Molecular conformation of gonadoliberin using two-dimensional NMR spectroscopy

Complete resonance assignments of the proton NMR spectrum of gonadoliberin (in its native amide and free acid forms) have been obtained using two-dimensional nuclear magnetic resonance spectroscopy under three different environmental conditions, namely, dimethyl sulphoxide solution, aqueous solution and lipid-bound form in model membranes. The proton chemical shifts in the three cases have been compared to derive information about inherent conformational characteristics of the molecule. It has been inferred that the molecule possesses no short-range or long-range order under any of the three solvent conditions. However, there is a nonspecific increase in the linewidths when gonadoliberin is bound to model membranes, indicating a reduced internal motion in the molecule due to lipid-peptide interactions

Site-specific fluorescence dynamics in an RNA 'thermometer' reveals the role of ribosome binding in its temperature-sensitive switch function

RNA thermometers control the translation of several heat shock and virulence genes by their temperature-sensitive structural transitions. Changes in the structure and dynamics of MiniROSE RNA, which regulates translation in the temperature range of 20–45°C, were studied by site specifically replacing seven adenine residues with the fluorescent analog, 2-aminopurine (2-AP), one at a time. Dynamic fluorescence observables of 2-AP-labeled RNAs were compared in their free versus ribosome-bound states for the first time. Noticeably, position dependence of fluorescence observables, which was prominent at 20°C, was persistent even at 45°C, suggesting the persistence of structural integrity up to 45°C. Interestingly, position-dependent dispersion of fluorescence lifetime and quenching constant at 45°C was ablated in ribosome-bound state, when compared to those at 20°C, underscoring loss of structural integrity at 45°C, in ribosome-bound RNA. Significant increase in the value of mean lifetime for 2-AP corresponding to Shine–Dalgarno sequences, when the temperature was raised from 20 to 45°C, to values seen in the presence of urea at 45°C was a strong indicator of melting of the 3D structure of MiniROSE RNA at 45°C, only when it was ribosome bound. Taken all together, we propose a model where we invoke that ribosome binding of the RNA thermometer critically regulates temperature sensing functions in MiniROSE RNA

Structural characterization of a novel KH-domain containing plant chloroplast endonuclease

Chlamydomonas reinhardtii is a single celled alga that undergoes apoptosis in response to UV-C irradiation. UVI31+, a novel UV-inducible DNA endonuclease in C. reinhardtii, which normally localizes near cell wall and pyrenoid regions, gets redistributed into punctate foci within the whole chloroplast, away from the pyrenoid, upon UV-stress. Solution NMR structure of the first putative UV inducible endonuclease UVI31+ revealed an alpha(1)-beta(1)-beta(2)-alpha(2)-alpha(3)-beta(3) fold similar to BolA and type II KH-domain ubiquitous protein families. Three alpha-helices of UVI31+ constitute one side of the protein surface, which are packed to the other side, made of three-stranded beta-sheet, with intervening hydrophobic residues. A twenty-three residues long polypeptide stretch (D54-H76) connecting beta(1) and beta(2) strands is found to be highly flexible. Interestingly, UVI31+ recognizes the DNA primarily through its beta-sheet. We propose that the catalytic triad residues involving Ser114, His95 and Thr116 facilitate DNA endonuclease activity of UVI31+. Further, decreased endonuclease activity of the S114A mutant is consistent with the direct participation of Ser114 in the catalysis. This study provides the first structural description of a plant chloroplast endonuclease that is regulated by UV-stress response

¹H, ¹³C and ¹⁵N NMR assignments of a mutant of UV inducible transcript (S55A-UVI31+) from Chlamydomonas reinhardtii

Almost complete sequence specific 1H, 13C and 15N resonance assignments of a mutant of UV inducible transcript (S55A-UVI31+) from Chlamydomonas reinhardtii are reported, as a prelude to its structural and functional characterization. Site-directed mutagenesis of uvi31+ was carried out using complementary mutation harbouring oligonucleotides for S55A mutant. The resulting mutant was sequenced and then S55A mutant of uvi31+ cDNA were expressed in E. coli, and purification were carried out from where the protein was purified to high homogeneity. The point mutation S55A in UVI31+ results in a significant enhancement in its DNA endonuclease activity as compared to its wild type protein

