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A solid phase assay for human immunodeficiency virus (HIV) protease using an immobilized substrate, Affi Gel 10-Gly-Gly-Gly-Gly-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-[3H]Gly-OH has been devised. The Tyr-Pro bond of the substrate was hydrolyzed by the protease, releasing the radiolabeled cleavage product, Pro-Ile-Val-Gln-[3H]Gly-OH, into the supernatant. The pH optimum was found to be 6.0, and a high ionic strength was required for maximal activity. The solid phase assay is usable for convenient monitoring of purification procedures, and rapid screening of inhibitors of HIV protease.

Background: 1 H and 15 N transverse relaxation measurements on perdeuterated proteins are ideally suited for detecting backbone conformational fluctuations on the millisecond–microsecond timescale. The identification of conformational exchange on this timescale by measuring the relaxation of both 1 H and 15 N holds great promise for the elucidation of functionally relevant conformational changes in proteins. Results: We measured the transverse 1 H and 15 N relaxation rates of backbone amides of HIV-1 protease in its free and inhibitor-bound forms. An analysis of these rates, obtained as a function of the effective rotating frame field, provided information about the timescale of structural fluctuations in several regions of the protein. The flaps that cover the active site of the inhibitor-bound protein undergo significant changes of backbone ( φ , ψ ) angles, on the 100 μ s timescale, in the free protein. In addition, the intermonomer β -sheet interface of the bound form, which from protease structure studies appears to be rigid, was found to fluctuate on the millisecond timescale. Conclusions: We present a working model of the flap-opening mechanism in free HIV-1 protease which involves a transition from a semi-open to an open conformation that is facilitated by interaction of the Phe53 ring with the substrate. We also identify a surprising fluctuation of the β -sheet intermonomer interface that suggests a structural requirement for maturation of the protease. Thus, slow conformational fluctuations identified by 1 H and 15 N transverse relaxation measurements can be related to the biological functions of proteins.

Ac-Lys-Ala-Ser-Gln-Asn-Phe(NO2)-Pro-Val-Val-NH2 (peptide I) and Thr-Phe-Gln-Ala-Phe(NO2)-Pro-Leu-Arg-Glu-Ala (peptide II) undergo hydrolysis between the p-nitrophenylalanyl and prolyl residues catalyzed by the proteases of HIV-1 and AMV, respectively. The specific hydrolyses of peptides I and II are accompanied by a decrease in their uv absorption at 269 nm (delta epsilon = 1000) and an increase at 316 nm (delta epsilon = 600). The use of microspectrophotometric cells allows continuous uv measurements on a volume (60 to 120 microliters) comparable to that required for the HPLC point assay currently used. At the highest substrate concentration possible under the assay conditions, good first-order kinetics were observed with both proteases, and the values of Vmax/Km were obtained.

The 297bp gene coding for the HIV-1 protease was chemically synthesized and expressed in E. coli. Single amino acid substitutions (Arg 87 - greater than Lys; Arg 87 - greater than Glu) were introduced in the C-terminally located conserved region GlyArgAsn of the protease gene in the wild-type clone. The products of the mutant and the wild-type clones were expressed at approximately similar levels at 30 minutes of induction but the mutant protease proteins accumulated as a function of time of induction unlike the wild-type protease which declined after 60 minutes. The mutants were completely devoid of proteolytic activity as determined in assays employing as substrates a synthetic nonapeptide and a gag related recombinant polyprotein.

The 297bp HIV-1 protease gene was constructed from five discrete synthetic fragments and expressed in E.coli . A soluble protein product of 11.5 Kd was detected by immunoblotting using protease specific antisera. A quantitative assay system, utilizing a synthetic nonapeptide spanning the cleavage site between p17–p24 in the gag polyprotein, was used to measure the specific protease activity in crude extracts. The protease hydrolyzed tyrosyl-proline bonds with an approximate specific activity of 43 pmoles/min/μg of total protein. The chemical synthesis of the protease gene and it's expression provides a feasible method for rapid mutant analysis, important for structure-function studies and rational design of potential inhibitors.

The phosphoprotein p53 has been investigated mainly because of its relationship with tumorigenic transformation. In this communication, we report that, during the embryonal development of mouse and chicken, there is a decline in the steady-state levels of the p53 protein and an equal decline in p53 mRNA. During the development of the chicken, the relative rates of p53 transcription appear to be constant. p53 mRNA is relatively stable (half-life greater than 12 h) in both chicken and mouse embryos. We conclude that (i) the down-modulation of p53 mRNA (and of protein) during embryonal development has been well conserved during the evolution of the vertebrate, implying that the p53 protein may have a function in embryonal development; and (ii) the mechanism of control is apparently mainly on a post-transcriptional level.

