Interactions of amidated acids with heparin

Raman and NMR studies are performed to characterize the solution structures of complexes between heparin and a group of amidated acids, which act as delivery agents that facilitate the gastrointestinal absorption of orally administered heparin. At concentrations typically employed for the oral drug delivery of heparin, the contact points between heparin complexed with the delivery agents include points near the OH groups of heparin. The results suggest that heparin interacts rather nonspecifically with the amidated acids as monomers and with self-associated complexes of the delivery agents. It is also found that the carboxyl groups of at least one of the bioactive delivery agents easily protonates when it forms complexes with itself or heparin. This attribute may be one reason why this class of compounds is effective in the oral delivery of heparin.     

Hydrogen bond interactions of G proteins with the guanine ring moiety of guanine nucleotides.

We have utilized Raman difference spectroscopy to investigate hydrogen bonding interactions of the guanine moiety in guanine nucleotides with the binding site of two G proteins, EF-Tu (elongation factor Tu from Escherichia coli) and the c-Harvey ras protein, p21 (the gene product of the human c-H-ras proto-oncogene). Raman spectra of proteins complexed with GDP (guanosine 5' diphosphate), IDP (inosine 5' diphosphate), 6-thio-GDP, and 6-18O-GDP were measured, and the various difference spectra were determined. These were compared to the difference spectra obtained in solution, revealing vibrational features of the nucleotide that are altered upon binding. Specifically, we observed significant frequency shifts in the vibrational modes associated with the 6-keto and 2-amino positions of the guanine group of GDP and IDP that result from hydrogen bonding interactions between these groups and the two proteins. These shifts are interpreted as being proportional to the local energy of interaction (delta H) between the two groups and protein residues at the nucleotide binding site. Consistent with the tight binding between the nucleotides and the two proteins, the shifts indicate that the enthalpic interactions are stronger between these two polar groups and protein than with water. In general, the spectral shifts provide a rationale for the stronger binding of GDP and IDP with p21 compared to EF-Tu. Despite the structural similarity of the binding sites of EF-Tu and p21, the strengths of the observed hydrogen bonds at the 6-keto and 2-amino positions vary substantially, by up to a factor of 2.(ABSTRACT TRUNCATED AT 250 WORDS)     

A microsatellite linkage map of Drosophila mojavensis

Background

Drosophila mojavensishas been a model system for genetic studies of ecological adaptation and speciation. However, despite its use for over half a century, no linkage map has been produced for this species or its close relatives.

Results

We have developed and mapped 90 microsatellites in D. mojavensis, and we present a detailed recombinational linkage map of 34 of these microsatellites. A slight excess of repetitive sequence was observed on the X-chromosome relative to the autosomes, and the linkage groups have a greater recombinational length than the homologous D. melanogaster chromosome arms. We also confirmed the conservation of Muller's elements in 23 sequences between D. melanogaster and D. mojavensis.

Conclusions

The microsatellite primer sequences and localizations are presented here and made available to the public. This map will facilitate future quantitative trait locus mapping studies of phenotypes involved in adaptation or reproductive isolation using this species.     

Characterization of the genetic diversity of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> in São Paulo city,Brazil

Background

Tuberculosis is a major health problem in São Paulo, Brazil, which is the most populous and one of the most cosmopolitan cities in South America. To characterize the genetic diversity of Mycobacterium tuberculosis in the population of this city, the genotyping techniques of spoligotyping and MIRU were applied to 93 isolates collected in two consecutive years from 93 different tuberculosis patients residing in São Paulo city and attending the Clemente Ferreira Institute (the reference clinic for the treatment of tuberculosis).

Findings

Spoligotyping generated 53 different spoligotype patterns. Fifty-one isolates (54.8%) were grouped into 13 spoligotyping clusters. Seventy- two strains (77.4%) showed spoligotypes described in the international databases (SpolDB4, SITVIT), and 21 (22.6%) showed unidentified patterns. The most frequent spoligotype families were Latin American Mediterranean (LAM) (26 isolates), followed by the T family (24 isolates) and Haarlem (H) (11 isolates), which together accounted for 65.4% of all the isolates. These three families represent the major genotypes found in Africa, Central America, South America and Europe. Six Spoligo-International-types (designated SITs by the database) comprised 51.8% (37/72) of all the identified spoligotypes (SIT53, SIT50, SIT42, SIT60, SIT17 and SIT1). Other SITs found in this study indicated the great genetic diversity of M. tuberculosis, reflecting the remarkable ethnic diversity of São Paulo city inhabitants. The MIRU technique was more discriminatory and did not identify any genetic clusters with 100% similarity among the 93 isolates. The allelic analysis showed that MIRU loci 26, 40, 23 and 10 were the most discriminatory. When MIRU and spoligotyping techniques were combined, all isolates grouped in the 13 spoligotyping clusters were separated.

Conclusions

Our data indicated the genomic stability of over 50% of spoligotypes identified in São Paulo and the great genetic diversity of M. tuberculosis isolates in the remaining SITs, reflecting the large ethnic mix of the São Paulo city inhabitants. The results also indicated that in this city, M. tuberculosis isolates acquired drug resistance independently of genotype and that resistance was more dependent on the selective pressure of treatment failure and the environmental circumstances of patients.

