Exposure of mammalian cells to UV radiation alters gene expression and cell cycle progression; some of these responses may ensure survival or serve as mutation-avoidance mechanisms, lessening the consequences of UV-induced DNA damage. We showed previously that UV irradiation increases phosphorylation of the p34 subunit of human replication protein A (RPA) and that this hyperphosphorylation correlated with loss of activity of the DNA replication complex. To characterize further the role of RPA hyperphosphorylation in the cellular response to UV irradiation and to determine which protein kinases might be involved, we identified by phosphopeptide analysis the sites phosphorylated in the p34 subunit of RPA (RPA-p34) from HeLa cells before and after exposure to 30 J/m2 UV light. In unirradiated HeLa cells, RPA-p34 is phosphorylated primarily at Ser-23 and Ser-29. At least four of the eight serines and one threonine in the N-terminal 33 residues of RPA-p34 can become phosphorylated after UV irradiation. Two of these sites (Ser-23 and Ser-29) are known to be sites phosphorylated by Cdc2 kinase; two others (Thr-21 and Ser-33) are consensus sites for the DNA-dependent protein kinase (DNA-PK); the fifth site (Ser-11, -12, or -13) does not correspond to the (Ser/Thr)-Gln DNA-PK consensus. All five can be phosphorylated in vitro by incubating purified RPA with purified DNA-PK. Two additional sites, probably Ser-4 and Ser-8, are phosphorylated in vivo after UV irradiation and in vitro by purified DNA-PK. The capacity of purified DNA-PK to phosphorylate many of these same sites on RPA-p34 in vitro implicates DNA-PK or a kinase with similar specificity in the UV-induced hyperphosphorylation of RPA in vivo.

The Xeroderma pigmentosum group D (XPD) protein is an essential participant in nucleotide excision repair and basal transcription. There is evidence that three common polymorphisms of the XPD gene (C156A, Asp312Asn, and Lys751Gln) may be associated with differential DNA repair activity. Because increased DNA repair plays an important role in chemoresistance to platinum-based compounds, we assessed the aforementioned polymorphisms in 73 patients with metastatic colorectal cancer and determined their outcome to 5-fluorouracil/oxaliplatin. Among those tested for the Lys751Gln polymorphism, 24% (5 of 21) patients with the Lys/Lys genotype responded, versus 10% (4 of 39) and 10% (1 of 10) of those with the Lys/Gln and Gln/Gln genotypes (P = 0.015). The median survival for those with the Lys/Lys genotype was 17.4 (95% CI 7.9, 26.5) versus 12.8 (95% CI 8.5, 25.9) and 3.3 (95% CI 1.4, 6.5) months for patients with the Lys/Gln and Gln/Gln respectively (P = 0.002). The polymorphisms C156A and Asp312Asn of the XPD gene were not associated with response to 5-fluorouracil/oxaliplatin nor with survival. However, a linkage was observed between the Lys751 allele and the C156 allele (P = 0.028), and between the Lys751Lys genotype and the Asp312Asp genotype (P < 0.001). We conclude that XPD Lys751Gln polymorphism may be an important marker in the prediction of clinical outcome to platinum-based chemotherapy.

BACKGROUND

Autoimmune thyroid diseases (AITD), including Graves' disease and Hashimoto's thyroiditis, arise due to complex interactions between environmental and genetic factors. There are sound data coming from epidemiological, family, and twin studies demonstrating a strong genetic influence on the development of AITD. In this review we summarize the new findings on the genetic susceptibility to AITD focusing on emerging mechanisms of susceptibility.

CONCLUSIONS

Candidate gene analysis, whole-genome linkage screening, genome-wide association studies, and whole-genome sequencing are the major technologies that have advanced this field, leading to the identification of at least seven genes whose variants have been associated with AITD. One of the major ones is the HLA-DR gene locus. Recently, it was shown that substitution of the neutral amino acids Ala or Gln with arginine at position beta 74 in the HLA-DR peptide-binding pocket is key to the etiology of both Graves' disease and Hashimoto's thyroiditis. Several other genes have also been shown to confer susceptibility to AITD. These can be classified into two groups: (i) immune regulatory genes (cytotoxic T lymphocyte-associated protein 4, CD40, protein tyrosine phosphatase-22, and CD25) and (ii) thyroid-specific genes (thyroglobulin and thyrotropin receptor genes). The influence of individual genes on the development of AITD when assessed in a population appears to be weaker than would be expected from the data showing strong genetic susceptibility to AITD. Two possible mechanisms explaining this discrepancy are gene-gene interactions and subset effects.