Stimulation of β-adrenoreceptors in rat parotid acinar cells in vitro by the β-adrenergic agonist isoproterenol induces steady-state levels of c- fos mRNA and c- fos protein in these cells. A dramatic increase in the steady-state levels of c- fos mRNA was observed at 60 min, followed by a decrease at 2 h with a second peak at 4 h. c- fos induction in rat parotid acinar cells in vitro seems to be mediated by cAMP. Increased levels of p53 and c- myc mRNA were detected only at 60 min. c- abl and c- sis were also induced by isoproterenol but in a pattern different from that seen with c- fos . c- abl was the only oncogene in rat parotid gland which showed increased expression after chronic isoproterenol treatment of rats. In rat parotid acinar cells we observed no correlation between DNA synthesis and c- fos induction.

Stimulation of β-adrenoreceptors in the RSMT-A5 epithelial cell line is accompanied by an early and transient increase in the expression of the proto-oncogene c-fos. Maximal induction was at 30 min, returning to basal levels after 2 h. Similar results were obtained when cells were incubated with 8-bromo-cAMP. The induction of c-fos is specific since the expression of p53, a transformation-related gene, is not modulated by isoproterenol or 8-bromo-cAMP. The increase in c-fos gene expression is not associated with proliferative activity in these epithelial cells.

Major stages of Dictyostelium development are regulated by secreted, extracellular cAMP through activation of a serpentine receptor family. During early development, oscillations of extracellular cAMP mobilize cells for aggregation; later, continuous exposure to higher extracellular cAMP concentrations downregulates early gene expression and promotes cytodifferentiation and cell-specific gene expression. The cAMP receptor 1 gene CAR1 has two promoters that are differentially responsive to these extracellular cAMP stimuli. The early CAR1 promoter is induced by nM pulses of cAMP, which in turn are generated by CAR1-dependent activation of adenylyl cyclase (AC). Higher, non-fluctuating concentrations of cAMP will adapt this AC stimulus-response, repress the activated early promoter and induce the dormant late promoter. We now identify a critical element of the pulse-induced CAR1 promoter and a nuclear factor with sequence-specific interaction. Mutation of four nucleotides within the element prevents both in vitro protein binding and in vivo expression of an otherwise fully active early CAR1 promoter and multimerization of the wild-type, but not mutant, sequence will confer cAMP regulation to a quiescent heterologous promoter. These cis and trans elements, thus, constitute a part of the molecular response to the cAMP transmembrane signal cascade that regulates early development of Dictyostelium.

The transition-path is the tiny fraction of an equilibrium molecular trajectory when a transition occurs between two states, and appears as an instantaneous jump in the measured signal in single molecule force or fluorescence experiments. Transition-paths are readily observed in atomistic molecular dynamics simulations for systems with fast kinetics, but have never been observed experimentally for any system in the condensed phase. The importance of the transition-path in protein folding is that it contains all the mechanistic information on how a protein folds and unfolds and is predicted from both theory and simulations to be heterogeneous. As a first step toward observing transition-paths in protein folding, we previously estimated an upper bound of ∼200 microseconds for the transition-path time of protein G using single molecule FRET spectroscopy, 10,000 times shorter than the average unfolded-state waiting-time of ∼2 seconds (Chung et al., PNAS 2009). The biggest obstacle to resolving a transition-path is to detect a sufficient number of photons during a single transition-path. To overcome this problem, we employed a fully-automated data acquisition system to collect a very large number of photon trajectories at high illumination intensities, and carried out a collective photon-by-photon analysis of the transitions between the folded and unfolded states using a maximum likelihood method (Chung et al., JPC A 2011). We determined a transition-path time of ∼2 microseconds for a WW domain that folds in ∼100 microseconds and an upper bound of ∼15 microseconds for protein GB1 that folds in ∼2 seconds. The transition-path times for the two proteins differ by less than 10-fold while the folding rates differ by a factor of 20,000. This result shows that a slow-folding protein can fold almost as fast as a fast-folding protein when folding actually occurs!

