The structure of a short fragment of the human HIV-1 membrane glycoprotein gp41 has been examined using a combination of parallel tempering molecular dynamics (PTMD) and far UV circular dichroism spectroscopy. The aim is to resolve conflicting reports on the solution state conformational bias in this membrane proximal domain spanning the epitope for the 2F5 monoclonal antibody. We conclude that gp41(659-671) exhibits conformational plasticity in which competing folding propensities are present and can be influenced by local microenvironment. Contrary to previous reports, the 3(10) helix does not emerge as a dominant motif from either simulation or experiment, and this peptide is therefore not a model system for this fold type. Other fold groups such as turn motifs are identifiable at elevated temperatures in the PTMD trajectories and are potentially relevant in antibody binding. Helical populations in pure water are significantly overestimated according to the CHARMM parametrization. However, circular dichroism (CD) data show that helices are promoted in membrane mimetic solvents. As this is a membrane proximal peptide, the helical motif may well have physiological significance.

The flexibility of the Membrane Proximal Region (MPR) of the HIV-1 gp41 envelope glycoprotein is believed to be relevant to its biological function. Its conformational bias is potentially influenced by the various environmental conditions experienced during viral fusion. Using a combination of Circular Dichroism and Molecular Dynamics simulations, we show that a very short MPR fragment gp41(659-671) spanning the 2F5 monoclonal antibody epitope, exhibits autonomous helical folding in the presence of membrane mimicking SDS micelles and the extent of which can be tuned by pH variation: Specifically, the peptide shows no defined fold type at basic pH but is helical at physiological and lower pH environments. By contrast, no such control of helical folding by pH is observed in aqueous solutions in the absence of SDS. Instead, the experimental data imply that unfolded structures persist and that pH has negligible influence on conformational bias. We also explore the pronounced sensitivity to standard empirical potentials and conclude that AMBER-ff03 provides a reasonably accurate description of the solution state structure and is therefore a good choice for future exploration of membrane-induced phenomena.

Membrane-mediated structural modulation in two short fragments of the human HIV-1 envelope protein gp41 is demonstrated. Derived from the C-terminal membrane proximal external (MPE) and N-terminal fusion peptide proximal (FPP) regions, these peptides are widely separated in the primary sequence but form tertiary contacts during the intermediate (hemifusion) phase of HIV infection. The structural perturbations observed at the membrane interface offer evidence of rudimentary regulatory mechanisms operating in the free peptides which may be relevant in the biological system. No such regulatory phenomena were observed for the individual peptides in a membrane environment or between the peptides in aqueous solutions. Structure determination is made using a combination of circular and linear dichroism spectroscopy (supported by calorimetric measurements) and molecular dynamics simulations. Specifically, we show that these peptides interact locally without the conformational support of helical heptad repeat regions in native gp41 and that the modulation is not mutual with the FPP peptide operating as a primary regulator of the MPE-FPP interactions in the hemifusion phase.

Human chorionic gonadotropin (hCG) is an important biomarker in pregnancy and oncology, where it is routinely detected and quantified by specific immunoassays. Intelligent epitope selection is essential to achieving the required assay performance. We present binding affinity measurements demonstrating that a typical ␤3-loop-specific monoclonal antibody (8G5) is highly selective in competitive immunoassays and distinguishes between hCG␤ 66 -80 and the closely related luteinizing hormone (LH) fragment LH␤ 86 -100 , which differ only by a single amino acid residue. A combination of optical spectroscopic measurements and atomistic computer simulations on these free peptides reveals differences in turn type stabilized by specific hydrogen bonding motifs. We propose that these structural differences are the basis for the observed selectivity in the full protein.The glycoprotein human chorionic gonadotropin (hCG), 2 is a heterodimer consisting of an ␣-and ␤-chain (Fig. 1a) in noncovalent association. Essential in pregnancy and detectable within a few days of fertilization, it has become one of the most frequently assayed hormones, and simple, one-step, antibodybased measurements of hCG are now standard in pregnancy testing. At this early stage of pregnancy, the trophoblast cells of the preimplantation embryo produce a hyperglycosylated form of the hormone, which drives embryonic implantation in the uterine wall (2). Subsequently, hCG regulation by syncytiotrophoblast cells of the placenta promotes and maintains progesterone secretion from the corpus luteum. In other applications, therapeutically injected hCG is the key to in vitro fertilization, where it is used to trigger ovulation and, in males, used to promote testosterone production.The role of hCG as a tumor marker in a variety of cancers is of great clinical significance (3, 4), and quantitative, specific hCG tests are important in the diagnosis and management of hCGsecreting cancers (5). However, the design criteria of hCG cancer assays differ from those required for pregnancy tests. Specifically, different sensitivity ranges are involved, and, for oncology applications, the antibodies used must be able to detect various modified forms of hCG with equal affinity (6). The role of hCG assays in the diagnosis and management of malignant trophoblastic disease (choriocarcinoma) is one of the great success stories of oncology (7,8); it is the ideal tumor marker, always present when choriocarcinoma cells exist, and in quantities directly related to the number of those cells. The correct use of hCG assays in combination with appropriate therapy has led to a survival rate approaching 100% in this otherwise aggressive cancer.All hCG diagnostic tests are carried out as immunoassays, based on specific antibodies that are able to distinguish hCG from three other, closely related, members of the glycoprotein hormone family (luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH)) (9). The ␣-chains of these four hormones are identica...