CONCLUSIONS

Significant progress has been made in our understanding of the immunogenetic mechanisms leading to thyroid autoimmunity. For the first time we are beginning to unravel these mechanisms at the molecular level. It is hoped that these new data will be translated into novel therapies and prevention strategies in AITD, such as costimulatory blockade.

Sugar kinases, stress-70 proteins, and actin belong to a superfamily defined by a fold consisting of two domains with the topology beta beta beta alpha beta alpha beta alpha. These enzymes catalyze ATP phosphoryl transfer or hydrolysis coupled to a large conformational change in which the two domains close around the nucleotide. The beta 1-beta 2 turns of each domain form hydrogen bonds with ATP phosphates, and conserved Asp, Glu or Gln residues coordinate Mg2+ or Ca2+ through bound waters. The activity of superfamily members is regulated by various effectors, some of which act by promoting or inhibiting the conformational change. Nucleotide hydrolysis eliminates interdomain bridging interactions between the second beta 1-beta 2 turn and the ATP gamma-phosphate. This is proposed to destabilize the closed conformation and affect the orientation of the two domains, which might in turn regulate the activity of kinase oligomers, stress-70 protein-protein complexes, and actin filaments.

Various DNA alterations can be caused by exposure to environmental and endogenous carcinogens. Most of these alterations, if not repaired, can result in genetic instability, mutagenesis and cell death. DNA repair mechanisms are important for maintaining DNA integrity and preventing carcinogenesis. Recent lung cancer studies have focused on identifying the effects of single nucleotide polymorphisms (SNPs) in candidate genes, among which DNA repair genes are increasingly being studied. Genetic variations in DNA repair genes are thought to modulate DNA repair capacity and are suggested to be related to lung cancer risk. We identified a sufficient number of epidemiologic studies on lung cancer to conduct a meta-analysis for genetic polymorphisms in nucleotide excision repair pathway genes, focusing on xeroderma pigmentosum group A (XPA), excision repair cross complementing group 1 (ERCC1), ERCC2/XPD, ERCC4/XPF and ERCC5/XPG. We found an increased risk of lung cancer among subjects carrying the ERCC2 751Gln/Gln genotype (odds ratio (OR) = 1.30, 95% confidence interval (CI) = 1.14 - 1.49). We found a protective effect of the XPA 23G/G genotype (OR = 0.75, 95% CI = 0.59 - 0.95). Considering the data available, it can be conjectured that if there is any risk association between a single SNP and lung cancer, the risk fluctuation will probably be minimal. Advances in the identification of new polymorphisms and in high-throughput genotyping techniques will facilitate the analysis of multiple genes in multiple DNA repair pathways. Therefore, it is likely that the defining feature of future epidemiologic studies will be the simultaneous analysis of large samples.

Heparanase is an endo-beta-d-glucuronidase that degrades heparan sulfate in the extracellular matrix and on the cell surface. Human proheparanase is produced as a latent protein of 543 amino acids whose activation involves excision of an internal linker segment (Ser(110)-Gln(157)), yielding the active heterodimer composed of 8- and 50-kDa subunits. Applying cathepsin L knock-out tissues and cultured fibroblasts, as well as cathepsin L gene silencing and overexpression strategies, we demonstrate, for the first time, that removal of the linker peptide and conversion of proheparanase into its active 8 + 50-kDa form is brought about predominantly by cathepsin L. Excision of a 10-amino acid peptide located at the C terminus of the linker segment between two functional cathepsin L cleavage sites (Y156Q and Y146Q) was critical for activation of proheparanase. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry demonstrates that the entire linker segment is susceptible to multiple endocleavages by cathepsin L, generating small peptides. Mass spectrometry demonstrated further that an active 8-kDa subunit can be generated by several alternative adjacent endocleavages, yielding the precise 8-kDa subunit and/or slightly elongated forms. Altogether, the mode of action presented here demonstrates that processing and activation of proheparanase can be brought about solely by cathepsin L. The critical involvement of cathepsin L in proheparanase processing and activation offers new strategies for inhibiting the prometastatic, proangiogenic, and proinflammatory activities of heparanase.

