New nucleotide analogues with enhanced signal properties.

We describe synthesis and testing of a novel type of dye-modified nucleotides which we call macromolecular nucleotides (m-Nucs). Macromolecular nucleotides comprise a nucleotide moiety, a macromolecular linear linker, and a large macromolecular ligand carrying multiple fluorescent dyes. With incorporation of the nucleotide moiety into the growing nucleic acid strand during enzymatic synthesis, the macromolecular ligand together with the coupled dyes is bound to the nucleic acid. By the use of this new class of modified nucleotides, signals from multiple dye molecules can be obtained after a single enzymatic incorporation event. The modified nucleotides are considered especially useful in the fields of nanobiotechnology, where signal stability and intensity is a limiting factor.

Discovery and optimization of a natural HIV-1 entry inhibitor targeting the gp41 fusion peptide.

A variety of molecules in human blood have been implicated in the inhibition of HIV-1. However, it remained elusive which circulating natural compounds are most effective in controlling viral replication in vivo. To identify natural HIV-1 inhibitors we screened a comprehensive peptide library generated from human hemofiltrate. The most potent fraction contained a 20-residue peptide, designated VIRUS-INHIBITORY PEPTIDE (VIRIP), corresponding to the C-proximal region of alpha1-antitrypsin, the most abundant circulating serine protease inhibitor. We found that VIRIP inhibits a wide variety of HIV-1 strains including those resistant to current antiretroviral drugs. Further analysis demonstrated that VIRIP blocks HIV-1 entry by interacting with the gp41 fusion peptide and showed that a few amino acid changes increase its antiretroviral potency by two orders of magnitude. Thus, as a highly specific natural inhibitor of the HIV-1 gp41 fusion peptide, VIRIP may lead to the development of another class of antiretroviral drugs.

NMR analysis of carbohydrate-protein interactions.

Carbohydrate-protein interactions are frequently characterized by dissociation constants in the microM to mM range. This is normally associated with fast dissociation rates of the corresponding complexes, in turn leading to fast exchange on the nuclear magnetic resonance (NMR) chemical shift time scale and on the NMR relaxation time scale. Therefore, NMR experiments that take advantage of fast exchange are well suited to study carbohydrate-protein interactions. In general, it is possible to analyze ligand binding by observing either protein signals or ligand resonances. Because most receptor proteins to which carbohydrates bind are rather large with molecular weights significantly exceeding 30 kDa, the analysis of the corresponding protein spectra is not trivial, and only very few studies have been addressing this issue so far. We, therefore, focus on NMR experiments that employ observation of free ligand, that is, carbohydrate signals to analyze the bound state. Two types of NMR experiments have been extremely valuable to analyze carbohydrate-protein interactions at atomic resolution. Whereas transferred nuclear Overhauser effect (NOE) experiments deliver bioactive conformations of carbohydrates binding to proteins, saturation transfer difference (STD) NMR spectra provide binding epitopes and valuable information about the binding thermodynamics and kinetics. We demonstrate the power of a combined transfer NOE/STD NMR approach for the analysis of carbohydrate-protein complexes using selected examples.

Blood group B galactosyltransferase: insights into substrate binding from NMR experiments.

The biosynthesis of human blood group B antigens is accomplished by a highly specific galactosyltransferase (GTB). On the basis of NMR experiments, we propose a "molecular tweezers mechanism" that accounts for the exquisite stereoselectivity of donor substrate selection. Transferred NOE experiments for the first time reveal the bioactive conformation of the donor substrate UDP-galactose (UDP-Gal) and of its enzymatically inactive analogue, UDP-glucose (UDP-Glc). Both bind to GTB in a folded conformation that is sparsely populated in solution, whereas acceptor ligands bind in a conformation that predominates in solution. The bound conformations of UDP-Gal and UDP-Glc are identical within experimental error. Therefore, GTB must discriminate between the two activated sugars on the basis of a hitherto unknown transition state that can only be formed in the case of UDP-Gal. A full relaxation and exchange matrix analysis of STD NMR experiments reveals that acceptor substrates dissociate significantly faster (k(off) > 100 Hz) from the binding pocket than donor substrates (k(off) approximately 10 Hz). STD NMR experiments also directly show that proper recognition of the hexopyranose rings of the UDP sugars requires bivalent metal cations. At the same time, this analysis furnishes the complete three-dimensional structure of the enzyme with its bound donor substrate UDP-Gal on the basis of a prior crystal structure analysis. We propose that, upon acceptor binding, GTB uses the Asp 302 and Glu 303 side chains as "molecular tweezers" to promote bound UDP-Gal but not UDP-Glc into a transition state that leads to product formation.

