Small Backbone Research Articles

Peptide chemical ligation inside living cells: in vivo generation of a circular protein domain

Here we describe the first example of a peptide chemical ligation reaction performed inside a living cell. A cell-based native chemical ligation approach was developed and used to generate a circular version of the N-terminal Src homology 3 (SH3) domain from the murine c-Crk adapter protein inside Escherichia coli cells. The in vivo cyclization reaction was extremely efficient and the resulting circular protein domain was fully biologically active and able to adopt the native SH3 folded structure. This work represents an important step towards the in vivo generation of small backbone cyclic peptides for use in basic biological research.

How do substrates enter and products exit the buried active site of cytochrome P450cam? 1. Random expulsion molecular dynamics investigation of ligand access channels and mechanisms

Cytochrome P450s form a ubiquitous protein family with functions including the synthesis and degradation of many physiologically important compounds and the degradation of xenobiotics. Cytochrome P450cam from Pseudomonas putida has provided a paradigm for the structural understanding of cytochrome P450s. However, the mechanism by which camphor, the natural substrate of cytochrome P450cam, accesses the buried active site is a long-standing puzzle. While there is recent crystallographic and simulation evidence for opening of a substrate-access channel in cytochrome P450BM-3, for cytochrome P450cam, no such conformational changes have been observed either in different crystal structures or by standard molecular dynamics simulations. Here, a novel simulation method, random expulsion molecular dynamics, is presented, in which substrate-exit channels from the buried active site are found by imposing an artificial randomly oriented force on the substrate, in addition to the standard molecular dynamics force field. The random expulsion molecular dynamics method was tested in simulations of the substrate-bound structure of cytochrome P450BM-3, and then applied to complexes of cytochrome P450cam with different substrates and with product. Three pathways were identified, one of which corresponds to a channel proposed earlier on the basis of crystallographic and site-directed mutagenesis data. Exit via the water-filled channel, which was previously suggested to be a product exit channel, was not observed. The pathways obtained by the random expulsion molecular dynamics method match well with thermal motion pathways obtained by an analysis of crystallographic B-factors. In contrast to large backbone motions (up to 4 Å) observed in cytochrome P450BM-3 for the exit of palmitoleic acid, passage of camphor through cytochrome P450cam only requires small backbone motions (less than 2.4 Å) in conjunction with side-chain rotations. Concomitantly, in almost all the exit trajectories, salt-links that have been proposed to act as ionic tethers between secondary structure elements of the protein, are perturbed.

DoMCoSAR: a novel approach for establishing the docking mode that is consistent with the structure-activity relationship. Application to HIV-1 protease inhibitors and VEGF receptor tyrosine kinase inhibitors.

DoMCoSAR is a novel approach for statistically determining the docking mode that is consistent with a structure-activity relationship. The approach establishes the binding mode for the compounds in a chemical series with the assumption that all molecules exhibit the same binding mode. It involves three stages. In the first stage all molecules that belong to a given chemical series are docked to the active site of the protein target. The only bias used in the docking at this stage involves the location of the protein binding site. Coordinates of the common substructure (CS) that results from the unbiased docking are then clustered to establish the major substructure docking modes. In the second stage all molecules are docked to the major docking modes (MDMs) with constraints based on the common substructure. The third stage generates, for the major docking modes, interaction-based descriptors that include electrostatic, VDW, strain, and solvation contributions. The problem of docking mode evaluation is now reduced to the question of which descriptor set is more predictive. To establish a quantitative comparison of the descriptor sets associated with the major docking modes, we use 50 instances of random 4-fold cross-validation. For each 4-fold cross-validation the predictive squared correlation coefficient (R(2)) is computed. t-Tests are applied to establish significance of the differences in mean R(2) for one docking mode versus another. We test the methodology on two test cases: HIV-1 protease inhibitors (Holloway et al. J. Med. Chem. 1995, 38, 305-317) and vascular endothelial growth factor (VEGF) receptor tyrosine kinase oxoindoles (Sun et al. J. Med. Chem. 1998, 41, 2588-2603). For both test cases there is statistically significant preference for the binding mode consistent with the X-ray structure. The appeal of this methodology is that researchers gain the objectivity of statistical justification for the selected docking mode. The methodology is relatively insensitive to subtle variations of the protein structure that include, but are not limited to, side chain and small backbone rearrangement during binding. In addition, predictive models that result from the approach can be used to further optimize chemical series.