Ligand binding may involve a wide range of structural changes in the receptor protein, from hinge movement of entire domains to small side-chain rearrangements in the binding pocket residues. The analysis of side chain flexibility gives insights valuable to improve docking algorithms and can provide an index of amino-acid side-chain flexibility potentially useful in molecular biology and protein engineering studies. In this study we analyzed side-chain rearrangements upon ligand binding. We constructed two non-redundant databases (980 and 353 entries) of "paired" protein structures in complexed (holo-protein) and uncomplexed (apo-protein) forms from the PDB macromolecular structural database. The number and identity of binding pocket residues that undergo side-chain conformational changes were determined. We show that, in general, only a small number of residues in the pocket undergo such changes (e.g., approximately 85% of cases show changes in three residues or less). The flexibility scale has the following order: Lys>> Arg, Gln, Met>> Glu, Ile, Leu>> Asn, Thr, Val, Tyr, Ser, His, Asp>> Cys, Trp, Phe; thus, Lys side chains in binding pockets flex 25 times more often then do the Phe side chains. Normalizing for the number of flexible dihedral bonds in each amino acid attenuates the scale somewhat, however, the clear trend of large, polar amino acids being more flexible in the pocket than aromatic ones remains. We found no correlation between backbone movement of a residue upon ligand binding and the flexibility of its side chain. These results are relevant to 1. Reduction of search space in docking algorithms by inclusion of side-chain flexibility for a limited number of binding pocket residues; and 2. Utilization of the amino acid flexibility scale in protein engineering studies to alter the flexibility of binding pockets.

Huntington's disease (HD) is a dominant neurodegenerative disease caused by polyglutamine (polyQ) expansion in the protein huntingtin (htt). HD pathogenesis appears to involve the production of mutated N-terminal htt, cytoplasmic and nuclear aggregation of htt, and abnormal activity of htt interactor proteins essential to neuronal survival. Before cell death, neuronal dysfunction may be an important step of HD pathogenesis. To explore polyQ-mediated neuronal toxicity, we expressed the first 57 amino acids of human htt containing normal [19 Gln residues (Glns)] and expanded (88 or 128 Glns) polyQ fused to fluorescent marker proteins in the six touch receptor neurons of Caenorhabditis elegans. Expanded polyQ produced touch insensitivity in young adults. Noticeably, only 28 +/- 6% of animals with 128 Glns were touch sensitive in the tail, as mediated by the PLM neurons. Similar perinuclear deposits and faint nuclear accumulation of fusion proteins with 19, 88, and 128 Glns were observed. In contrast, significant deposits and morphological abnormalities in PLM cell axons were observed with expanded polyQ (128 Glns) and partially correlated with touch insensitivity. PLM cell death was not detected in young or old adults. These animals indicate that significant neuronal dysfunction without cell death may be induced by expanded polyQ and may correlate with axonal insults, and not cell body aggregates. These animals also provide a suitable model to perform in vivo suppression of polyQ-mediated neuronal dysfunction.

Adenosine diphosphate ribosyl transferase (ADPRT) and X-ray repair cross-complementing 1 (XRCC1) are two major DNA base excision repair (BER) proteins and act interactively in stimulating and executing BER processes. Polymorphisms of ADPRT Val762Ala and XRCC1 Arg399Gln have been associated with altered protein function and BER activity. This case-control study examined the contribution of these two polymorphisms, alone and in combination, or in interaction with smoking, to the risk of developing lung cancer. We estimated the risk of lung cancer associated with these polymorphisms in 1,000 cases and 1,000 cancer-free controls using logistic regression models. Subjects having the ADPRT Ala/Ala genotype had an odds ratio (OR) of 1.68 [95% confidence interval (95% CI), 1.27-2.23] compared with those having the Val/Val genotype. A greater than multiplicative joint effect between the ADPRT polymorphism and smoking was observed. The ORs (95% CI) of the Ala/Ala genotype for nonsmokers and smokers who smoked < or =16, 16 to 28, or >28 pack-years were 1.13 (0.79-1.62), 1.35 (0.68-2.70), 2.46 (1.35-4.51) or 17.09 (8.15-35.83), respectively (P trend test < 0.001). Gene-gene interaction of ADPRT and XRCC1 polymorphisms increased risk of lung cancer in a supermultiplicative manner (OR for the presence of both ADPRT 762Ala/Ala and XRCC1 399Gln/Gln genotypes, 5.91; 95% CI, 2.09-16.72), although the XRCC1 polymorphism itself was not associated with the risk. In conclusion, the ADPRT Val762Ala polymorphism plays an important role in smoking-related lung cancer and the XRCC1 Arg399Gln polymorphism may serve as a risk modifier.

