometry is developed for de novo amino acid sequencing of peptides for the purpose of protein identification. It is based on a multi-pass analysis of product ion data using a rigorous data extraction and sequence interpretation protocol in the initial pass. The extraction/interpretation algorithm becomes more relaxed in subsequent passes, considering more of the fragment ions, and potentially more sequence candidates. The possible peptide sequences generated by the algorithm are scored according to those sequences which best explain the fragment ion spectrum. These sequences are searched against a protein database using a BLAST search engine to find likely protein candidates. The method is also suitable for locating and determining protein modifications, and can be applied to de novo interpretation of peptide fragment ions in the tandem mass (MS/MS) spectrum produced from a mixture of two peptides having similar nominal mass, but different sequences. Using a known protein, bovine serum albumin, as an example, it is illustrated that this method is rapid and efficient for MS/MS spectral interpretation. This method combined with BLAST programs is then applied to search homologies and to generate information on post-translational modifications of an unknown protein isolated from shark cartilage that does not have a complete genome or proteome database.     

Application and Structural Analysis of Triazole‐Bridged Disulfide Mimetics in Cyclic Peptides

Ruthenium‐catalysed azide–alkyne cycloaddition (RuAAC) provides access to 1,5‐disubstituted 1,2,3‐triazole motifs in peptide engineering applications. However, investigation of this motif as a disulfide mimetic in cyclic peptides has been limited, and the structural consequences remain to be studied. We report synthetic strategies to install various triazole linkages into cyclic peptides through backbone cyclisation and RuAAC cross‐linking reactions. These linkages were evaluated in four serine protease inhibitors based on sunflower trypsin inhibitor‐1. NMR and X‐ray crystallography revealed exceptional consensus of bridging distance and backbone conformations (RMSD<0.5 Å) of the triazole linkages compared to the parent disulfide molecules. The triazole‐bridged peptides also displayed superior half‐lives in liver S9 stability assays compared to disulfide‐bridged peptides. This work establishes a foundation for the application of 1,5‐disubstituted 1,2,3‐triazoles as disulfide mimetics.     

Mass Spectrometry Analysis of 2-Nitrophenylhydrazine Carboxy Derivatized Peptides

Peptides with two or more basic residues, including those with post-translational modifications (PTMs), such as methylation and phosphorylation, can be highly hydrophilic and, therefore, are often difficult to be retained on a reversed-phase (RP) column. In addition, these highly hydrophilic peptides may carry two or more positive charges, which often fragment poorly upon collisionally activated dissociation (CAD), resulting in few sequence-specific ions. C-terminal rearrangement may also occur during CAD. Furthermore, some PTMs are labile and tend to be lost when subjected to CAD as is the case with phosphorylation on serine or threonine. To overcome the difficulties of separation, detection, and fragmentation of highly hydrophilic peptides, we report here the effect of carboxy group derivatization with 2-nitrophenylhydrazine (this strategy will be called NPHylation for simplicity). NPHylation significantly increases the hydrophobicity of the peptides, eliminates C-terminal rearrangement in all cases, and offers enhanced sensitivity in some cases. In addition, the CAD spectra of the resulting NPHylated peptides carry more sequence-specific ions due to significant reduction of sequence scrambling as observed for peptide EHAGVISVL. Furthermore, the different carboxy derivatives of this peptide undergo sequence scrambling to varying degrees, which clearly demonstrates that the C-terminus has a profound effect on peptide fragmentation. Finally, sequence scrambling is a charge dependent phenomenon, which affects CAD of doubly charged peptides far more than their singly charged counterparts.     

Comparison between RGD-peptide-modified titanium and borosilicate surfaces

The use of synthetic peptides containing adhesive sequences, such as the Arg-Gly-Asp (RGD) motif, represents a promising strategy to control biological interactions at the cell–material interface. These peptides are known to improve the tissue–material contact owing to highly specific binding to cellular membrane receptors known as integrins, thereby promoting the adhesion, migration and proliferation of cells. The peptides were coupled to borosilicate glass and titanium surfaces using silanisation chemistry. A tryptophan residue was incorporated into the amino acid sequences of selected peptides to facilitate the detection of the covalently bound peptides. Successful peptide immobilisation was proven by fluorimetric measurements. The confocal imaging analysis suggests a homogeneous distribution of the immobilised peptide across the biomaterial surface. In vitro cell proliferation assays were employed to compare the adhesion potentials of the well-known RGD-containing peptides GRGDSP, GRADSP and RGDS to the three peptides designed by our group. The results demonstrate that the RGD sequence is not necessarily required to enhance the adhesion of cells to non-biological surfaces. Moreover, it is shown that the number of adhering cells can be increased by changes in the peptide hydrophobicity. Changes in the cytoskeleton are observed depending on the type of RGD-peptide modification.