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.     

Scaling and the Smoluchowski equations

The Smoluchowski equations, which describe coalescence growth, take into account combination reactions between a j-mer and a k-mer to form a (j+k)-mer, but not breakup of larger clusters to smaller ones. All combination reactions are assumed to be second order, with rate constants K(jk). The K(jk) are said to scale if K(lambda j,gamma k) = lambda(mu)gamma(nu)K(jk) for j < or = k. It can then be shown that, for large k, the number density or population of k-mers is given by Ak(a)e(-bk), where A is a normalization constant (a function of a, b, and time), a = -(mu+nu), and b(mu+nu-1) depends linearly on time. We prove this in a simple, transparent manner. We also discuss the origin of odd-even population oscillations for small k. A common scaling arises from the ballistic model, which assumes that the velocity of a k-mer is proportional to 1/square root of m(k) (Maxwell distribution), i.e., thermal equilibrium. This does not hold for the nascent distribution of clusters produced from monomers by reactive collisions. By direct calculation, invoking conservation of momentum in collisions, we show that, for this distribution, velocities are proportional to m(k)(-0.577). This leads to mu+nu = 0.090, intermediate between the ballistic (0.167) and diffusive (0.000) results. These results are discussed in light of the existence of systems in the experimental literature which apparently correspond to very negative values of mu+nu.     

Secondary Structure Libraries for Artificial Evolution Experiments

Methods of artificial evolution such as SELEX and in vitro selection have made it possible to isolate RNA and DNA motifs with a wide range of functions from large random sequence libraries. Once the primary sequence of a functional motif is known, the sequence space around it can be comprehensively explored using a combination of random mutagenesis and selection. However, methods to explore the sequence space of a secondary structure are not as well characterized. Here we address this question by describing a method to construct libraries in a single synthesis which are enriched for sequences with the potential to form a specific secondary structure, such as that of an aptamer, ribozyme, or deoxyribozyme. Although interactions such as base pairs cannot be encoded in a library using conventional DNA synthesizers, it is possible to modulate the probability that two positions will have the potential to pair by biasing the nucleotide composition at these positions. Here we show how to maximize this probability for each of the possible ways to encode a pair (in this study defined as A-U or U-A or C-G or G-C or G.U or U.G). We then use these optimized coding schemes to calculate the number of different variants of model stems and secondary structures expected to occur in a library for a series of structures in which the number of pairs and the extent of conservation of unpaired positions is systematically varied. Our calculations reveal a tradeoff between maximizing the probability of forming a pair and maximizing the number of possible variants of a desired secondary structure that can occur in the library. They also indicate that the optimal coding strategy for a library depends on the complexity of the motif being characterized. Because this approach provides a simple way to generate libraries enriched for sequences with the potential to form a specific secondary structure, we anticipate that it should be useful for the optimization and structural characterization of functional nucleic acid motifs.     

Centromeric Alpha‐Satellite DNA Adopts Dimeric i‐Motif Structures Capped by AT Hoogsteen Base Pairs

Human centromeric alpha‐satellite DNA is composed of tandem arrays of two types of 171 bp monomers; type A and type B. The differences between these types are concentrated in a 17 bp region of the monomer called the A/B box. Here, we have determined the solution structure of the C‐rich strand of the two main variants of the human alpha‐satellite A box. We show that, under acidic conditions, the C‐rich strands of two A boxes self‐recognize and form a head‐to‐tail dimeric i‐motif stabilized by four intercalated hemi‐protonated C:C⁺ base pairs. Interestingly, the stack of C:C⁺ base pairs is capped by T:T and Hoogsteen A:T base pairs. The two main variants of the A box adopt a similar three‐dimensional structure, although the residues involved in the formation of the i‐motif core are different in each case. Together with previous studies showing that the B box (known as the CENP‐B box) also forms dimeric i‐motif structures, our finding of this non‐canonical structure in the A box shows that centromeric alpha satellites in all human chromosomes are able to form i‐motifs, which consequently raises the possibility that these structures may play a role in the structural organization of the centromere.     

The A53T mutation is key in defining the differences in the aggregation kinetics of human and mouse α-synuclein

Despite a 95% sequence similarity, the aggregation of human and mouse α-synuclein is remarkably different, as the human form is slower than the mouse form in forming fibrils but is associated with Parkinson's disease in both humans and transgenic mice. Here, the amino acid code underlying these differences is investigated by comparing the lag times, growth rates, and secondary structure propensities of a systematic series of eight human-mouse chimeras. Fluorescence analysis of these variants shows that the A53T substitution dominates the growth kinetics, while the lag phase is affected by a combination of the A53T and S87N substitutions. The secondary structure propensities derived from an NMR chemical shift analysis of the monomeric forms of the human-mouse variants enable us to establish a link between the changes in the conformational properties in the region of position 53 upon mutation and the corresponding changes in growth rates. These results suggest that the presence of an alanine residue at position 53 may be an evolutionary adaptation to minimize Parkinson's disease in humans and indicates that effective drug development efforts may be directed to target this N-terminal region of the sequence.     

