A combined force field of molecular mechanics and solvation free energy is tested by carrying out... more A combined force field of molecular mechanics and solvation free energy is tested by carrying out energy minimization and molecular dynamics on several conformations of the alanyl dipeptide. Our results are qualitatively consistent with previous experimental and computational studies, in that the addition of solvation energy stabilizes the C5 conformation of the alanyl dipeptide relative to the C,.
Entry of proteins into membranes and transmembrane ion channel formation are two fundamental aspe... more Entry of proteins into membranes and transmembrane ion channel formation are two fundamental aspects of membrane biology. The ion channel forming colicins beautifully exemplify both properties. Recent results delineate the structure of a whole colicin; coupled with new biophysical studies, a mechanism for insertion is proposed.
Highly ordered two-dimensional (2-D) crystals of Escherichia cofi-expressed bacteriorhodopsin ana... more Highly ordered two-dimensional (2-D) crystals of Escherichia cofi-expressed bacteriorhodopsin analog (e-bR) and its D96N variant (e-D96N) reconstituted in Halobacterium halobium lipids have been obtained by starting with the opsin protein purified in the denaturing detergent sodium dodecyl sulfate. These crystals embedded in glucose show electron diffraction in projection to better than 3.0 A at room temperature. This is the first instance that expressed bR or a variant has been crystallized in 2-D arrays showing such high order. The crystal lattice is homologous to that in wild-type bR (w-bR) in purple membranes (PM) and permit high resolution analyses of the structure of the functionally impaired D96N variant. The e-bR crystal is isomorphous to that in PM with an overall averaged fractional change of 12.7% (26-3.6-A resolution) in the projection structure factors. The projection difference Fourier map e-bR-PM at 3.6-A resolution indicates small conformational changes equivalent to movement of-<7 C-atoms distributed within and in the neighborhood of the protein envelope. This result shows that relative to w-bR there are no global structural rearrangements in e-bR at this 3.6 A resolution level. The e-D96N crystal is isomorphous to the e-bR crystal with a smaller (9.2%) overall averaged fractional change in the structure factors. The significant structural differences between e-D96N and e-bR are concentrated at high resolution (5-3.6 A); however, these changes are small as quantified from the 3.6 A resolution e-D96N-e-bR Fourier difference map. The difference map showed no statistically significant peaks or valleys within 5 A in projection from the site of D96 substitution on helix C. Elsewhere within the protein envelope the integrated measure of peaks or valleys was <-3 C-atom equivalents. Thus, our results show that for the isosteric substitution of Asp96 by Asn, the molecular conformation of bR in its ground state is essentially unaltered. Therefore, the known effect of D96N on the slowed M412 decay is not due to ground-state structural perturbations.
Channel-forming colicins exist in at least two different membrane-associated conformations: a vol... more Channel-forming colicins exist in at least two different membrane-associated conformations: a voltage-independent closed-channel state and a voltage-dependent open-channel state. In a voltageindependent membrane-associated conformation, we find that two major regions of colicin Ia are protected from pepsin proteolysis after association with negatively charged membranes. In contrast, colicin Ia is rapidly and completely proteolyzed in the absence of membranes. The major protected region includes an electrophysiologically defined C-terminal channel-forming domain as well as 96 residues upstream of this region. Approximately 100 residues spanning Ala79-wArgl89 within the N-terminal domain are protected as well. The first N-terminal76 residues of colicin Ia and a large region which includes much of the putative central receptor-binding domain are not protected from proteolysis. Both N-and C-termini of protected peptides have been identified using a combination of gel electrophoresis, N-terminal sequencing, and mass spectrometry, thereby defining specific residues that are located on the outside of the lipid bilayer. These data suggest a role for regions other than the electrophysiologically defined C-terminal channel-forming domain in membrane insertion and channel formation. This work was supported by National Institutes of Health Grant GM-24485 t0R.M.S. andNIH GrantsRROl614andES04705 t0A.L.B. S.F.M. was the recipient of a Claypole Graduate Fellowship and a UCSF Mentorship Award.
Acta Crystallographica Section A Foundations of Crystallography, 2002
HIV-1 integrase catalyzes the insertion of viral DNA into the human chromosome, and as such, it i... more HIV-1 integrase catalyzes the insertion of viral DNA into the human chromosome, and as such, it is a target for the development of new anti-HIV drugs. Structural studies of HIV-1 integrase have been limited due to its insolubility. We have engineered a soluble, functional integrase by introducing five point mutations, and have solved the structure of the viral DNA binding core and C-terminal domains to 2.8 Å resolution. The Y-shaped, dimeric molecule reveals a putative DNA binding region consisting of residues contributed by both monomers of the dimer. This implies that a dimer is the minimal DNA binding unit. A kink at T210 occurs at a proteolytic cleavage site, suggesting a functional flexibility in the molecule that may be crucial for integration. The C-terminal domain is an SH3-like fold, and provides the majority of the crystal contacts, consistent with the role of the domain in oligomerization of integrase. Based on this structure, we are outlining new strategies to discover novel drug leads targeting integrase.
An iterative Fourier method is applied to solving and refining the electron density profile proje... more An iterative Fourier method is applied to solving and refining the electron density profile projected onto the line perpendicular to a membrane surface. Solutions to the continuous X-ray scattering pattern derived from swelling of multilayer systems or from membrane dispersions can be obtained by this technique. The method deals directly with the observed structure factors and does not rely on deconvolution of the Patterson function. We used this method previously to derive the electron density profile for acetylcholine receptor membranes (Ross et al., 1977). The present paper is an analysis of the theoretical basis for the procedure. In addition, the technique is tested on artificially generated continuous-scattering data, on the data for frog sciatic nerve myelin derived from swelling experiments by Worthington and McIntosh (1974), and on the data for purple membrane (Blaurock and Stoeckenius, 1971). Although the method applies to asymmetric membranes, the special case of centrosymmetric profiles is also shown to be solvable by the same technique. The limitations of the method and the boundary conditions that limit the degeneracy of the solution are analyzed.
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Papers by Robert Stroud