Horea Porumb

Self-association and Domains of Interactions of an Amphipathic Helix Peptide Inhibitor of HIV-1 Integrase Assessed by Analytical Ultracentrifugation and NMR Experiments in Trifluoroethanol/H2O Mixtures

EAA26 (VESMNEELKKIIAQVRAQAEHLKTAY) is a better inhibitor of human immunodeficiency virus, type 1, integrase than its parent Lys-159, reproducing the enzyme segment 147–175 with a nonpolar-polar/charged residue periodicity defined by four helical heptads (abcdefg) prone to collapse into a coiled-coil. Circular dichroism, nuclear magnetic resonance, sedimentation equilibrium, and chemical cross-linking were used to analyze EAA26 in various trifluoroethanol/H2O mixtures. In pure water the helix content is weak but increases regularly up to 50–60% trifluoroethanol. In contrast the multimerization follows a bell-shaped curve with monomers in pure water, tetramers at 10% trifluoroethanol, and dimers at 40% trifluoroethanol. All suggest that interhelical interactions between apolar side chains are required for the coiled-coil formation of EAA26 and subsist at medium trifluoroethanol concentration. The NH temperature coefficients measured by nuclear magnetic resonance show that at low trifluoroethanol concentration the amide groups buried in the hydrophobic interior of four α-helix bundles are weakly accessible to trifluoroethanol and are only weakly subject to its hydrogen bond strengthening effect. The increased accessibility of trifluoroethanol to buried amide groups at higher trifluoroethanol concentration entails the reduction of the hydrophobic interactions and the conversion of helix tetramers into helix dimers, the latter displaying a smaller hydrophobic interface. The better inhibitory activity of EAA26 compared with Lys-159 could arise from its better propensity to form a helix bundle structure with the biologically important helical part of the 147–175 segment in integrase.

Circular dichroism signatures of features simultaneously present in structured guanine-rich oligonucleotides: A combined spectroscopic and electrophoretic approach

In order to identify possible signatures of the most typical structures adopted by guanine‐rich oligonucleotides, we submitted them to the crossed fire of circular dichroism (CD) and electrophoresis. These signatures show up in the circular dichroism spectra even when simultaneously present within the same molecule. Guanine‐rich oligonucleotides, when structured, manifest themselves by CD contributions around 260 or 295 nm. For instance, positive bands at 264 nm and 295 nm, respectively, signal the parallel and antiparallel guanine quartets, while a positive band around 261 nm may indicate the presence of a (parallel?) Hoogsteen duplex. A positive band at 264 nm may also reflect the presence of rigidly and unusually oriented GpT and TpG steps within loops. The signatures are additive with those of other structural features of the same molecule, such as hairpins or Watson‐Crick duplexes, whose bands are observed at 280 nm.

Conformations of three-stranded DNA structures formed in presence and in absence of the RecA protein.

Using FTIR and UV spectroscopies, we have studied the structures of three-stranded DNA complexes (TSC) having two identical strands, containing all four bases, in parallel orientation. In the first system, an intermolecular TSC is formed by the addition of the third strand (ssDNA) previously coated with RecA protein to an hairpin duplex (dsDNA), in presence of ATP gamma S. In the second one, the formation of an intramolecular triplex is forced by folding back twice on itself an oligonucleotide. The sequences of the three strands are the same in both systems. The formation of the RecA-TSC, which accommodates all four bases, is evidenced by gel retardation assay, and by its biphasic melting profile observed by UV spectroscopy. Using FTIR spectroscopy, N-type sugars are detected in this structure. This shows that in the RecA-TSC studied in presence of the protein, the nucleic acid part adopts an extended form, in agreement with the model proposed by Zhurkin et al. (1,2) and electron microscopy observations (3-6). In contrast, the RecA-free intramolecular triplex in a non extended form has S-type sugars.

An unusual helix turn helix motif in the catalytic core of HIV-1 integrase binds viral DNA and LEDGF.

Background Integrase (IN) of the type 1 human immunodeficiency virus (HIV-1) catalyzes the integration of viral DNA into host cellular DNA. We identified a bi-helix motif (residues 149–186) in the crystal structure of the catalytic core (CC) of the IN-Phe185Lys variant that consists of the α4 and α5 helices connected by a 3 to 5-residue turn. The motif is embedded in a large array of interactions that stabilize the monomer and the dimer. Principal Findings We describe the conformational and binding properties of the corresponding synthetic peptide. This displays features of the protein motif structure thanks to the mutual intramolecular interactions of the α4 and α5 helices that maintain the fold. The main properties are the binding to: 1- the processing-attachment site at the LTR (long terminal repeat) ends of virus DNA with a Kd (dissociation constant) in the sub-micromolar range; 2- the whole IN enzyme; and 3- the IN binding domain (IBD) but not the IBD-Asp366Asn variant of LEDGF (lens epidermal derived growth factor) lacking the essential Asp366 residue. In our motif, in contrast to the conventional HTH (helix-turn-helix), it is the N terminal helix (α4) which has the role of DNA recognition helix, while the C terminal helix (α5) would rather contribute to the motif stabilization by interactions with the α4 helix. Conclusion The motif, termed HTHi (i, for inverted) emerges as a central piece of the IN structure and function. It could therefore represent an attractive target in the search for inhibitors working at the DNA-IN, IN-IN and IN-LEDGF interfaces.

Parallel and antiparallel triple helices with G,A-containing third strands

A triple helix, formed by a 13 nucleotide (nt) all‐purine oligonucleotide, containing six contiguous guanines, oriented parallel to a homopurine strand present in the polypurine tract of Friend leukemia virus, was obtained in 0.1 M LiCl. Its dissociation constant at 25oC, given by electrophoretic titration, of the order of 50 nM, is at least ten times lower than that of the corresponding antiparallel triplex formed on the same target. At 4oC, the parallel orientation of the homopurine strands is favored to the point that the guanine block of 6 nt, present in the ‘antiparallel’ oligonucleotide, attaches in a parallel fashion to the corresponding block in the target strand, to generate a partial, parallel triplex, that coexists with the antiparallel one.

A spin-labeled derivative of Procion brilliant blue MX-R: Synthesis and interaction with pig heart nucleoside diphosphate kinase

ESR Nucleoside diphosphate kinase Anthraquinone trimme dye Differential spectroscopy Phospholransferase active site