Promoter-Controlled Synthesis and Conformational Analysis of Cyclic Mannosides up to a 32-mer

Cyclodextrins are widely used as carriers of small molecules for drug delivery owing to their remarkable host properties and excellent biocompatibility. However, cyclic oligosaccharides with different sizes and shapes are limited. Cycloglycosylation of ultra-large bifunctional saccharide precursors is challenging due to the constrained conformational spaces. Herein we report a promoter-controlled cycloglycosylation approach for the synthesis of cyclic α-(1→6)-linked mannosides up to a 32-mer. Cycloglycosylation of the bifunctional thioglycosides and (Z)-ynenoates was found to be highly dependent on the promoters. In particular, a sufficient amount of a gold(I) complex played a key role in the proper preorganization of the ultra-large cyclic transition state, providing a cyclic 32-mer polymannoside, which represents the largest synthetic cyclic polysaccharide to date. NMR experiments and a computational study revealed that the cyclic 2-mer, 4-mer, 8-mer, 16-mer, and 32-mer mannosides adopted different conformational states and shapes.     

Structural Basis for the Identification of an i‐Motif Tetraplex Core with a Parallel‐Duplex Junction as a Structural Motif in CCG Triplet Repeats

CCG triplet repeats can fold into tetraplex structures, which are associated with the expansion of (CCG)_n trinucleotide sequences in certain neurological diseases. These structures are stabilized by intertwining i‐motifs. However, the structural basis for tetraplex i‐motif formation in CCG triplet repeats remains largely unknown. We report the first crystal structure of a CCG‐repeat sequence, which shows that two dT(CCG)₃A strands can associate to form a tetraplex structure with an i‐motif core containing four C:C⁺ pairs flanked by two G:G homopurine base pairs as a structural motif. The tetraplex core is attached to a short parallel‐stranded duplex. Each hairpin itself contains a central CCG loop in which the nucleotides are flipped out and stabilized by stacking interactions. The helical twists between adjacent cytosine residues of this structure in the i‐motif core have an average value of 30°, which is greater than those previously reported for i‐motif structures.     

Identification of potential small molecule peptidomimetics similar to motifs in proteins

Protein-protein interactions are central to most biological processes and represent a large and important class of targets for human therapeutics. Small molecules containing peptide substituents may mimic regions of interacting proteins and inhibit their interactions. We set out to develop efficient methods to screen for similarities between known peptide structures within proteins and small molecules. We developed a method to rank peptide-compound similarities, that is restricted to small linear motifs in proteins, and to compounds containing amino acid substituents. Application to a search of the PubChem database (5.4 million compounds) using all short motifs on accessible surface areas in a nonredundant set of 11 488 peptides from the protein structure database PDB demonstrated the feasibility of the method for high throughput comparisons and the availability of compounds with comparable substituents: over 6 million compound-peptide pairs shared at least three amino acid substituents, approximately 100 000 of which had an rmsd score of less than 1 A. A Z-score function was developed that compares matches of a compound to different instances of the peptide motif in PDB, providing an appropriate scoring function for comparison among peptide-compound similarities involving different numbers of atoms (while simultaneously enriching for similarities that are likely to be more specific for the protein of interest). We applied the method to searches of known short protein motifs against the National Cancer Institute Developmental Therapeutic Program compound database, identifying a known true positive.     

Computing motif correlations in proteins

Protein motifs, which are specific regions and conserved regions, are found by comparing multiple protein sequences. These conserved regions in general play an important role in protein functions and protein folds, for example, for their binding properties or enzymatic activities. The aim here is to find the existence correlations of protein motifs. The knowledge of protein motif/domain sharing should be important in shedding new light on the biologic functions of proteins and offering a basis in analyzing the evolution in the human genome or other genomes. The protein sequences used here are obtained from the PIR-NREF database and the protein motifs are retrieved from the PROSITE database. We apply data mining approach to discover the occurrence correlations of motif in protein sequences. The correlation of motifs mined can be used in evolution analyses and protein structure prediction. We discuss the latter, i.e., protein structure prediction in this study. The correlations mined are stored and maintained in a database system. The database is now available at http://bioinfo.csie.ncu.edu.tw/ProMotif/.     

Evolution of the dUTPase gene of mammalian and avian herpesviruses

Sequences of dUTPases encoded by Alpha- and Gammaherpesviruses resemble other dUTPases in their possession of five conserved motifs, but differ in having greater chain lengths (about twice as long) and in the location of Motif 3 at an N terminal location relative to the other motifs. It was proposed that the herpesvirus gene arose by intragenic duplication of a standard dUTPase coding sequence and subsequent loss of one copy of each motif from the double length chain, and that the resulting enzyme was active as a monomer. With knowledge of the trimeric 3D structure of standard dUTPases, it is possible to suggest transformations that occurred in evolutionary development of the herpesvirus dUTPase. The distinct location of Motif 3 can indeed be seen to be consistent with it contributing to a single intramolecular active site with the other motifs. Separately, the occurrence in herpesvirus dUTPases of around 20 to 40 additional residues between Motifs 4 and 5 allows the C-terminal Motif 5 to reach the active site intramolecularly. The driving force behind these evolutionary changes remains obscure. We speculate that they may have allowed acquisition of a novel, presently unknown function by the protein. Consistent with this idea is the observation that in Alpha- and Gammaherpesvirus dUTPases the original locus of Motif 3 is occupied by a distinct conserved sequence (Motif 6); perhaps this element constitutes part of a separate functional capability. Notably, the apparently orthologous protein in Betaherpesviruses lacks the standard motifs while Motif 6 is still present.