The effect of a room temperature ionic liquid (RTIL) on the conformational dynamics of a protein, human serum albumin (HSA), is studied by fluorescence correlation spectroscopy (FCS). For this, the protein was covalently labeled by a fluorophore, 7-dimethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). On addition of a RTIL ([pmim][Br]) to the native protein, the diffusion coefficient (D(t)) decreases and the hydrodynamic radius (R(h)) increases. This suggests that the RTIL ([pmim][Br]) acts as a denaturant when the protein is in the native state. However, addition of [pmim][Br] to a protein denatured by GdnHCl causes an increases in D(t) and decrease in R(h). This suggests that in the presence of GdnHCl addition of RTIL helps the protein to refold. In the native state, the conformational dynamics of protein is described by three distinct time constants: ~3.6 ± 0.7, ~29 ± 4.5, and 133 ± 23 μs. The faster components (~3.6 ± 0.7 and ~29 ± 4.5 μs) are ascribed to chain dynamics of the protein, while the slowest component (133 μs) is responsible for interchain interaction or concerted motion. On addition of [pmim][Br], the conformational dynamics of HSA becomes slower (~5.1 ± 1, ~43.5 ± 2.8, and ~311 ± 2.3 μs in the presence of 1.5 M [pmim][Br]). The time constants for the protein denatured by 6 M GdnHCl are 3.2 ± 0.4, 34 ± 6, and 207 ± 38 μs. When 1.5 M [pmim][Br] is added to the denatured protein (in 6 M GdnHCl), the time constants become ~5 ± 1, ~41 ± 10, and ~230 ± 45 μs. The lifetime histogram shows that, on addition of GdnHCl to HSA, the contribution of the shorter lifetime component decreases and vanishes at 6 M GdnHCl. The shorter lifetime component immediately reappears after addition of RTIL to unfolded HSA. This suggests recoiling of the unfolded protein by RTIL.

There are various conflicting hypotheses regarding the origins of the tribal groups of India, who belong to three major language groups--Austro-Asiatic, Dravidian and Tibeto-Burman. To test some of the major hypotheses we designed a genetic study in which we sampled tribal populations belonging to all the three language groups. We used a set of autosomal DNA markers, mtDNA restriction-site polymorphisms (RSPs) and mtDNA hypervariable segment-1 (HVS-1) sequence polymorphisms in this study. Using the unlinked autosomal markers we found that there is a fair correspondence between linguistic and genomic affinities among the Indian tribal groups. We reconstructed mtDNA RSP haplotypes and found that there is extensive haplotype sharing among all tribal populations. However, there is very little sharing of mtDNA HVS-1 sequences across populations, and none across language groups. Haplogroup M is ubiquitous, and the subcluster U2i of haplogroup U occurs in a high frequency. Our analyses of haplogroup and HVS-1 sequence data provides evidence in support of the hypothesis that the Austro-Asiatic speakers are the most ancient inhabitants of India. Our data also support the earlier finding that some of the western Eurasian haplogroups found in India may have been present in India prior to the entry of Aryan speakers. However, we do not find compelling evidence to support the theory that haplogroup M was brought into India on an "out of Africa" wave of migration through a southern exit route from Ethiopia. On the contrary, our data raise the possibility that this haplogroup arose in India and was later carried to East Africa from India.

Protein synthesis has an overall error rate of approximately 10 −4 for each mRNA codon translated. The fidelity of translation is mainly determined by two events: synthesis of cognate amino acid:tRNA pairs by aminoacyl-tRNA synthetases (aaRSs) and accurate selection of aminoacyl-tRNAs (aa-tRNAs) by the ribosome. To ensure faithful aa-tRNA synthesis, many aaRSs employ a proofreading ("editing") activity, such as phenylalanyl-tRNA synthetases (PheRS) that hydrolyze mischarged Tyr-tRNA Phe . Eukaryotes maintain two distinct PheRS enzymes, a cytoplasmic (ctPheRS) and an organellar form. CtPheRS is similar to bacterial enzymes in that it consists of a heterotetramer in which the α-subunits contain the active site and the β-subunits harbor the editing site. In contrast, mitochondrial PheRS (mtPheRS) is an α-subunit monomer that does not edit Tyr-tRNA Phe , and a comparable transacting activity does not exist in organelles. Although mtPheRS does not edit, it is extremely specific as only one Tyr-tRNA Phe is synthesized for every ∼7; 300 Phe-tRNA Phe , compatible with an error rate in translation of ∼10 −4 . When the error rate of mtPheRS was increased 17-fold, the corresponding strain could not grow on respiratory media and the mitochondrial genome was rapidly lost. In contrast, error-prone mtPheRS, editing-deficient ctPheRS, and their wildtype counterparts all supported cytoplasmic protein synthesis and cell growth. These striking differences reveal unexpectedly divergent requirements for quality control in different cell compartments and suggest that the limits of translational accuracy may be largely determined by cellular physiology.aminoacyl-tRNA synthetase | protein synthesis | tRNA

Elevated homocysteine (Hcy) levels are observed in two apparently unrelated diseases: neural-tube defects (NTD) and premature vascular disease. Defective human methionine synthase (MS) could result in elevated Hcy levels. We sequenced the coding region of MS in 8 hyperhomocysteinaemic patients (4 NTD patients and 4 patients with pregnancies complicated by spiral arterial disease, SAD). We identified only one mutation resulting in an amino acid substitution: an A-->G transition at bp 2756, converting an aspartic acid (D919) into a glycine (G). We screened genomic DNA for the presence of this mutation in 56 NTD patients, 69 mothers of children with NTD, 108 SAD patients and 364 controls. There was no increased prevalence of the GG and AG genotypes in NTD patients, their mothers or SAD patients. The D919G mutation does not seem to be a risk factor for NTD or vascular disease. We then examined the mean Hcy levels for each MS genotype. There was no correlation between GG- or AG-genotype and Hcy levels. The D919G mutation is thus a fairly prevalent, and probably benign polymorphism. This study, though limited, provides no evidence for a major involvement of MS in the aetiology of homocysteine-related diseases such as NTD or vascular disease.

