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The backbone structure and dynamics of the 30 N-terminal residues of HIV-1 gp41 in membrane-mimicking environments is investigated using NMR spectroscopy and (super 15)N- and (super 15)N, (super 13)C, (super 2)H-labeled peptides. It is found that residues external to the micelle exhibit enhanced picosecond to nanosecond time scale dynamics and their mobility increases with distance from the micelle.

The structure of anti-tumor and anti-HIV-1 plant protein MAP30, a ribosome-inactivating protein, has been investigated and structural insights into multiple functions gained. MAP30 comes from Momordica charantia, bitter melon, the extracts of which have long been used as therapeutic agents. The protein is among the largest single-chain proteins the solution structures of which have been found without a prior X-ray crystal structure. It behaves as would a DNA glycosylase/apurinic lyase.

Proton relaxation of a perdeuterated protein, HIV-1 protease, bound to a protonated inhibitor DMP323 has been investigated. The contributions of (super1)H-(super1)H dipolar interactions to the relaxation of the amide protons are significantly reduced by perdeuteration. The overall relaxation rate is further reduced by the ROESY R(sub1p) experiment. A reduction in the proton spin-flip rate in the perdeuterated protein allowed measurement of (super15)N R(sub2) values, based on the CPMG method.

The amide backbone dynamics were studied by correlated 1H-15N NMR spectroscopy to elucidate HIV-1 protease-inhibitor reactions. The dynamics of HIV-1 protease complexed to KNI-272, a potent, asymmetric inhibitor which lifts the chemical shift degeneracy of the protease monomers, were determined. More than 90% of the backbone amide sites were found to be highly ordered with the squared order parameter, averaged over all measured residues.

The binding interactions between HIV-1 protease and the peptidomimetic transition state analog inhibitor KNI-221 in water were examined via 2D water/NOESY and water/ROESY experiments. The results revealed the presence of well-ordered water molecules bound to the protease/KNI-221 complex which have long residence times and short interproton distances to the Ile50/150, Ala28/128 and Asp29/129 amide protons.

The water-nuclear Overhauser enhancement spectroscopy (NOESY) and water-rotating frame NOESY (ROESY) spectra of the protease/DMP323 complex exhibited the large water-proton distances that characterizes H2O hydration. The 430/480 symmetry-related water molecules exhibited short residence times and internuclear distances that are higher the 2.5 angstrom. The local fluctuations that were exhibited by the protease/DMP323 complex also indicated the presence of H-D solvent exchange in inhibitor hydroxyl protons.

NMR spectroscopic surveys of monomer specific signal assignments for amino acid residues in the potent HIV-1 protease inhibitor KNI-272 provide evidence of distinct protonation states of Asp25/125, and throw light on interactions that induce inhibitor binding. The ionization states of the Asp carboxyl groups is determined through measurements of the pD dependence of the Asp carboxyl carbon chemical shifts as well as the effect of H/D isotope upon these shifts. The asymmetric nature of KNI-272 gives rise to distinct NMR spectra for the two protease monomers.

The structure of a peptide fragment of the HIV-1 envelope protein gp41(residues 282 - 304), when bound to micelles and bicelles is compared. The detergent micelles can induce significant strain in attached helical peptides and confirm that small bicelles are well suited for studying the detailed peptide structure in a biologically more relevant environment.

The rate of monomer interchange in a ligand-bound homodimeric protein from NOESY cross peaks has been determined from the HIV protease/KNI-529 complex. It was found that the cross-peaks result from a global process, which affects the exchange of each pair of residues in similar fashion. The exchange rate strongly depended on temperature, increasing from ca. 0.2 to 6 s (super -1) as the temperature increased from 25 to 45 degrees Celsius. In addition, structural information or sequential assignments are required to distinguish dipolar from exchange cross-peaks.

A four dimensional (4D) version of the NOESY experiment can be used effectively to gain numerous H(super N)-H(super N) NOE interactions in a N-terminal deletion mutant of the HIV-1 protein nef. Calculations suggest that 4D H(super N)-H(super N) NOESY on perdeuterated proteins provides high standard spectra up to correlation times of approximately 25 ns. Hence, it may become feasible to apply this method for getting structural information on slowly tumbling proteins.