BACKGROUND

In-vitro studies have suggested that polymorphisms of the beta 2-adrenoceptor may influence the desensitisation induced by beta 2-agonists. We investigated the influence of beta 2-AR polymorphism on the development of bronchodilator desensitisation in asthma patients.

METHODS

We carried out an analysis of 22 moderately severe stable asthmatics, mean age 38 years, FEV1 63% of predicted and FEF25-75 38% of predicted, who received a median inhaled corticosteroid dose of 1000 micrograms/day. Patients were randomly assigned inhaled placebo or inhaled formoterol 24 micrograms bid for 4 weeks each in a crossover study. Bronchodilator dose-response curves were made at the end of each treatment period by use of cumulative doses of formoterol (6-108 micrograms) with FEV1 and FEF25-75 measured 30 min after each dose, and up to 6 h after the last dose. We calculated the degree of bronchodilator desensitisation by comparing the dose-response (for maximum and 6 h) after placebo with that after formoterol, and expressed this degree as a percentage of placebo response. Patients were divided into groups according to genotype at codon 16: homozygous Arg 16 (n = 4), heterozygous Arg 16/Gly 16 (n = 8), and homozygous Gly 16 (n = 10). At codon 27: homozygous Gln 27 (n = 5), heterozygous Gln 27/Glu 27 (n = 11), and homozygous Glu 27 (n = 6).

RESULTS

We found a significantly (p < 0.05) greater degree of bronchodilator desensitisation with homozygous Gly 16 than with homozygous Arg 16 for maximal FEV1 response: -8% (Arg 16) vs 46% (Gly 16); and for maximal FEF25-75 response: -32% (Arg 16) vs 74% (Gly 16; 95% CI 15-92% and 49-164%, respectively). Bronchodilator responses at 6 h were also significantly (p < 0.05) different for FEV1 and FEF25-75 when Arg 16 and Gly 16 were compared and values for heterozygous Arg 16/Gly 16 were intermediate. There was significantly greater desensitisation with Glu 27 than with Gln 27 for maximal FEF25-75 response: -7% (Gln 27) vs 68% (Glu 27), p = 0.05; and for 6 h FEF25-75 response: 43% (Gln 27) vs 93% (Glu 27), p < 0.05 (95% CI 2-147% and 5-94%, respectively). All patients who were homozygous Glu 27 were also homozygous Gly 16.

CONCLUSIONS

We have found preliminary evidence that beta 2-adrenoceptor polymorphism is associated with altered beta 2-adrenoceptor expression in asthma patients. The homozygous Gly-16 form was significantly more prone to bronchodilator desensitisation than Arg 16, with the influence of Gly 16 dominating over any putative protective effects of Glu 27.

Mammalian ras oncogenes encode polypeptides of relative molecular mass (Mr) 21,000 (p21) which bind GTP and GDP. Oncogenic ras-encoded proteins differ from their normal homologues by an amino acid substitution for Gly 12, Ala 59 or Gln 61. Recently, we and others have observed that normal p21, encoded by the Ha-ras gene, has a GTP hydrolytic activity that is reduced by the oncogenic substitutions Val 12 or Thr 59. The yeast Saccharomyces cerevisiae contains two ras-related genes, RASsc1 and RASsc2, the expression of either of which is sufficient for viability. RASsc1 and RASsc2 encode proteins of 309 (SC1) and 322 (SC2) residues which are 62% homologous to mammalian p21 in their 172-amino acid N-terminal sequences. We report here that the N-terminal domain of SC1 binds GTP and GDP and has a GTP hydrolytic activity that is reduced in the variants SC1[Thr 66] and SC1[Leu 68] which are analogous to oncogenic Ha[Thr 59] and Ha[Leu 61], respectively. These results suggest that yeast and mammalian ras proteins have similar biochemical and possibly biological functions.