4-(N-bromoacetylamino)-phthalimide (I) is used as a new solvation probe for protein and microemulsions. The photophysics of the probe 4-(N-bromoacetylamino)-phthalimide (I) is dramatically different from that of the parent compound, 4-aminophthalimide (4-AP). The solvation dynamics of I in an AOT microemulsion is similar to that of 4-AP in microemulsions. Solvation dynamics in the vicinity of a protein glutaminyl-tRNA synthetase (GlnRS) is studied by covalently attaching I to the protein. The solvation dynamics of the proteinbound probe is described by a very fast component of 40 ps and another of 580 ps.

UDP-glucuronosyltransferase (UGT) isozymes catalyze detoxification of numerous chemical toxins present in our daily diet and environment by conjugation to glucuronic acid. The special properties and enzymatic mechanism(s) that enable endoplasmic reticulum-bound UGT isozymes to convert innumerable structurally diverse lipophiles to excretable glucuronides are unknown. Inhibition of cellular UGT1A7 and UGT1A10 activities and of [ 33 P]orthophosphate incorporation into immunoprecipitable proteins after exposure to curcumin or calphostin-C indicated that the isozymes are phosphorylated. Furthermore, inhibition of UGT phosphorylation and activity by treatment with PKC-specific inhibitor peptide supported PKC involvement. Coimmunoprecipitation, colocalization by means of immunofluorescence, and crosslinking studies of PKC and UGT1A7His revealed that the proteins reside within 11.4 Å of each other. Moreover, mutation of three PKC sites in each UGT isozyme demonstrated that T73A͞G and T202A͞G caused null activity, whereas S432G-UGT1A7 caused a major shift of its pH-8.5 optimum to 6.4 with new substrate selections, including 17␤-estradiol. S432G-UGT1A10 exhibited a minor pH shift without substrate alterations. PKC involvement was confirmed by the demonstration that PKC overexpression enhanced activity of UGT1A7 but not of its S432 mutant and the conversion of 17␤-[ 14 C]estradiol by S432G-UGT1A7 but not by UGT1A7. Consistent with these observations, treatment of UGT1A7-transfected cells with PKC-specific inhibitor peptide or general PKC inhibitors increased 17␤-estradiol catalysis between 5-and 11-fold, with parallel decreases in phosphoserine-432. Here, we report a mechanism involving PKC-mediated phosphorylation of UGT such that phosphoserine͞threonine regulates substrate specificity in response to chemical exposures, which possibly confers survival benefit.PKC ͉ PKC inhibitors ͉ detoxifying enzymes ͉ UDP-glucuronosyltransferase mutants ͉ immunofluorescence M ammalian UDP-glucuronosyltransferase (UGT) isozymes detoxify numerous lipid-soluble chemicals, including metabolites, such as neurotoxic bilirubin, as well as dietary and environmental agents͞toxins and medicines (1). UGT facilitates detoxification of chemicals by attaching glucuronic acid molecules to form water-soluble, readily excretable glucuronides. Failure to attach glucuronic acid (glucuronidation) to toxins allows high levels to accumulate in tissues, causing deleterious effects, including mutations of genes and the development of cancer (2). Mutations in UGT1 genes lead to lethal hyperbilirubinemic Crigler-Najjar disease (3) and to toxic tissue levels of the commonly used analgesic acetaminophen (Tylenol) in rats (4). The fact that a limited number of UGT isozymes facilitate excretion of a vast number of structurally diverse chemicals suggests a mechanism evolved to confer flexibility on an isozyme to metabolize multiple toxins. Because UGTs are bound to membranes of the endoplasmic reticulum (ER) that cause difficulties associated with purification and s...

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Solvation dynamics in the molten globule state of a protein, glutaminyl-tRNA synthetase (GlnRS), has been studied using both a noncovalent probe (bis-ANS) and a covalent probe 4-(N-thioacetylamino)-phthalimide. In the native state of GlnRS, bis-ANS exhibits an average solvation time (〈τ S 〉) of 1400 ps, which is 12 times longer than that for the covalent probe (120 ps). The difference in the solvation times for the two probes in the native state of the protein is ascribed to different locations of the probes. The covalent probe resides close to the protein surface and experiences fast relaxation of the water molecules. The noncovalent probe penetrates deeper inside the protein and displays slower relaxation in the buried region. In the molten globule state, 〈τ S 〉 is 400 ps for the noncovalent probe and 250 ps for the covalent probe. Evidently, in the molten globule state, 〈τ S 〉 is much longer than that the longest component of the solvation dynamics (∼1 ps) in bulk water. This shows that, in the compact molten globule state, the protein retains considerable residual structure.