We propose that arginine side chains often play a previously unappreciated general structural role in the maintenance of tertiary structure in proteins, wherein the positively charged guanidinium group forms multiple hydrogen bonds to backbone carbonyl oxygens. Using as a criterion for a "structural" arginine one that forms 4 or more hydrogen bonds to 3 or more backbone carbonyl oxygens, we have used molecular graphics to locate arginines of interest in 4 proteins: Arg 180 in Thermus thermophilus manganese superoxide dismutase, Arg 254 in human carbonic anhydrase 11, Arg 31 in Streptomyces rubiginosusxylose isomerase, and Arg 313 in Rhodospirillum rubrum ribulose-l,S-bisphosphate carboxylase/oxygenase. Arg 180 helps to mold the active site channel of superoxide dismutase, whereas in each of the other enzymes the structural arginine is buried in the "mantle" (i.e., inside, but near the surface) of the protein interior well removed from the active site, where it makes 5 hydrogen bonds to 4 backbone carbonyl oxygens. Using a more relaxed criterion of 3 or more hydrogen bonds to 2 or more backbone carbonyl oxygens, arginines that play a potentially important structural role were found in yeast enolase, Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase, bacteriophage T4 and human lysozymes, Enteromorpha prolifera plastocyanin, HIV-1 protease, Trypanosoma brucei brucei and yeast triosephosphate isomerases, and Escherichia coli trp aporepressor (but not trp repressor or the trp repressor/operator complex). In addition to helping form the active site funnel in superoxide dismutase, the structural arginines found in this study play such diverse roles as stapling together 3 strands of backbone from different regions of the primary sequence, and tying a-helix to a-helix, &turn to 6-turn, and subunit to subunit.Keywords: backbone carbonyl oxygens; buried arginine; carbonic anhydrase; enolase; glyceraldehyde-3-phosphate dehydrogenase; HIV-1 protease; lysozyme; manganese superoxide dismutase; multiple hydrogen bonds; plastocyanin; ribulose-l,5-bisphosphate carboxylase/oxygenase; structural arginine; triosephosphate isomerase; trp aporepressor; xylose isomeraseThe guanidinium group of arginine is the most polar of all the common amino acid side chains found in proteins (Wolfenden, 1983) and is thus the residue most likely to be found on the surface in an aqueous environment. The side chain almost always has a pK, 112 and the positively charged planar guanidinium group can be a hydrogen donor in the formation of up to 5 hydrogen bonds (Fig. 1). Arginine plays a major role in the binding of negatively charged substrates, cofactors, and effectors to

The single-strand overhang present at all known telomeres plays a critical role in mediating both the capping and telomerase regulation functions of telomeres. The telomere end-binding proteins, Cdc13 in S. cerevisiae, Pot1 in higher eukaryotes, and TEBP in the ciliated protozoa O. nova, exhibit sequence-specific binding to their respective single-strand overhangs. S. cerevisiae telomeres are composed of a heterogeneous mixture of GT-rich telomeric sequence, unlike in higher eukaryotes which have a simple repeat that is maintained with high fidelity. In yeast, the telomeric overhang is recognized by the essential protein Cdc13, which coordinates end-capping and telomerase activities at the telomere. The Cdc13-DNA-binding domain (Cdc13-DBD) binds these telomere sequences with high affinity (3 pM) and sequence specificity. To better understand the basis for this remarkable recognition, we have investigated the binding of the Cdc13-DBD to a series of altered DNA substrates. Although an 11-mer of GT-rich sequence is required for full binding affinity, only 3 of these 11 bases are recognized with high specificity. This specificity differs from that observed in the other known telomere end-binding proteins, but is well suited to the specific role of Cdc13 at yeast telomeres. These studies expand our understanding of telomere recognition by the Cdc13-DBD and of the unique molecular recognition properties of ssDNA binding.Recognition of single-strand DNA (ssDNA) is essential for many fundamental cellular activities, including recombination, repair, replication, transcription, translation, and telomere maintenance. Structural and biochemical studies of the proteins that mediate ssDNA-binding activity have provided key insights into the mechanism of recognition. Generally, small domains containing mixed α/β topologies, such as the OB-fold and KH domain, are used for ssDNA recognition, and binding is achieved through base and backbone interactions that are completely distinct from those used in double-strand DNA recognition (1-5). In many biological contexts DNA needs to be uniformly recognized, thus it is appropriate for this recognition to be non-sequence specific. However, in a few cases, including telomere maintenance, transcription, and viral packaging, it is imperative that recognition be exquisitely sequence specific (3,(6)(7)(8).Telomeres, the DNA, RNA and protein complexes at the ends of chromosomes, play important roles in many essential cellular functions. They regulate the proliferative lifetime of the cell and protect chromosomes from degradation or end-to-end fusion, as well as participate in meiotic segregation and chromatic silencing (reviewed in (9-13)). Telomeres are critical to the maintenance of the genome and normal development. Thus, telomere malfunction can have dramatic adverse impacts on human health, leading to cancer, aging disorders and increased human mortality (14)(15)(16)(17)(18)(19). Therefore, it is essential to understand the mechanisms through which telomeres are properly maintain...

