Critical events in the life cycle of herpes simplex virus (HSV) are the binding of cytoplasmic capsids to cellular organelles and subsequent envelopment. Work from several laboratories suggests that these events occur as a result of a network of partially redundant interactions among the capsid surface, tegument components, and cytoplasmic tails of virally encoded glycoproteins. Consistent with this model, we previously showed that tegument protein VP16 can specifically interact with the cytoplasmic tail of envelope protein gH in vitro and in vivo when fused to glutathione S-transferase and to green fluorescent protein, respectively. In both instances, this association was strikingly temperature dependent: binding occurred only at 37°C and not at lower temperatures. Here we demonstrate that virally expressed full-length gH and VP16 can be coimmunoprecipitated from HSV-infected cells and that this association is also critically dependent upon the physiological temperature. To investigate the basis of this temperature requirement, we performed one-and two-dimensional nuclear magnetic resonance spectroscopy on peptides with the sequence of the gH tail. We found that the gH tail is disorganized at temperatures permissive for binding but becomes structured at lower temperatures. Furthermore, a mutated tail unable to adopt this rigid conformation binds VP16 even at 4°C. We hypothesize that the gH tail is unstructured under physiological conditions in order to maximize the number of potential tegument partners with which it may associate. Being initially disordered, the gH tail may adopt one of several induced conformations as it associates with VP16 or alternative components of the tegument, maximizing redundancy during particle assembly.Herpes simplex virus (HSV) type 1 (HSV-1) is an alphaherpesvirus with a 150-kb double-stranded genome packaged within an icosahedral capsid. The capsid is surrounded by an amorphous protein layer known as the tegument, which is in turn surrounded by an envelope derived from host cell organelle membranes. Within the envelope are multiple virally encoded glycoproteins, which function in many aspects of the viral life cycle, such as host membrane attachment and fusion, immune evasion, and prevention of apoptosis (23, 28). The tegument is composed of at least 19 known viral polypeptide species, which perform multiple functions during the life cycle of the virus (28).Several studies have indicated that viral envelopment and tegumentation occur at late Golgi or post-Golgi compartments, such as the trans-Golgi network and endosomes (4,6,15,16,23,25,31,33). However, the molecular details that drive envelopment are still poorly understood. It is thought that a network of interactions among viral glycoproteins present in the host organelle membranes, tegument proteins, and capsid proteins is critical for this process. In this model, tegument proteins serve as a bridge, binding to the cytoplasmic tails of viral glycoproteins, each other, and capsid proteins, thus recruiting capsids to the site o...

NMR structure determination of helical integral membrane proteins is one of the most challenging undertakings in modern structural biology. The solubilizing detergent micelle or phospholipid bicelle adds significantly to the overall size of the system, often requiring perdeuteration to obtain useable spectra. However, perdeuteration prevents structure determination using traditional NOE analysis. Residual dipolar coupling constraints are an attractive complement to NOE distance constraints, but alignment methods are limited to strained polyacrylamide gels due to the incompatibility of the solubilizing detergent or lipid with alignment media such as bicelles or phage particles. We demonstrate the use of lanthanide ions bound to the protein through a small thiol-linked metal chelator as a robust method for partial alignment of membrane proteins. This method provides multiple alignment orientations depending on the ion bound, and permits RDC measurement of multiple bond vectors. We demonstrate that using this method a large number of RDC's can be measured using 3dimensional NMR methods where alignment using strained polyacrylamide gels results in fewer peaks due to drastic line-broadening.

Pectate lyase C (pelC) was the first protein in which the parallel beta-helix structure was recognized. The unique features of parallel beta-helix-containing proteins-a relatively simple topology and unusual interactions among side chains-make pelC an interesting protein to study with respect to protein folding. In this paper, we report studies of the unfolding equilibrium of pelC. PelC is unfolded reversibly by gdn-HCl at pH 7 and 5, as monitored by far- and near-UV CD and fluorescence. The coincidence of these spectroscopically detected transitions is consistent with a two-state transition at pH 7, but the three probes are not coincident at pH 5. No evidence was found for a loosely folded intermediate in the transition region at pH 5. At pH 7, the for unfolding is 12.2 kcal/mol, with the midpoint of the transition at 0.99 M gdn-HCl and m = 12.3 kcal/(mol.M). Thus, pelC is unusually stable and has an m value that is much larger than for typical globular proteins. Thermal denaturation of pelC has been studied by differential scanning calorimetry (DSC) and by CD. Although thermal denaturation is not reversible, valid thermodynamic data can be obtained for the unfolding transition. DeltaH(van't Hoff)/DeltaH(cal) is less than 1 for pHs between 5 and 8, with a maximum value of 0.91 at pH 7 decreasing to 0.85 at pH 8 and to 0.68 at pH 5. At all pHs studied, the excess heat capacity can be deconvoluted into two components corresponding to two-state transitions that are nearly coincident at pH 7, but deviate more at higher and lower pH. Thus, pelC appears to consist of two domains that interact strongly and unfold in a cooperative fashion at pH 7, but the cooperativity decreases at higher and lower pH. The crystal structure of pelC shows no obvious domain structure, however.