     

Is the PTPase-vanadate complex a true transition state analogue?

Vanadate can often bind to phosphoryl transfer enzymes to form a trigonal-bipyramidal structure at the active site. The enzyme-vanadate dissociation constants in these enzymes are much lower than those for phosphate. Therefore, enzyme-bound vanadate moieties are often considered as transition state analogues. To test whether the enzyme-vanadate complex is a true transition state analogue beyond the simple geometry and binding affinity arguments and whether the bond orders of the VO bonds in the complex approach those of the PO bonds in the transition state, the binding properties of vanadate in the Yersinia protein-tyrosine phosphatase (PTPase) and its T410A, D356N, W354A, R409K, and D356A mutants have been studied by steady-state kinetic measurements and by difference Raman measurements. The results of the kinetic measurements show no correlation between K(I) and kcat or kcat/K(m) in these mutants. In addition, our analysis of the Raman data shows that the bond order change of the nonbridging V--O bonds in the vanadate complexes does not correlate with the kinetic parameters in a number of PTPase variants as predicted by the transition state binding paradigm. Furthermore, the ionization state of the bound vanadate moiety is not invariant across the PTPase variants studied, and the average bond order of the nonbridging V--O bonds decreased by 0.06-0.07 valence unit in the wild type and all of the mutant PTPases, either in dianionic or in monoanionic form. Thus the complex would resemble an associative transition state, contrary to the previously determined dissociative structure of the transition state. Therefore, it is concluded that vanadate is not a true transition state analogue for the PTPase reactions.     

Mek1 Down Regulates Rad51 Activity during Yeast Meiosis by Phosphorylation of Hed1

During meiosis, programmed double strand breaks (DSBs) are repaired preferentially between homologs to generate crossovers that promote proper chromosome segregation at Meiosis I. In many organisms, there are two strand exchange proteins, Rad51 and the meiosis-specific Dmc1, required for interhomolog (IH) bias. This bias requires the presence, but not the strand exchange activity of Rad51, while Dmc1 is responsible for the bulk of meiotic recombination. How these activities are regulated is less well established. In dmc1Δ mutants, Rad51 is actively inhibited, thereby resulting in prophase arrest due to unrepaired DSBs triggering the meiotic recombination checkpoint. This inhibition is dependent upon the meiosis-specific kinase Mek1 and occurs through two different mechanisms that prevent complex formation with the Rad51 accessory factor Rad54: (i) phosphorylation of Rad54 by Mek1 and (ii) binding of Rad51 by the meiosis-specific protein Hed1. An open question has been why inhibition of Mek1 affects Hed1 repression of Rad51. This work shows that Hed1 is a direct substrate of Mek1. Phosphorylation of Hed1 at threonine 40 helps suppress Rad51 activity in dmc1Δ mutants by promoting Hed1 protein stability. Rad51-mediated recombination occurring in the absence of Hed1 phosphorylation results in a significant increase in non-exchange chromosomes despite wild-type levels of crossovers, confirming previous results indicating a defect in crossover assurance. We propose that Rad51 function in meiosis is regulated in part by the coordinated phosphorylation of Rad54 and Hed1 by Mek1.     

Active site loop motion in triosephosphate isomerase: T-jump relaxation spectroscopy of thermal activation

As for many enzymes, the enzymatic pathway of triosephosphate isomerase (TIM) includes the partially rate determining motion of an active site loop (loop 6, residues 166-176), which must remain closed during chemistry but must open just before product release. The motion of this loop was monitored using laser induced temperature-jump relaxation spectroscopy at nanosecond to millisecond time resolution. Trp168 in the hinge of the mobile loop served as a fluorophore reporter in a mutant of the yeast enzyme. The opening rate was studied as a function of the concentration of glycerol 3-phosphate, a substrate surrogate. Monoexponential kinetics were observed; assuming a simple two-step ligand release mechanism involving an encounter complex intermediate, the time scales of loop opening and closing were derived. The opening rate of the loop at 25 degrees C was determined to be 2500 +/- 1000 s(-1), in remarkable agreement with solution and solid state NMR measurements. The closing rate at the same temperature was 46,700 +/- 1800 s(-1). The rates were also studied as a function of the sample temperature following the jump. Enthalpies of activation of the loop motion, DeltaH(close) and DeltaH(open), were estimated to be 13.8 and 14.1 kcal/mol, respectively. The enthalpy of dissociation estimated from the kinetic studies is in reasonable agreement with steady-state values. Moreover, the enthalpy was dissected, for the first time, into components associated with ion binding and with protein conformational change. The enthalpy of the release reaction appeared to have a substantial contribution from the dissociation of the ligand from the encounter complex, found to be endothermic at 6 kcal/mol. In contrast, the population ratio of the open to closed loop conformations is found to favor the closed conformation but to be substantially less temperature dependent than the release step. Preliminary data of other ligands show that G3P behavior resembles that of the substrate but differs from 2-phosphoglycolate, a tight binding inhibitor, and phosphate. This study represents one of the first detailed comparisons between NMR and fluorescence based probes of protein motion and results in good agreement between the methods. The data in aggregate support a model in which the rate of the loop opening for TIM is dependent on the ligand and results in opening rates in the presence of the product that are comparable to enzymatic throughput, kcat.