Fragment-based screening of the donor substrate specificity of human blood group B galactosyltransferase using saturation transfer difference NMR.

Saturation transfer difference NMR experiments on human blood group B alpha-(1,3)-galactosyltransferase (GTB) for the first time provide a comprehensive set of binding epitopes of donor substrate analogs in relation to the natural donor UDP-Gal. This study revealed that the enzyme binds several UDP-activated sugars, including UDP-Glc, UDP-GlcNAc, and UDP-GalNAc. In all cases, UDP is the dominant binding epitope. To identify the minimum requirements for specific binding, a detailed analysis utilizing a fragment-based approach was employed. The binding of donor substrate to GTB is essentially controlled by the base as a "molecular anchor." Uracil represents the smallest fragment that is recognized, whereas CDP, AMP, and GDP do not exhibit any significant binding affinity for the enzyme. The ribose and beta-phosphate moieties increase the affinity of the ligands, whereas the pyranose sugar apparently weakens the binding, although this part of the molecule controls the specificity of the enzyme. Accordingly, UDP represents the best binder. The binding affinities of UDP-Gal, UDP-Glc, and UMP are about the same, but lower than that of UDP. Furthermore, we observed that beta-D-galactose and alpha-D-galactose bind weakly to GTB. Whereas beta-D-galactose binds to the acceptor and donor sites, it is suggested that alpha-D-galactose occupies a third hitherto unknown binding pocket. Finally, our experiments revealed that modulation of enzymatic activity by metal ions critically depends on the total enzyme concentration, raising the question as to which of the bivalent metal cations Mg(2+) and Mn(2+) is more relevant under physiological conditions.

Refinement of the conformation of UDP-galactose bound to galactosyltransferase using the STD NMR intensity-restrained CORCEMA optimization.

The STD NMR technique has originally been described as a tool for screening large compound libraries to identify the lead compounds that are specific to target proteins of interest. The application of this technique in the qualitative epitope mapping of ligands weakly binding to proteins, virus capsid shells, and nucleic acids has also been described. Here we describe the application of the STD NMR intensity-restrained CORCEMA optimization (SICO) procedure for refining the bound conformation of UDP-galactose in galactosyltransferase complex using STD NMR intensities recorded at 500 MHz as the experimental constraints. A comparison of the SICO structure for the bound UDP-galactose in solution with that in the crystal structure for this complex shows some differences in ligand torsion angles and V253 side-chain orientation in the protein. This work describes the first application of an STD NMR intensity-restrained CORCEMA optimization procedure for refining the torsion angles of a bound ligand structure. This method is likely to be useful in structure-based drug design programs since most initial lead compounds generally exhibit weak affinity (millimolar to micromolar) to target proteins of pharmaceutical interest, and the bound conformation of these lead compounds in the protein binding pocket can be determined by the CORCEMA-ST refinement.

Molecular Recognition of UDP-Gal by ß-1,4-Galactosyltransferase T1.

Saturation transfer difference (STD) NMR experiments reveal the binding epitopes of UDP-Gal and UDP-Glc bound to the glycosyltransferase ß4Gal-T1. Whereas the enzyme recognizes the galactose residue in UDP-Gal, it does not make any close contacts with the glucose residue in UDP-Glc. This observation explains why ß4Gal-T1 binds to UDP-Glc but is unable to transfer glucose to an acceptor substrate.