Combining nucleoside analogues to achieve recognition of oligopurine tracts by triplex-forming oligonucleotides at physiological pH

We have used DNase I footprinting to examine DNA triple helix formation at a 12 base pair oligopurine.oligopyrimidine sequence, using oligonucleotides that contain combinations of 2'-aminoethoxy-5-(3-aminoprop-1-ynyl)uridine (bis-amino-U, BAU) and 3-methyl-2-aminopyridine (MeP) in place of T and C, respectively. This combination acts cooperatively to enable high affinity triple helix formation at physiological pH. The affinity depends on the number of substitutions and their arrangement; oligonucleotides in which these analogues are evenly distributed throughout the third strand bind much better than those in which they are clustered together.     

DNA melting profiles from a matrix method

In this article we give a new method for the calculation of DNA melting profiles. Based on the matrix formulation of the DNA partition function, the method relies for its efficiency on the fact that the required matrices are very sparse, essentially reducing matrix multiplication to vector multiplication and thus making the computer time required to treat a DNA molecule containing N base pairs proportional to N(2). A key ingredient in the method is the result that multiplication by the inverse matrix can also be reduced to vector multiplication. The task of calculating the melting profile for the entire genome is further reduced by treating regions of the molecule between helix-plateaus, thus breaking the molecule up into independent parts that can each be treated individually. The method is easily modified to incorporate changes in the assignment of statistical weights to the different structural features of DNA. We illustrate the method using the genome of Haemophilus influenzae.     

Electrostatics of ion stabilization in a ClC chloride channel homologue from Escherichia coli

The structural determinants of electrostatics of ion stabilization within EcClC, a ClC-type chloride channel homologue from Escherichia coli, are studied using a continuum dielectric approximation. Specifically, the ion occupancy is investigated in the wild-type protein and a mutant thereof, and the contribution to the electrostatic binding free energy of local and non-local interactions is characterized at the single-residue level. This analysis shows that, in spite of the desolvation cost and the strong ion-ion repulsion, all previously reported binding sites can be occupied simultaneously. The stabilizing effect of the protein arises from hydrogen bonding as well as from longer-range favorable interactions, such as with the strictly conserved Lys131 side-chain. The latter is involved in the stabilization of the conserved GSGIP motif that delimits two of the binding sites. Interestingly, an additional low-affinity binding site, mediated by a structurally analogous motif including the side-chain of Arg340, can be identified on the extracellular side of the permeation pathway. Finally, it is shown that, in contrast to K-channels, and in analogy to the SBP/PBP sulfate/phosphate-binding proteins, the contribution of helix macrodipoles to chloride binding in EcClC is only marginal.     

Side-chain contributions to membrane protein structure and stability

The molecular forces that stabilize membrane protein structure are poorly understood. To investigate these forces we introduced alanine substitutions at 24 positions in the B helix of bacteriorhodopsin and examined their effects on structure and stability. Although most of the results can be rationalized in terms of the folded structure, there are a number of surprises. (1) We find a remarkably high frequency of stabilizing mutations (17%), indicating that membrane proteins are not highly optimized for stability. (2) Helix B is kinked, with the kink centered around Pro50. The P50A mutation has no effect on stability, however, and a crystal structure reveals that the helix remains bent, indicating that tertiary contacts dominate in the distortion of this helix. (3) We find that the protein is stabilized by about 1kcal/mol for every 38A(2) of surface area buried, which is quite similar to soluble proteins in spite of their dramatically different environments. (4) We find little energetic difference, on average, in the burial of apolar surface or polar surface area, implying that van der Waals packing is the dominant force that drives membrane protein folding.     

Helix packing moments reveal diversity and conservation in membrane protein structure

Helical membrane proteins are more tightly packed and the packing interactions are more diverse than those found in helical soluble proteins. Based on a linear correlation between amino acid packing values and interhelical propensity, we propose the concept of a helix packing moment to predict the orientation of helices in helical membrane proteins and membrane protein complexes. We show that the helix packing moment correlates with the helix interfaces of helix dimers of single pass membrane proteins of known structure. Helix packing moments are also shown to help identify the packing interfaces in membrane proteins with multiple transmembrane helices, where a single helix can have multiple contact surfaces. Analyses are described on class A G protein-coupled receptors (GPCRs) with seven transmembrane helices. We show that the helix packing moments are conserved across the class A family of GPCRs and correspond to key structural contacts in rhodopsin. These contacts are distinct from the highly conserved signature motifs of GPCRs and have not previously been recognized. The specific amino acid types involved in these contacts, however, are not necessarily conserved between subfamilies of GPCRs, indicating that the same protein architecture can be supported by a diverse set of interactions. In GPCRs, as well as membrane channels and transporters, amino acid residues with small side-chains (Gly, Ala, Ser, Cys) allow tight helix packing by mediating strong van der Waals interactions between helices. Closely packed helices, in turn, facilitate interhelical hydrogen bonding of both weakly polar (Ser, Thr, Cys) and strongly polar (Asn, Gln, Glu, Asp, His, Arg, Lys) amino acid residues. We propose the use of the helix packing moment as a complementary tool to the helical hydrophobic moment in the analysis of transmembrane sequences.     

