Activated Conformations of the ras-gene-Encoded p21 Protein. 1. An Energy-Refined Structure for the Normal p21 Protein Complexed with GDP

Abstract

A complete three-dimensional structure for the ras-gene-encoded p21 protein with Gly 12 and Gin 61, bound to GDP, has been constructed in four stages using the available α-carbon coordinates as deposited in the Brookhaven National Laboratories Protein Data Bank. No all-atom structure has been made available despite the fact that the first crystallographic structure for the p21 protein was reported almost four years ago. In the p21 protein, if amino acid substitutions are made at any one of a number of different positions in the amino acid sequence, the protein becomes permanently activated and causes malignant transformation of normal cells or, in some cell lines, differentiation and maturation. For example, all amino acids except Gly and Pro at position 12 result in an oncogenic protein; all amino acids except Gin, Glu and Pro at position 61 likewise cause malignant transformation of cells. We have constructed our all-atom structure of the non-oncogenic protein from the x-ray structure in order to determine how oncogenic amino acid substitutions affect the three-dimensional structure of this protein. In Stage 1 we generated a poly-alanine backbone (except at Gly and Pro residues) through the α-carbon structure, requiring the individual Ala, Pro or Gly residues to conform to standard amino acid geometry and to form trans-planar peptide bonds. Since no a-carbon coordinates for residues 60–65 have been determined these residues were modeled by generating them in the extended conformation and then subjecting them to molecular dynamics using the computer application DISCOVER and energy minimization using DISCOVER and the ECEPP (Empirical Conformational Energies for Peptides Program). In Stage 2, the positions of residues that are homologous to corresponding residues of bacterial elongation factor Tu (EF-Tu) to which p21 bears an overall 40% sequence homology, were determined from their corresponding positions in a high-resolution structure of EF-Tu. Non-homologous loops were taken from the structure generated in Stage 1 and were placed between the appropriate homologous segments so as to connect them. In Stage 3, all bad contacts that occurred in this resulting structure were removed, and the coordinates of the α-carbon atoms were forced to superimpose as closely as possible on the corresponding atoms of the reference (x-ray) structure. Then the side chain positions of residues of the nonhomologous loop regions were modeled using a combination of molecular dynamics and energy minimization using DISCOVER and ECEPP respectively. All of the residues of the structure were then allowed to move under restrained energy minimization where the restraints were gradually removed. In Stage 4, the nucleotide GOP was added to the model and further energy minimization was carried out. The energy of the protein-GOP complex was minimized by allowing the atoms of GOP to move with the protein held fixed and then by allowing both the nucleotide and the residues of the protein to move together. The reconstructed model agrees with the published features of the p21 protein-GOP complex including the hydrogen bonding scheme, the distribution of backbone dihedral angles, the residues contacting the nucleotide, and the orientation of loops with respect to one another in the protein. The structure also agrees with one that was predicted previously (Chen, J.M. et al., J. Biomol. Struct. Dynamics 6, 850–875 (1989)). In our molecular dynamics-energy minimization procedures, we also have been able to place all residues except Ala 66, which occurs in a poorly-defined region crystallographically, in local single residue minima, including residues reported to be in high energy regions in the x-ray structure. The constructed model can explain observed physical phenomena such as autophosphorylation by GTP on Thr 59 in proteins containing Thr in place of Ala 59.     

Down-regulation of calponin destabilizes actin cytoskeletal structure in A7r5 cells

The effects of changes in the expression levels of h1 calponin (CaP) on actin cytoskeletal organization were studied in control and phorbol-ester-treated A7r5 smooth muscle cells. Protein association and expression in control and stimulated A7r5 smooth muscle cells were evaluated by Western blotting, laser scanning confocal microscopy (LSCM), and fluorescence resonance energy transfer (FRET) microscopy in cells treated with either 2 x 10(-6 ) mol/L TGF-beta 1 or 2 x 10(-)5 mol/L PDGF-BB to alter h1 calponin expression. Single immunostained samples showed that CaP and alpha-actin, localized in fibers in unstimulated control A7r5 smooth muscle cells, were translocated to podosomes following treatment with phorbol-12,13-dibutyrate (PDBu). Confocal colocalization imaging and FRET analysis both indicated substantial association of CaP with alpha-actin in stress fibers of control cells and in podosomes of PDBu-treated cells. PKC alpha, which showed evidence of only slight association with CaP in control cells, exhibited markedly increased (293%) association in PDBu-contracted cells. Platelet-derived growth factor (PDGF)-BB down-regulated CaP to non-detectable levels, whereas transforming growth factor (TGF)-beta 1 up-regulated (424%) the expression of CaP without affecting the levels of alpha-actin or PKC alpha. PDGF-BB resulted in a significant loss in alpha-actin stress fibers (-47%) and reduced podosome formation (-69%). By comparison, TGF-beta 1 had no effect on stress fibers in control cells but also reduced (-70%) podosome formation. The results suggest that CaP could play a major role in the stabilization of actin stress fibers in resting cells and may contribute to podosome formation in PDBu-treated cells.     