Overexpression and purification of isotopically labeled Escherichia coli MutH for NMR studies

MutH is one of the enzymes involved in the methyl directed -GATC-based DNA repair system. We report a significantly optimized protocol to prepare isotopically (15N and/or 13C) labeled MutH in minimal medium with high yields for NMR studies. Under the various conditions that we have standardized for the affinity purification of His(6) MutH, the yield of the purified MutH has been estimated to be 35-40 mg of protein from 1liter of M9 minimal media. The yield, thus, obtained by this method is significantly higher than those of previously reported methods. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy analysis revealed that the protein was pure and existed essentially in a monomeric form. Uniformly 15N-labeled protein, thus, produced has been characterized by recording a sensitivity enhanced 2D [15N]-[1H] HSQC spectrum. The dispersion seen in 15N-1H cross-peaks indicates the presence of a well-ordered structure for MutH and proper folding of the purified protein. The spectrum confirms further the existence of MutH as a monomer

Klenow exo^-, as opposed to exo⁺, traverses through G–G:C triplex by melting G–G base pairs

G–G base-paired hairpin DNA structures on template strands offer potential "road-blocks" to a traversing polymerase. Klenow polymerase (exo+) pauses while replicating through G–G base-paired hairpin DNA due to the generation of G–G:C triplex. However, exonuclease-deficient Klenow traverses through de novo generated G–G:C triplexes leading to full-length C:G duplexes. Alleviation of such road-blocks by exo- Klenow ensues faster at lower Mg2+, a kinetic effect consistent with the role of Mg2+ in stabilizing G–G:C triplex fold. The ability of exonuclease-deficient polymerase to go past the de novo generated G–G:C triplexes suggests that the "idling" of exo+ polymerase at G–G road-block is due to the reiterative polymerase/exonuclease action. The full-length replication product carrying a Cn–Gn duplex at one end is further "expanded" by exo- Klenow through C-strand "slippage" leading to the generation of C+–G:C triplex, which is exemplified by the premature arrest of the same at low pH that further stabilizes the C+–G:C triplex

Complete backbone assignment of a Ca<SUP>2+</SUP>-binding protein of the βγ-crystallin superfamily from Methanosarcina acetivorans, at two denaturant concentrations

We report here almost complete backbone assignment of a Ca2+-binding protein of the &#946;&#947;-crystallin superfamily from Methanosarcina acetivorans, at two denaturant (GdmCl) concentrations, using double and triple resonance experiments. These NMR assignments will be useful to understand the unfolding path of this protein

¹H, ¹³C and ¹⁵N NMR assignments of Mg²⁺ bound form of UV inducible transcript protein (UVI31+) from Chlamydomonas reinhardtii

Almost complete sequence specific ¹H, ¹³C and ¹⁵N resonance assignments of Mg²⁺ bound form of UV inducible transcript protein (UVI31+) from Chlamydomonas reinhardtii are reported, as a prelude to its structural and functional characterization

NMR assignment of M-crystallin: a novel Ca<SUP>2+</SUP> binding protein of the βγ-crystallin superfamily from Methanosarcina acetivorans

This article does not have an abstract

Structure of Ca2+-binding protein-6 from Entamoeba histolytica and its involvement in trophozoite proliferation regulation.

Cell cycle of Entamoeba histolytica, the etiological agent of amoebiasis, follows a novel pathway, which includes nuclear division without the nuclear membrane disassembly. We report a nuclear localized Ca2+-binding protein from E. histolytica (abbreviated hereafter as EhCaBP6), which is associated with microtubules. We determined the 3D solution NMR structure of EhCaBP6, and identified one unusual, one canonical and two non-canonical cryptic EF-hand motifs. The cryptic EF-II and EF-IV pair with the Ca2+-binding EF-I and EF-III, respectively, to form a two-domain structure similar to Calmodulin and Centrin proteins. Downregulation of EhCaBP6 affects cell proliferation by causing delays in transition from G1 to S phase, and inhibition of DNA synthesis and cytokinesis. We also demonstrate that EhCaBP6 modulates microtubule dynamics by increasing the rate of tubulin polymerization. Our results, including structural inferences, suggest that EhCaBP6 is an unusual CaBP involved in regulating cell proliferation in E. histolytica similar to nuclear Calmodulin