The complete amino acid sequence of human salivary statherin, a peptide which strongly inhibits precipitation from supersaturated calcium phosphate solutions, and therefore stabilizes supersaturated saliva, has been determined. The NH2-terminal half of this Mr=5380 (43 amino acids) polypeptide was determined by automated Edman degradations (liquid phase) on native statherin. The peptide was digested separately with trypsin, chymotrypsin, and Staphylococcus aureus protease, and the resulting peptides were purified by gel filtration. Manual Edman degradations on purified peptide fragments yielded peptides that completed the amino acid sequence through the penultimate COOH-terminal residue. These analyses, together with carboxypeptidase digestion of native statherin and of peptide fragments of statherin, established the complete sequence of the molecule. The 2 serine residues (positions 2 and 3) in statherin were identified as phosphoserine. The amino acid sequence of human salivary statherin is striking in a number of ways. The NH2-terminal one-third is highly polar and includes three polar dipeptides: H2PO3-Ser-Ser-H2PO3-Arg-Arg-, and Glu-Glu-. The COOH-terminal two-thirds of the molecule is hydrophobic, containing several repeating dipeptides: four of -Gn-Pro-, three of -Tyr-Gln-, two of -Gly-Tyr-, two of-Gln-Tyr-, and two of the tetrapeptide sequence -Pro-Tyr-Gln-Pro-. Unusual cleavage sites in the statherin sequence obtained with chymotrypsin and S. aureus protease were also noted.

A peptide of 21 amino acids with the sequence Arg-Ile-Leu-Ala-Val-Glu-Arg-Tyr-Leu-Lys-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp- Gly-Cys - Ser encoded by a segment in the env gene of human T-lymphotropic virus type III (HTLV-III), corresponding to amino acids 586-606 of the precursor envelope glycoprotein, has been synthesized by the Merrifield solid-phase method. Combined serological and chemical analyses of this peptide and related peptides have revealed the importance of certain amino acid residues in the antigenic determinant of the relevant peptide. Enzyme immunoassay (EIA) employing this peptide as an antigen adsorbent was shown to reproducibly detect antibodies in sera of patients with HTLV-III infection. This assay provided positive results with all sera that were reactive with gp41 envelope protein of HTLV-III in electrophoretic immunoblot analysis. Thus far, no false-positive sera have been encountered in control populations. Our EIA with this peptide as the coating antigen is shown to have advantages over that with the whole HTLV-III virus as an immunoadsorbent.

The ga2 mutant of Arabidopsis thaliana is a gibberellin-deficient dwarf. Previous biochemical studies have suggested that the ga2 mutant is impaired in the conversion of copalyl diphosphate to ent-kaurene, which is catalyzed by ent-kaurene synthase (KS). Overexpression of the previously isolated KS cDNA from pumpkin (Cucurbita maxima) (CmKS) in the ga2 mutant was able to complement the mutant phenotype. A genomic clone coding for KS, AtKS, was isolated from A. thaliana using CmKS cDNA as a heterologous probe. The corresponding A. thaliana cDNA was isolated and expressed in Escherichia coli as a fusion protein. The fusion protein showed enzymatic activity that converted [3H]copalyl diphosphate to [3H]ent-kaurene. The recombinant AtKS protein derived from the ga2-1 mutant is truncated by 14 kD at the C-terminal end and does not contain significant KS activity in vitro. Sequence analysis revealed that a C-2099 to T base substitution, which converts Gln-678 codon to a stop codon, is present in the AtKS cDNA from the ga2-1 mutant. Taken together, our results show that the GA2 locus encodes KS.

The formation of amyloid-like aggregates by expanded polyglutamine (polyGln) sequences is suspected to play a critical role in the neuropathology of Huntington's disease and other expanded CAG-repeat diseases. To probe the folding of the polyGln sequence in the aggregate, we replaced Gln-Gln pairs at different sequence intervals with Pro-Gly pairs, elements that are compatible with beta-turn formation and incompatible with beta-extended chain. We find that PGQ9 and PGQ10, peptides consisting of four Q9 or Q10 elements interspersed with PG elements, undergo spontaneous aggregation as efficiently as a Q45 sequence, whereas the corresponding PGQ7 and PGQ8 peptides aggregate much less readily. Furthermore, a PDGQ9 sequence containing d-prolines aggregates more efficiently than the peptide with l-prolines, consistent with beta-turn formation in aggregate structure. Introduction of one additional Pro residue in the center of a Q9 element within PGQ9 completely blocks the peptide's ability to aggregate. This strongly suggests that the Q9 elements are required to be in extended chain for efficient aggregation to occur. We determined the critical nucleus for aggregation nucleation of the PGQ9 peptide to be one, a result identical to that for unbroken polyGln sequences. The PGQN peptide aggregates are structurally quite similar to Q45 aggregates, as judged by heterologous seeding aggregation kinetics, recognition by an anti-polyGln aggregate antibody, and electron microscopy. The results suggest that polyGln aggregate structure consists of alternating elements of extended chain and turn. In the future it should be possible to conduct detailed and interpretable mutational studies in the PGQ9 background.