Modeling and estimation of replication fitness of human immunodeficiency virus type 1 in vitro experiments by using a growth competition assay

Growth competition assays have been developed to quantify the relative fitnesses of human immunodeficiency virus (HIV-1) mutants. In this article we develop mathematical models to describe viral/cellular dynamic interactions in the assay experiment, from which new competitive fitness indices or parameters are defined. These indices include the log fitness ratio (LFR), the log relative fitness (LRF), and the production rate ratio (PRR). From the population genetics perspective, we clarify the confusion and correct the inconsistency in the definition of relative fitness in the literature of HIV-1 viral fitness. The LFR and LRF are easier to estimate from the experimental data than the PRR, which was misleadingly defined as the relative fitness in recent HIV-1 research literature. Calculation and estimation methods based on two data points and multiple data points were proposed and were carefully studied. In particular, we suggest using both standard linear regression (method of least squares) and a measurement error model approach for more-accurate estimates of competitive fitness parameters from multiple data points. The developed methodologies are generally applicable to any growth competition assays. A user-friendly computational tool also has been developed and is publicly available on the World Wide Web at http://www.urmc.rochester.edu/bstools/vfitness/virusfitness.htm.     

Quantitative trait loci for hip dysplasia in a crossbreed canine pedigree

Canine hip dysplasia is a common developmental inherited trait characterized by hip laxity, subluxation or incongruity of the femoral head and acetabulum in affected hips. The inheritance pattern is complex and the mutations contributing to trait expression are unknown. In the study reported here, 240 microsatellite markers distributed in 38 autosomes and the X chromosome were genotyped on 152 dogs from three generations of a crossbred pedigree based on trait-free Greyhound and dysplastic Labrador Retriever founders. Interval mapping was undertaken to map the QTL underlying the quantitative dysplastic traits of maximum passive hip laxity (the distraction index), the dorsolateral subluxation score, and the Norberg angle. Permutation testing was used to derive the chromosome-wide level of significance at p < 0.05 for each QTL. Chromosomes 4, 9, 10, 11 (p < 0.01), 16, 20, 22, 25, 29 (p < 0.01), 30, 35, and 37 harbor putative QTL for one or more traits. Successful detection of QTL was due to the crossbreed pedigree, multiple-trait measurements, control of environmental background, and marked advancement in canine mapping tools.     

Effect of Sodium Metabisulphite and Storage Temperature on the Survival of Vibrio cholerae on Prawns (Penaeus monodon)

Summary Sodium metabisulphite is traditionally used to control a non-microbiological spoilage symptom of prawns known as blackspot. This study was undertaken to evaluate the effect of sodium metabisulphite, at levels currently used to control blackspot, on the survival of V. cholerae on prawns during simulated commercial storage. Fresh prawns (Penaeus monodon) were divided into two batches, one of which was exposed to seawater while the other was treated with sodium metabisulphite (1%) in seawater. Two V. cholerae O1 strains were inoculated onto the prawns in separate experiments. Each of the batches were further sub-divided and stored either under chilled conditions (1.5 °C) for 14 days or under frozen conditions (−25 °C) for 49 days. At appropriate time intervals whole prawns were sampled and dilutions plated onto TCBS Cholera medium to determine numbers of V. cholerae. Experiments were performed in triplicate and uninoculated controls were included. Results indicated a significant (P < 0.05) decrease in numbers of V. cholerae on both chilled (~4 log units) and frozen (~2 log units) prawns over the storage period. A significantly (P < 0.05) greater decrease in numbers of the pathogen (up to 1.8 log units) on chilled sodium-metabisulphite-treated, as compared to chilled untreated, prawns was apparent. Sodium metabisulphite had a negligible effect on numbers of V. cholerae on frozen prawns. The use of sodium metabisulphite could be optimized and used to enhance the safety of chilled seafood in cholera-endemic countries.     

The genetic variants of group-specific component (vitamin D-binding protein) possess different binding characteristics for immobilized Cibacron Blue 3-GA.

The elution profiles of several variants of the Gc protein have been studied after chromatography on immobilized Cibacron Blue 3-GA. The allele products belonging to the Gcl type were retarded and eluted with a Ve/Vo at 1.5, as previously reported for the Gcl-1 phenotype [Chapuis-Cellier, Gianazza & Arnaud (1982) Biochim. Biophys. Acta 709, 353-357]. The allele products belonging to the Gc2 type were further retarded (Ve/Vo at 2.6), and both Gcl and Gc2 allele products were clearly separated in heterozygous individuals. This observation allows the isolation and purification of Gc variants in heterozygous individuals which carry the combination Gcl variant-Gc2, Gcl-Gc2 variant, or Gcl variant-Gc2 variant. In contrast, the corresponding holoproteins did not bind to the gel and were eluted in the void volume. This suggests that the interaction of Gc with immobilized Cibacron Blue 3-GA involves the binding site of the protein for 25-hydroxycholecalciferol and that the dye behaves as a 'pseudoligand' for the protein. In addition, our data suggest that the different elution profiles of the variants could reflect a different affinity of the gene products for the dye.     

An androgen-inducible expression system for Saccharomyces cerevisiae

A novel controllable expression system for Saccharomyces cerevisiae has been developed. Expression of the gene encoding the human androgen receptor, from a strong yeast promoter, results in transactivation of a hybrid promoter carrying androgen-responsive sequences such that a target gene may be expressed in an androgen-dependent manner. By selection of an appropriate combination of androgen receptor level, target-gene copy number and concentration of the androgenic ligand, dihydrotestosterone, the expression level can be set within a 1400-fold range with no detectable effect on normal cell growth.