Virulence of the gastric pathogen Helicobacter pylori (Hp) is directly linked to the pathogen's ability to glycosylate proteins; for example, Hp flagellin proteins are heavily glycosylated with the unusual nine-carbon sugar pseudaminic acid, and this modification is absolutely essential for Hp to synthesize functional flagella and colonize the host's stomach. Although Hp's glycans are linked to pathogenesis, Hp's glycome remains poorly understood; only the two flagellin glycoproteins have been firmly characterized in Hp. Evidence from our laboratory suggests that Hp synthesizes a large number of as-yet unidentified glycoproteins. Here we set out to discover Hp's glycoproteins by coupling glycan metabolic labeling with mass spectrometry analysis. An assessment of the subcellular distribution of azide-labeled proteins by Western blot analysis indicated that glycoproteins are present throughout Hp and may therefore serve diverse functions. To identify these species, Hp's azide-labeled glycoproteins were tagged via Staudinger ligation, enriched by tandem affinity chromatography, and analyzed by multidimensional protein identification technology. Direct comparison of enriched azide-labeled glycoproteins with a mock-enriched control by both SDS-PAGE and mass spectrometry-based analyses confirmed the selective enrichment of azide-labeled glycoproteins. We identified 125 candidate glycoproteins with diverse biological functions, including those linked with pathogenesis. Mass spectrometry analyses of enriched azide-labeled glycoproteins before and after cleavage of O-linked glycans revealed the presence of Staudinger ligation-glycan adducts in samples only after beta-elimination, confirming the synthesis of O-linked glycoproteins in Hp. Finally, the secreted colonization factors urease alpha and urease beta were biochemically validated as glycosylated proteins via Western blot analysis as well as by mass spectrometry analysis of cleaved glycan products. These data set the stage for the development of glycosylation-based therapeutic strategies, such as new vaccines based on natively glycosylated Hp proteins, to eradicate Hp infection. Broadly, this report validates metabolic labeling as an effective and efficient approach for the identification of bacterial glycoproteins.

The substitution of methionines with leucines within the interior of a protein is expected to increase stability both because of a more favorable solvent transfer term as well as the reduced entropic cost of holding a leucine side chain in a defined position. Together, these two terms are expected to contribute about 1.4 kcal/mol to protein stability for each Met + Leu substitution when fully buried. At the same time, this expected beneficial effect may be offset by steric factors due to differences in the shape of leucine and methionine. To investigate the interplay between these factors, all methionines in T4 lysozyme except at the amino-terminus were individually replaced with leucine. Of these mutants, M106L and M120L have stabilities 0.5 kcal/mol higher than wild-type T4 lysozyme, while M6L is significantly destabilized (-2.8 kcal/mol). M102L, described previously, is also destabilized (-0.9 kcal/mol). Based on this limited sample it appears that methionine-to-leucine substitutions can increase protein stability but only in a situation where the methionine side chain is fully or partially buried, yet allows the introduction of the leucine without concomitant steric interference. The variants, together with methionine-to-lysine substitutions at the same sites, follow the general pattern that substitutions at rigid, internal sites tend to be most destabilizing, whereas replacements at more solvent-exposed sites are better tolerated.