Amino acid propensities are position-dependent throughout the length of alpha-helices

The 20 commonly occurring amino acids have been shown to have distinct position-dependent, helix-forming propensities near the ends of alpha-helices. Here, we show that the amino acids also have very strong position-dependent propensities throughout the length of a helix. Most helices are amphiphilic, and they have a strong tendency to both begin and end on the solvent-inaccessible face of the helix. These position-specific propensities should provide valuable parameters to guide de novo protein design, and should allow more precise prediction of helical topology in natural proteins.     

ExSer: A standalone tool to mine protein data bank (PDB) for secondary structural elements

Detailed structural analysis of protein necessitates investigation at primary, secondary and tertiary levels, respectively. Insight into protein secondary structures pave way for understanding the type of secondary structural elements involved (α-helices, β-strands etc.), the amino acid sequence that encode the secondary structural elements, number of residues, length and, percentage composition of the respective elements in the protein. Here we present a standalone tool entitled "ExSer" which facilitate an automated extraction of the amino acid sequence that encode for the secondary structural regions of a protein from the protein data bank (PDB) file. AVAILABILITY: ExSer is freely downloadable from http://code.google.com/p/tool-exser/     

Ribosomes Lacking Protein S20 Are Defective in mRNA Binding and Subunit Association

The functional significance of ribosomal proteins is still relatively unclear. Here, we examined the role of small subunit protein S20 in translation using both in vivo and in vitro techniques. By means of lambda red recombineering, the rpsT gene, encoding S20, was removed from the chromosome of Salmonella enterica var. Typhimurium LT2 to produce a ΔS20 strain that grew markedly slower than the wild type while maintaining a wild-type rate of peptide elongation. Removal of S20 conferred a significant reduction in growth rate that was eliminated upon expression of the rpsT gene on a high-copy-number plasmid. The in vitro phenotype of mutant ribosomes was investigated using a translation system composed of highly active, purified components from Escherichia coli. Deletion of S20 conferred two types of initiation defects to the 30S subunit: (i) a significant reduction in the rate of mRNA binding and (ii) a drastic decrease in the yield of 70S complexes caused by an impairment in association with the 50S subunit. Both of these impairments were partially relieved by an extended incubation time with mRNA, fMet-tRNA^fMet, and initiation factors, indicating that absence of S20 disturbs the structural integrity of 30S subunits. Considering the topographical location of S20 in complete 30S subunits, the molecular mechanism by which it affects mRNA binding and subunit docking is not entirely obvious. We speculate that its interaction with helix 44 of the 16S ribosomal RNA is crucial for optimal ribosome function.     

Higher order structure of short collagen model peptides attached to dendrimers and linear polymers

Collagen, which is used as a biomaterial, is the most abundant protein in mammals. We have previously reported that a dendrimer modified with collagen model peptides, (Gly‐Pro‐Pro)₅, formed a collagen‐like triple‐helical structure, showing thermal reversibility. In this study, various collagen‐mimic dendrimers of different generations and at different binding ratios were synthesized, to investigate the relationship between the peptide clustering effect and the higher order structure formation. The formation of the higher order structure was influenced by the binding ratios of the peptide to the dendrimer, but was not influenced by the dendrimer generation. A spacer, placed between the dendrimer terminal group and the peptide, negatively contributed to the formation of the higher order structure. The collagen model peptides were also attached to poly(allylamine) (PAA) and poly‐L ‐lysine (poly(Lys)) to compare them with the collagen‐mimic dendrimers. The PAA‐based collagen‐mimic compound, bearing more collagen model peptides than the dendrimer, exhibited a thermally stable higher order structure. In contrast, this was not observed for the collagen‐mimic polymers based on poly(Lys). Therefore, dendrimers and vinyl polymers act as a scaffold for collagen model peptides and subsequently induce higher order structures. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 640–648, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com