Nonintrusive TCP connection admission control for bandwidth management of an Internet access link

We describe our approach to monitoring and managing the bandwidth of an Internet edge link with a view toward certain quality of service objectives for the services it carries. Such a link could be, for example, a campus's Internet access link or a small ISP's backbone access link. We use SNMP polls and packet snooping to obtain traffic statistics, and TCP admission control for bandwidth management. Our implementation is completely nonintrusive: we use Ethernet packet capture in the promiscuous mode for traffic analysis, and IP masquerading for blocking new TCP connections. This approach has been implemented by us in a software system for traffic management. We first justify our approach with a simple analytical model. We give an overview of our software implementation, and discuss some implementation issues. Then we provide measurement results that show the effectiveness of the techniques.

Mutations improving the folding of phage P22 tailspike protein affect its receptor binding activity

Four previously isolated mutations in Salmonella phage P22 tailspike protein were used to study the relationship between protein stability, folding, and function. Tailspike protein binds and hydrolyzes the repetitive O-antigen structure in Salmonella lipopolysaccharide. Four mutations (V331G, V331A, A334V, A334I) are known to increase the folding efficiency, and two of them (at position 331) also increase the thermal stability of the protein. Octasaccharides comprising two repeating units of the O-antigens from two different Salmonella strains were employed to analyze the receptor binding function of the mutant proteins. Their endorhamnosidase enzymatic activity was assayed with the aid of a fluorescence-labeled dodecasaccharide. Both V331A and V331G were found to strongly affect O-antigen binding. Octasaccharide binding affinities of the mutant proteins are reduced tenfold and 200-fold, corresponding to a loss of 17 % and 36 % of the standard free energy of binding, respectively. Both mutations at position 334 affected O-antigen binding only slightly (ΔΔGB03≈1 kJ/mol), but these mutations reduce the thermal stability of the protein. The observed effects on the endoglycosidase activity are fully explained by the changes in substrate binding, suggesting that neither of the mutations affect the catalytic rate. Crystal structures of all four mutants were determined to a resolution of 2.0 Å. Except for the partly or completely missing side-chain, no significant changes compared to the wild-type protein structure were found for the mutants at position 331, whereas a small but significant backbone displacement around the mutation site in A334V and A334I may explain the observed thermal destabilization.

Peptides derived from HIV-1 vif: a non-substrate based novel type of HIV-1 protease inhibitors

The retroviral protease (PR) is absolutely essential for completion of human immunodeficiency virus multiplication cycle, and cannot be replaced by any cellular function. Thus PR, like reverse transcriptase, is an ideal target for the development of anti-AIDS therapy. A large number of human immunodeficiency virus type-1 (HIV-1) PR inhibitors have been developed, and several are currently used as anti-AIDS drugs. These inhibitors are mainly based on the natural PR cleavage sites within the viral Gag and Gag-Pol precursors. The major difficulty encountered while using anti-HIV therapeutic agents in patients has been the rapid emergence of drug-resistant viral strains. Most of the mutations which convert the PR into inhibitor-resistant are located within the substrate binding subsites of the enzyme. Recently, it has been shown that the HIV-1 auxiliary protein Vif, and especially the N-terminal half of Vif (N′-Vif) specifically interacts with the viral PR and inhibits its activity. We now show that efficient inhibition of HIV-1 PR activity can be achieved using Vif-derived peptides. Based on the above model we have performed peptide mapping of N′-Vif in order to find a small peptidic lead compound which inhibits PR activity. The screening revealed that peptides derived from two regions in Vif spanning from residues 30–65 and 78–98 inhibit PR activity in vitro, specifically bind HIV-PR and inhibit HIV-1 production in vivo. Further mapping of these regions revealed the lead compounds Vif81–88 and Vif88–98. These peptides specifically inhibit and bind HIV-1 PR, but do not affect pepsin and rous sarcoma virus protease. In contrast to other known PR inhibitors, these peptides are not substrate-based and their sequences do not resemble the sequences of the natural PR substrates (cleavage sites). Moreover, the Vif-derived peptides themselves are not cleaved by HIV-1 PR. Conversion of the lead peptides into small backbone cyclic peptidomimetics is taking place nowadays in order to turn these lead compounds into metabolically stable selective novel type of HIV-PR non-substrate-based inhibitors.