Addition of 8-anilino-1-naphthalenesulfonate (ANS) to acid-denatured pectate lyase C (pelC) leads to a large increase in the fluorescence quantum yield near 480 nm. The conventional interpretation of such an observation is that the ANS is binding to a partially folded intermediate such as a molten globule. Farultraviolet circular dichroism demonstrates that the enhanced fluorescence results from the induction of a partially folded protein species that adopts a large fraction of native-like secondary structure on binding ANS. Thus, ANS does not act as a probe to detect a partially folded species, but induces such a species. Near-ultraviolet circular dichroism suggests that ANS is bound to the protein in a specific conformation. The mechanism of ANS binding and structure induction was probed. The interaction of acid-unfolded pelC with several ANS analogs was investigated. The results strongly indicate that the combined effects of hydrophobic and electrostatic interactions account for the relatively high binding affinity of ANS for acidunfolded pelC. These results demonstrate the need for caution in interpreting enhancement of ANS fluorescence as evidence for the presence of molten globule or other partially folded protein intermediates.Keywords: Pectate lyase C; ANS; molten globule; electrostatic stabilization; circular dichroism ANS is an amphipathic dye, with hydrophobic naphthalene and phenyl groups, and a charged sulfonate group. It is frequently used for the investigation of equilibrium and kinetic protein folding intermediates (Semisotnov et al. 1987;Ptitsyn et al. 1990;Semisotnov et al. 1991). When ANS is bound to a protein in a nonpolar environment, there is a large increase in the fluorescence quantum yield (Stryer 1965;Turner and Brand 1968). ANS has been used extensively as a probe for protein folding intermediates, especially molten globules, because their partially structured nature provides access for ANS to bind exposed hydrophobic regions (Ptitsyn et al. 1990), whereas ANS has a very weak affinity for fully unfolded or folded proteins. ANS has also been widely used as a probe for kinetic intermediates in protein folding (Semisotnov et al. 1987;Ptitsyn et al. 1990). Partially folded transient species will bind ANS, and the time dependence of its emission is indicative of the lifetime of the species.Acid denaturation is a widely used method of inducing protein conformational transitions. The major cause of acid denaturation of proteins is the charge-charge repulsion of basic amino acid side chains that have uncompensated positive charges resulting from the neutralization of carboxylate groups. It has been shown that addition of excess acid to an acid-denatured protein can induce a significant fraction of secondary structure (Goto et al. 1990a). A similar result is obtained on the addition of neutral salts (Goto et al. 1990b From a dissertation submitted to the Academic Faculty of Colorado State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.Abbrev...

The folding mechanism of pectate lyase C (pelC) involves two slow phases that have been attributed to proline isomerization. To have a more detailed and complete understanding of the folding mechanism, experiments have been carried out to identify the prolyl-peptide bonds responsible for the slow kinetics. Site-directed mutagenesis has been used to mutate each of the prolines in pelC to alanine or valine. It has been determined that isomerization of the Leu219-Pro220 peptide bond is responsible for the slowest folding phase observed. The mutant P220A shows kinetic behavior that is identical to the wild-type protein except that the 46-s phase is eliminated. The Leu219-Pro220 peptide bond is cis in the native enzyme. An analysis of the free energy of unfolding of this mutant indicates that the mutation destabilizes the protein by about 4 kcal/mol. However, it appears that the major refolding pathways are unaltered. Further mutations were carried out in order to assign the peptide bond responsible for the 21-s folding phase in pelC. Mutation of the remaining 11 prolines, which are trans in the native enzyme, resulted in no significant changes in the kinetic folding behavior. The conclusion from these experiments is that the 21-s phase involves isomerization of more than one prolyl-peptide bond with similar activation energies.