A single pair of Glu and Lys residues has been placed at four different spacings, and in both orientations, in an otherwise neutral alanine-glutamine peptide helix, and the contribution to helix stability of the different Glu-Lys interactions has been measured. The contribution from the interaction of each charged side chain with the helix macrodipole has also been determined. A side-chain interaction between Gln and Glu, when the spacing is (i,i+4), has been detected and quantified. The interactions have been divided into contributions from hydrogen bonds (independent of the concentration of NaCl) and from electrostatic interactions (present in 10 mM NaCl, absent in 2.5 M NaCl). The major results are as follows: (1) The (i,i+3) and (i,i+4) Glu-Lys interactions are helix-stabilizing and are similar in strength to each other, regardless of the orientation of the side chains. (2) Hydrogen bonds provide the major contribution to these side-chain interactions, as shown by the following facts. First, the major part of the interaction observed in 10 mM NaCl, pH 7, is still present in 2.5 M NaCl. Second, the interaction found at pH 2 is equally as strong as that found in 2.5 M NaCl at pH 7. (3) The (i,i+4) Gln-Glu side-chain hydrogen bond is as strong as the hydrogen-bond component of the Glu-Lys interaction at both pH 2 and pH 7. The Gln-Glu interaction differs from the Glu-Lys interaction in being specific both for the orientation and the spacing of the residues. (4) No significant hydrogen-bonding interaction was found for the (i,i+1) or (i, i+2) Glu-Lys spacings, either at pH 2 or at pH 7, in 2.5 M NaCl. At 10 mM NaCl and pH 7, these spacings show a helix-destabilizing electrostatic interaction which probably results from stabilization of the coil conformation.

Natural scrapie has been viewed both as a recessive trait and as a contagious disease modulated by a host locus. To address this conundrum, we determined the structure of the sheep prion protein (PrP) gene, which contains three exons and extends over 20 kb of DNA. In the United States 86.4% of scrapie cases occur in Suffolk sheep, and within this breed 49 +/- 6% (+/- S.D., n = 69) of healthy animals carry one or more PrP alleles encoding Arg (R)-171. Four scrapie-affected sheep were homozygous for wild-type PrP open reading frames encoding the alternative Gln (Q)-171 allele. Analysis of additional cases revealed that all were Q/Q-171 homozygotes (n = 31), yielding a probability of 0.000004 that PrP genotype is unrelated to susceptibility. These data imply that homozygosity for Q-171 codons is necessary but not sufficient for the development of natural scrapie, echo reports of recessive manifestation, and parallel over-representation of PRNP codon 129 homozygotes in Creutzfeldt-Jakob disease of humans. Whereas progress has been substantial regarding experimental scrapie in rodents, the occurrence and spread of disease in flocks of sheep has remained enigmatic. Appreciation of the relationship between codon 171 genotype and susceptibility may help define the molecular basis of natural scrapie.

Multiconformation continuum electrostatics (MCCE) explores different conformational degrees of freedom in Monte Carlo calculations of protein residue and ligand pK(a)s. Explicit changes in side chain conformations throughout a titration create a position dependent, heterogeneous dielectric response giving a more accurate picture of coupled ionization and position changes. The MCCE2 methods for choosing a group of input heavy atom and proton positions are described. The pK(a)s calculated with different isosteric conformers, heavy atom rotamers and proton positions, with different degrees of optimization are tested against a curated group of 305 experimental pK(a)s in 33 proteins. QUICK calculations, with rotation around Asn and Gln termini, sampling His tautomers and torsion minimum hydroxyls yield an RMSD of 1.34 with 84% of the errors being <1.5 pH units. FULL calculations adding heavy atom rotamers and side chain optimization yield an RMSD of 0.90 with 90% of the errors <1.5 pH unit. Good results are also found for pK(a)s in the membrane protein bacteriorhodopsin. The inclusion of extra side chain positions distorts the dielectric boundary and also biases the calculated pK(a)s by creating more neutral than ionized conformers. Methods for correcting these errors are introduced. Calculations are compared with multiple X-ray and NMR derived structures in 36 soluble proteins. Calculations with X-ray structures give significantly better pK(a)s. Results with the default protein dielectric constant of 4 are as good as those using a value of 8. The MCCE2 program can be downloaded from http://www.sci.ccny.cuny.edu/~mcce.