We have measured folding/unfolding trajectories of single protein G (B1 domain) molecules, a simple two-state folder, by simultaneously measuring the fluorescence intensity, lifetime, and spectrum at various concentrations of denaturant. Protein molecules were labeled by a fluorescence resonance energy transfer (FRET) pair, Alexa Fluor 488 and Alexa Fluor 594 and were immobilized on a glass surface coated with polyethyleneglycol via streptavidin-biotin linkage. The vast majority of molecules (∼ 85%) exhibits simple two-state trajectories, with either high or low values of the FRET efficiency, corresponding to the folded and unfolded states, respectively, with unresolvable jumps between them. About 10% of the trajectories show transitions in the unfolded state that can be attributed to a ∼20 nm spectral shift of the donor, as revealed by measurements of their emission spectra. The mean FRET efficiency of immobilized molecules matches the value measured in free diffusion experiments. There is a distribution of these values beyond the width expected from shot noise, which can, however, be quantitatively accounted for by the distribution of acceptor lifetimes. In spite of these complications from photophysics, rate coefficients obtained from the exponential distribution of residence times in either the folded or unfolded state yield relaxation times that agree within a factor of 2 with those measured on the dye-labeled protein by stopped flow kinetics. In addition, no correlation is observed between the donor and acceptor intensity in the unfolded state from microseconds to seconds suggesting that structural averaging between unfolded conformations occurs on the nanosecond timescale, as expected from previous measurements by B. Schuler and coworkers (PNAS:104,2655,2007). All these results indicate that we have successfully immobilized the protein without significantly altering its structure, kinetics, or dynamics, and represent a major step forward toward the goal of “watching” individual molecules fold.

The first step of HIV infection involves the fusion of the viral and target cell membranes, a process mediated by the viral envelope glycoproteins, gp120 and gp41. The binding of gp120 to the cell surface receptors CD4 triggers a cascade of conformational changes that disrupt the gp41-gp120 interactions and allows the insertion of the N-terminal fusion peptide of gp41 into the host cell membrane. The gp41 trimer then rapidly folds into a 6-helix bundle that pulls together the fusion peptide, inserted in the host cell membrane and the transmembrane domain, located in the viral membrane. Very little structural information is known about the pre-fusion state of gp41 notwithstanding its critical importance for the design of fusion inhibitors. To investigate the dynamics and structural properties of such metastable states, we designed a set of protein constructs mimicking the extracellular ectodomain of gp41. We found that the secondary structure of gp41 ectodomain is pronouncedly impacted by the presence of phospholipid vesicles or detergents containing phosphatidyl choline head groups. Our NMR and multi-angle light scattering data attribute these changes to the transition between a trimer in the absence of detergent to a monomer in the presence of a membrane-mimicking environment. The structure of gp41 in a monomeric state was determined by state-of-the-art NMR techniques, including measurements of NOE distance restraints and residual dipolar couplings in weakly aligned solutions. 15N relaxation data provided a detailed view at the backbone dynamics of both the trimeric and monomeric conformations. This stable monomeric state may represent a crucial structural intermediate facilitating both the gp41 conformational change during fusion and the local apposition of the viral and cellular membranes.

The transition-path is the tiny fraction of an equilibrium, single-molecule trajectory when a transition occurs between two states. The importance of the transition-path in protein folding is that it contains all the mechanistic information on how a protein folds and unfolds. However, a transition-path has never been observed experimentally for any molecular system in the condensed phase because it is too fast to measure. Even determining the average transition-path time, 〈tTP〉, is challenging. Previously, we determined 〈tTP〉∼2μs for the all-β protein, FBP28 WW-domain (1/kF=100μs) and an upper bound of 〈tTP〉∼10μs for the much slower αβ-protein GB1 (1/kF =1s) by employing the Gopich-Szabo maximum likelihood analysis of photon trajectories in single-molecule FRET experiments and a kinetic model in which the lifetime of an additional state in a one-step discretization of the transition path corresponds to 〈tTP〉 (Chung et al., Science 2012). Surprisingly, the 〈tTP〉s for the two proteins differ by

A solution NMR study on the structure and dynamics of the homotrimeric gp41 complex, reconstituted in dodecyl phosphatidylcholine (DPC) micelles, is presented. Observed resonances were assigned to the fusion peptide (FP), N-terminal heptad repeat (NHR) and immuno-dominant loop region (IL), whereas the C-terminal heptad repeat (CHR) region, membrane proximal external region (MPER) and transmembrane helix (TM) remain completely invisible to solution NMR. 15N relaxation data reveal a high degree of intrinsic mobility: The a-helical fusion peptide (FP) exhibits high amplitude motion on the fast nanosecond time scale relative to the NHR region. The linker between NHR and CHR shows both fast and slow (microsecond) motions.Figure 1: Homotrimeric gp41 shows a high degree of intrinsic mobility. Our data are compatible with the protein switching on a microsecond time scale between the pre-hairpin intermediate, three-helical bundle state, and the late-fusion, anti-parallel six-helical bundle. MPER and TM are not shown since their conformation cannot be evaluated from the available solution NMR data. While MPER and TM are absent from the spectra, the FP shows the most intense resonances, which excludes a strong interaction between FP and TM region.View Large Image | View Hi-Res Image | Download PowerPoint Slide