Determination of pKa values of Asp and Glu residues in the protease/DMP-323 complex by NMR and X-ray crystallographic studies demonstrate the presence of protonated Asp25/25' carboxyl groups. This protonation occurs in the pD range 2 to 7 and carboxyl groups form strong hydrogen bonds with the inhibitor's diol hydroxyl groups, stabilizing the complex structure.

MAP30 is a 30 kDa single-stranded, type-I ribosome inactivating protein (RIP) possessing anti-tumor and anti-HIV activities. It binds both ribosomal RNA and the HIV-1 long-terminal repeat DNA. To understand the structural basis for MAP30 activities, we undertook the study of MAP30 by solution NMR spectroscopy. We report nearly complete 1H, 13C, and 15N chemical shift assignments of its 263 amino acids. Based upon an analysis of secondary 13C chemical shifts, 3JHNHA coupling constants, hydrogen exchange data, and nuclear Overhauser effect patterns, we find that the secondary structure and ²-sheet topology of MAP30 are very similar to those of the ricin A chain, a subunit of the well-known type-II RIP, even though two proteins display distinct activities. We therefore suggest that MAP30 and ricin A chain share a similar three-dimensional fold, and that the reported functional differences between two proteins arise primarily from differences in local three-dimensional structure and other structural properties such as surface electrostatic potentials. [ABSTRACT FROM PUBLISHER]

Hepatitis B virus (HBV) infection afflicts millions worldwide, causing cirrhosis and liver cancer. HBV e-antigen (HBeAg), a clinical marker for disease severity, is a soluble variant of the viral capsid protein. HBeAg is not required for viral replication but is implicated in establishing immune tolerance and chronic infection. The structure of recombinant e-antigen (rHBeAg) was recently determined, yet to date, the exact nature and quantitation of HBeAg still remain uncertain. Here, to further characterize HBeAg, we used phage display to produce a panel of chimeric rabbit/human monoclonal antibody fragments (both Fab and scFv) against rHBeAg. Several of the Fab/scFv, expressed in Escherichia coli, had unprecedentedly high binding affinities (Kd ~10-12 M) and high specificity. We used Fab/scFv in the context of an enzyme-linked immunosorbent assay (ELISA) for HBeAg quantification, which we compared with commercially available kits and verified with seroconversion panels, the WHO HBeAg standard, rHBeAg, and patient plasma samples. We found that the specificity and sensitivity are superior to those of existing commercial assays. To identify potential fine differences between rHBeAg and HBeAg, we used these Fabs in microscale immunoaffinity chromatography to purify HBeAg from individual patient plasmas. Western blotting and MS results indicated that rHBeAg and HBeAg are essentially structurally identical, although HBeAg from different patients exhibits minor carboxyl-terminal heterogeneity. We discuss several potential applications for the humanized Fab/scFv. [ABSTRACT FROM AUTHOR]

The core of skeletal muscle Z-discs consists of actin filaments from adjacent sarcomeres that are cross-linked by α-actinin homodimers. Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP)/Cypher interacts with α-actinin, myotilin, and other Z-disc proteins via the PDZ domain. However, these interactions are not sufficient to maintain the Z-disc structure. We show that ZASP directly interacts with skeletal actin filaments. The actin-binding domain is between the modular PDZ and LIM domains. This ZASP region is alternatively spliced so that each isoform has unique actin-binding domains. All ZASP isoforms contain the exon 6-encoded ZASP-like motif that is mutated in zaspopathy, a myofibrillar myopathy (MFM), whereas the exon 8-11 junction-encoded peptide is exclusive to the postnatal long ZASP isoform (ZASP-LΔex10). MFM is characterized by disruption of skeletal muscle Z-discs and accumulation of myofibrillar degradation products. Wild-type and mutant ZASP interact with α-actin, α-actinin, and myotilin. Expression of mutant, but not wild-type, ZASP leads to Z-disc disruption and F-actin accumulation in mouse skeletal muscle, as in MFM. Mutations in the actin-binding domain of ZASP-LΔex10, but not other isoforms, cause disruption of the actin cytoskeleton in muscle cells. These isoform-specific mutation effects highlight the essential role of the ZASP-LΔex10 isoform in F-actin organization. Our results show that MFM-associated ZASP mutations in the actin-binding domain have deleterious effects on the core structure of the Z-discs in skeletal muscle. [ABSTRACT FROM AUTHOR]