The cAMP-dependent protein kinase (PKA I and II) and the cAMP-stimulated GDP exchange factors (Epac1 and -2) are major cAMP effectors. The cAMP affinity of the PKA holoenzyme has not been determined previously. We found that cAMP bound to PKA I with a K(d) value (2.9 microM) similar to that of Epac1. In contrast, the free regulatory subunit of PKA type I (RI) had K(d) values in the low nanomolar range. The cAMP sites of RI therefore appear engineered to respond to physiological cAMP concentrations only when in the holoenzyme form, whereas Epac can respond in its free form. Epac is phylogenetically younger than PKA, and its functional cAMP site has presumably evolved from site B of PKA. A striking feature is the replacement of a conserved Glu in PKA by Gln (Epac1) or Lys (Epac2). We found that such a switch (E326Q) in site B of human RIalpha led to a 280-fold decreased cAMP affinity. A similar single switch early in Epac evolution could therefore have decreased the high cAMP affinity of the free regulatory subunit sufficiently to allow Epac to respond to physiologically relevant cAMP levels. Molecular dynamics simulations and cAMP analog mapping indicated that the E326Q switch led to flipping of Tyr-373, which normally stacks with the adenine ring of cAMP. Combined molecular dynamics simulation, GRID analysis, and cAMP analog mapping of wild-type and mutated BI and Epac1 revealed additional differences, independent of the Glu/Gln switch, between the binding sites, regarding space (roominess), hydrophobicity/polarity, and side chain flexibility. This helped explain the specificity of current cAMP analogs and, more importantly, lays a foundation for the generation of even more discriminative analogs.

A major source of inorganic nitrogen for rice plants grown in paddy soil is ammonium ions. The ammonium ions are actively taken up by the roots via ammonium transporters and subsequently assimilated into the amide residue of glutamine (Gln) by the reaction of glutamine synthetase (GS) in the roots. The Gln is converted into glutamate (Glu), which is a central amino acid for the synthesis of a number of amino acids, by the reaction of glutamate synthase (GOGAT). Although a small gene family for both GS and GOGAT is present in rice, ammonium-dependent and cell type-specific expression suggest that cytosolic GS1;2 and plastidic NADH-GOGAT1 are responsible for the primary assimilation of ammonium ions in the roots. In the plant top, approximately 80% of the total nitrogen in the panicle is remobilized through the phloem from senescing organs. Since the major form of nitrogen in the phloem sap is Gln, GS in the senescing organs and GOGAT in developing organs are important for nitrogen remobilization and reutilization, respectively. Recent work with a knock-out mutant of rice clearly showed that GS1;1 is responsible for this process. Overexpression studies together with age- and cell type-specific expression strongly suggest that NADH-GOGAT1 is important for the reutilization of transported Gln in developing organs. The overall process of nitrogen utilization within the plant is discussed.

Semliki Forest virus (SFV), an alphavirus, infects cells via a low pH-triggered membrane fusion reaction that takes place within the cellular endocytic pathway. Fusion is mediated by the heterotrimeric virus spike protein, which undergoes conformational changes upon exposure to low pH. The SFV E1 spike subunit contains a hydrophobic domain of 23 amino acids that is highly conserved among alphaviruses. This region is also homologous to a domain of the rotavirus outer capsid protein VP4. Mutagenesis of an SFV spike protein cDNA was used to evaluate the role of the E1 domain in membrane fusion. Mutant spike proteins were expressed in COS cells and assayed for cell-cell fusion activity. Four mutant phenotypes were identified: (i) substitution of Gln for Lys-79 or Leu for Met-88 had no effect on spike protein fusion activity; (ii) substitution of Ala for Asp-75, Ala for Gly-83, or Ala for Gly-91 shifted the pH threshold of fusion to a more acidic range; (iii) mutation of Pro-86 to Asp, Gly-91 to Pro, or deletion of amino acids 83 to 92 resulted in retention of the E1 subunit within the endoplasmic reticulum; and (iv) substitution of Asp for Gly-91 completely blocked cell-cell fusion activity without affecting spike protein assembly or transport. These results argue that the conserved hydrophobic domain of SFV E1 is closely involved in membrane fusion and suggest that the homologous region in rotavirus VP4 may be involved in the entry pathway of this nonenveloped virus.