Editorial

Cytochrome P450s form a ubiquitous protein family with functions including the synthesis and degradation of many physiologically important compounds and the degradation of xenobiotics. Cytochrome P450cam from Pseudomonas putida has provided a paradigm for the structural understanding of cytochrome P450s. However, the mechanism by which camphor, the natural substrate of cytochrome P450cam, accesses the buried active site is a long-standing puzzle. While there is recent crystallographic and simulation evidence for opening of a substrate-access channel in cytochrome P450BM-3, for cytochrome P450cam, no such conformational changes have been observed either in different crystal structures or by standard molecular dynamics simulations. Here, a novel simulation method, random expulsion molecular dynamics, is presented, in which substrate-exit channels from the buried active site are found by imposing an artificial randomly oriented force on the substrate, in addition to the standard molecular dynamics force field. The random expulsion molecular dynamics method was tested in simulations of the substrate-bound structure of cytochrome P450BM-3, and then applied to complexes of cytochrome P450cam with different substrates and with product. Three pathways were identified, one of which corresponds to a channel proposed earlier on the basis of crystallographic and site-directed mutagenesis data. Exit via the water-filled channel, which was previously suggested to be a product exit channel, was not observed. The pathways obtained by the random expulsion molecular dynamics method match well with thermal motion pathways obtained by an analysis of crystallographic B-factors. In contrast to large backbone motions (up to 4 Å) observed in cytochrome P450BM-3 for the exit of palmitoleic acid, passage of camphor through cytochrome P450cam only requires small backbone motions (less than 2.4 Å) in conjunction with side-chain rotations. Concomitantly, in almost all the exit trajectories, salt-links that have been proposed to act as ionic tethers between secondary structure elements of the protein, are perturbed.

Novel secretory proline-rich proteoglycans from rat parotid. Cloning and characterization by expression in AtT-20 cells.

We have isolated two cDNAs that encode backbones of proline-rich proteoglycans identified previously in parotids of isoproterenol-treated rats (Blair, A. E., Castle, A. M., and Castle, J. D. (1991) Am. J. Physiol. 30, C897-C905). The sequences and domain structure of these cDNAs are characteristic of acidic proline-rich proteins, and potential glycosaminoglycan attachment sites are present within the acidic N-terminal domain. When the cDNAs were individually expressed in a mouse pituitary cell line, AtT-20, the expressed proteins were modified to sulfated proteoglycans. As in parotid acinar cells, only a fraction of the larger backbone (M(r) = 38,000) is modified by glycosaminoglycan addition; however, most of the smaller backbone (M(r) = 30,000) appears modified. Both proteoglycans expressed in AtT-20 cells contain heparan sulfate and chondroitin sulfate, whereas the proteoglycan in parotid contained chondroitin sulfate exclusively. Both proteoglycans are targeted to the regulated secretory pathway in AtT-20 cells where they are stored more efficiently than a parotid basic proline-rich protein.

Characterization of the CAMPATH‐1 (CDw52) antigen: biochemical analysis and cDNA cloning reveal an unusually small peptide backbone

The CAMPATH-1 (CDw52) antigen has been purified from human spleen. The antigenic epitope is heat stable but sensitive to mild alkali treatment. Experiments with phosphatidylinositol-specific phospholipase C indicate that it is anchored by a glycosylphosphatidylinositol (GPI) anchor. An N-terminal sequence of 11 amino acids was determined, followed by an abrupt stop. Using short overlapping mixed oligonucleotide primers, cDNA synthesized from the mRNA of a human B cell line was amplified by the polymerase chain reaction. The product was used to isolate cDNA clones and the full amino acid sequence of the CAMPATH-1 antigen was deduced. It consists of 37 amino acid residues plus a 24-residue signal peptide. It has all the features expected for a GPI-anchored membrane protein except that the predicted mature protein is remarkably short, comprising no more than 18 residues and possibly as few as 12 (depending on the GPI linkage site). Potential attachment sites for carbohydrate are present and it is shown that the antigen contains N-linked oligosaccharide(s). This structure accounts for the known properties of the antigen, though the exact reasons why it is such a good target for cell lysis in vitro and in vivo are not yet clear.

Sorption and transport studies of water in Kapton polymide. I

Sorption isotherms and diffusion coefficients of water in a 0.3-mil Kapton polyimide film at 30, 45, and 60°C are reported. The data are well described by the dual mode sorption and transport models at low activities. At high penetrant activities, clustering of water is suggested by a Zimm–Lundberg analysis of the sorption data and the fact that the diffusion coefficient for water decreases with increasing external vapor activity. The effect of temperature on the diffusion coefficients at infinite dilution and the dual mode sorption parameters kD, b, and C are presented and discussed. The magnitude of the activation energy of the diffusion coefficient at infinite dilution, 5.4 kcal/mol, is smaller than the corresponding activation energy in more flexible chain polymers, perhaps suggesting that rather small backbone motions are associated with diffusion of water through the Kapton matrix. The predictions for the isosteric enthalpy of sorption from the dual mode model are presented and compared with the values determined from graphical analysis of the sorption isotherms performed independently without reference to the dual mode sorption model.