Structural comparison of a 15 residue peptide from the V3 loop of HIV-1IIIb and an O-glycosylated analogue

AbstractAs part of a program to study the effect of glycosylation on the three-dimensional structures of HIV-1IIIB V3 peptide constructs, we have examined the solution structures of a 15 residue peptide (RIQRGPGRAFVTIGK, P18IIIB), originally mapped as an epitope recognized by CD8+ Dd class I MHC-restricted murine cytotoxic T-lymphocytes (CTL), and an analogue (P18IIIB-g), O-glycosylated with an α-galactosamine on Thr-12, using NMR, circular dichroism and molecular modeling methods. Our studies show that the peptides sample mainly random conformations in aqueous solution near 25°C and become more ordered by the addition of trifluoroethanol. Upon decreasing the temperature to 5°C, a reverse turn is formed around the immunodominant tip (G5−R8). Glycosylation on T12 ‘tightens’ the turn slightly as suggested by NOE and CD analysis. In addition, the sugar has a defined conformation with respect to the peptide backbone and influences the local peptide conformation. These data suggest that simple glycosylation may influence the conformational equilibrium of a V3 peptide which contains a domain critical for antibody recognition and virus neutralization. We also show that the ability of cytotoxic T-lymphocytes (CTL) to lyse tumor cells presenting P18IIIB was completely abrogated by threonine glycosylation

Biophysical studies of DNA modified with conformationally constrained nucleotides: comparison of 2′-exo (north) and 3′-exo (south) ‘locked’ templates

The biophysical properties of oligodeoxyribonucleotides (ODNs) selectively modified with conformationally ‘locked’ bicyclo[3.1.0]hexane pseudosugars (Maier,M.A., Choi,Y., Gaus,H., Barchi,J.J. Jr, Marquez,V.E., Manoharan,M. (2004) Synthesis and characterization of oligonucleotides containing conformationally constrained bicyclo[3.1.0]hexane pseudosugar analogs Nucleic Acids Res., 32, 3642–3650) have been studied by various techniques. Six separate synthetic ODNs based on the Dickerson Drew dodecamer sequence (CGCGAAT*T*CGCG) were examined where each one (or both) of the thymidines (T*) were substituted with a bicyclic pseudosugar locked in either a North (2′-exo) or South (3′-exo) ring pucker. Circular dichroism spectroscopy, differential scanning calorimetry and 1H NMR spectroscopy were used to examine the duplex stability and conformational properties of the ODNs. Replacement of one or both thymidines with North-locked sugars (RNA-like) into the dodecamer did not greatly affect duplex formation or melt temperatures but distinct differences in thermodynamic parameters were observed. In contrast, incorporation of South-locked sugar derivatives that were predicted to stabilize this standard B-DNA, had the unexpected effect of causing a conformational equilibrium between different duplex forms at specific strand and salt concentrations. Our data and those of others suggest that although DNA can tolerate modifications with RNA-like (North) nucleotides, a more complicated spectrum of changes emerges with modifications restricted to South (DNA-like) puckers