We describe the de novo design and biophysical characterization of a model coiled-coil protein in which we have systematically substituted 20 different amino acid residues in the central "d" position. The model protein consists of two identical 38 residue polypeptide chains covalently linked at their N termini via a disulfide bridge. The hydrophobic core contained Val and Ile residues at positions "a" and Leu residues at positions "d". This core allowed for the formation of both two-stranded and three-stranded coiled-coils in benign buffer, depending on the substitution at position "d". The structure of each analog was analyzed by CD spectroscopy and their relative stability determined by chemical denaturation using GdnHCI (all analogs denatured from the two-stranded state). The oligomeric state(s) was determined by high-performance size-exclusion chromatography and sedimentation equilibrium analysis in benign medium. Our results showed a thermodynamic stability order (in order of decreasing stability) of: Leu, Met, Ile, Tyr, Phe, Val, Gln, Ala, Trp, Asn, His, Thr, Lys, Ser, Asp, Glu, Arg, Orn, and Gly. The Pro analog prevented coiled-coil formation. The overall stability range was 7.4 kcal/mol from the lowest to the highest analog, indicating the importance of the hydrophobic core and the dramatic effect a single substitution in the core can have upon the stability of the protein fold. In general, the side-chain contribution to the level of stability correlated with side-chain hydrophobicity. Molecular modelling studies, however, showed that packing effects could explain deviations from a direct correlation. In regards to oligomerization state, eight analogs demonstrated the ability to populate exclusively one oligomerization state in benign buffer (0.1 M KCl, 0.05 M K(2)PO(4)(pH 7)). Ile and Val (the beta-branched residues) induced the three-stranded oligomerization state, whereas Tyr, Lys, Arg, Orn, Glu and Asp induced the two-stranded state. Asn, Gln, Ser, Ala, Gly, Phe, Leu, Met and Trp analogs were indiscriminate and populated two-stranded and three-stranded states. Comparison of these results with similar substitutions in position "a" highlights the positional effects of individual residues in defining the stability and numbers of polypeptide chains occurring in a coiled-coil structure. Overall, these results in conjunction with other work now generate a relative thermodynamic stability scale for 19 naturally occurring amino acid residues in either an "a" or "d" position of a two-stranded coiled-coil. Thus, these results will aid in the de novo design of new coiled-coil structures, a better understanding of their structure/function relationships and the design of algorithms to predict the presence of coiled-coils within native protein sequences.

Although conformational changes are essential for the function of proteins, little is known about their structural dynamics at atomic level resolution. Myoglobin (Mb) is the paradigm to investigate conformational dynamics because it is a simple globular heme protein displaying a photosensitivity of the iron-ligand bond. Upon laser photodissociation of carboxymyoglobin Mb a nonequilibrium population of protein structures is generated that relaxes over a broad time range extending from picoseconds to milliseconds. This process is associated with migration of the ligand to cavities in the matrix and with a reduction in the geminate rebinding rate by several orders of magnitude. Here we report nanosecond time-resolved Laue diffraction data to 1.55-A resolution on a Mb mutant, which depicts the sequence of structural events associated with this extended relaxation. Motions of the distal E-helix, including the mutated residue Gln-64(E7), and of the CD-turn are found to lag significantly (100-300 ns) behind local rearrangements around the heme such as heme tilting, iron motion out of the heme plane, and swinging of the mutated residue Tyr-29(B10), all of which occur promptly (< or =3 ns). Over the same delayed time range, CO is observed to migrate from a cavity distal to the heme known to bind xenon (called Xe4) to another such cavity proximal to the heme (Xe1). We propose that the extended relaxation of the globin moiety reflects reequilibration among conformational substates known to play an essential role in controlling protein function.

We have previously shown that a number of platelet proteins become phosphorylated at tyrosine residues in response to platelet-activating agents. Here we present two lines of evidence implicating a platelet integrin, glycoprotein IIb-IIIa, in the regulation of a specific subset of these tyrosine phosphorylations. (i) Two peptides that inhibit the binding of fibrinogen and other ligands to gpIIb-IIIa, Arg-Gly-Asp-Ser and His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val, also inhibited the thrombin-induced tyrosine phosphorylation of this subset of proteins. The tetrapeptide Arg-Gly-Glu-Ser, which does not inhibit fibrinogen binding, did not inhibit thrombin-stimulated tyrosine phosphorylation. (ii) Platelets lacking gpIIb-IIIa (from a subject with Glanzmann thrombasthenia) did not undergo this subset of tyrosine phosphorylation in response to thrombin, although other serine, threonine, and tyrosine phosphorylations proceeded normally. These findings suggest a role for tyrosine-specific protein phosphorylation in integrin-mediated cell-matrix recognition.