Calcium binding to the tubulin-colchicine complex in the state of GTP in a BES buffer

Magnesium and calcium binding were assayed using the tubulin-colchicine complex in a BES buffer, in which the calcium binding to tubulin had been measured by Grisham et al. (Fed. Proc. 39, 2162). In the previous paper, an imidazole buffer was used as the buffer which does not chelate to calcium and which is substituted to phosphate buffer. The result of calcium binding measurement indicated the same binding constant between at pH 7.0 and at pH 6.5 in the absence of magnesium (1.08×10^ M at pH 7.0 and 1.10×10^ M at pH 6.5). Also, the calcium binding constant of the tubulin-colchicine complex was the same as that of tubulin in a BES buffer, pH 7.0. The increase of magnesium concentration inhibited calcium binding to the tubulin-colchicine complex. The affinity of calcium at pH 7.0 was lower than that at pH 6.5 in the presence of magnesium. This leads the effect of calcium on the polymerization of the tubulin-colchicine complex is small at pH 6.5 rather than at pH 7.0. The results obtained in the polymerization experiment were in good agreement with the results of the calcium binding experiment

Three Stages of Lysozyme Thermal Stabilization by High and Medium Charge Density Anions

Addition of high and medium charge density anions (phosphate, sulfate, and chloride) to lysozyme in pure water demonstrates three stages for stabilization of the protein structure. The first two stages have a minor impact on lysozyme stability and are probably associated with direct interaction of the ions with charged and partial charges on the protein’s surface. There is a clear transition between the second and third stages; in the case of sodium chloride, disodium sulfate and disodium hydrogen phosphate this is at 550, 210, and 120 mM, respectively. Stabilization of lysozyme can be explained by the free energy required to hydrate the protein as it unfolds. At low ion concentrations, the protein’s hydration layer is at equilibrium with the bulk water. After the transition, bulk water is depleted and the protein is competing for water with the ions. With competition for water between the protein and the ions at higher salt concentrations, the free energy required to hydrate the interior of the protein rises and it is this that stabilizes the protein structure

Allosteric Modulators of Steroid Hormone Receptors : Structural Dynamics and Gene Regulation

Peer reviewedPublisher PD

Circular dichroism of the “random” polypeptide chain

The circular dichroism (CD) spectrum of an unordered polypeptide chain does not correspond, as has been assumed heretofore, to that of a charged chain such as poly- L -glutamic acid or poly- L -lysine. The latter have been shown to have locally ordered structures with characteristic CD spectra. We have now obtained CD spectra of the unordered forms of the above synthetic, polypeptides, as well as of two fibrous proteins (collagen and feather keratin) and a globular protein (myoglobin). These spectra are all similar to that of unordered polyproline, having a negative band in the vicinity of 2000 mΜ and no additional bands at longer wavelengths. The lack of structural uniqueness of the unordered polypeptide chain is emphasized by these studies.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/37825/1/360080306_ftp.pd

Predicting Arabidopsis Freezing Tolerance and Heterosis in Freezing Tolerance from Metabolite Composition

Heterosis, or hybrid vigor, is one of the most important tools in plant breeding and has previously been demonstrated for plant freezing tolerance. Freezing tolerance is an important trait because it can limit the geographical distribution of plants and their agricultural yield. Plants from temperate climates increase in freezing tolerance during exposure to low, non-freezing temperatures in a process termed ‘cold acclimation’. Metabolite profiling has indicated a major reprogramming of plant metabolism in the cold, but it has remained unclear in previous studies which of these changes are related to freezing tolerance. In the present study, we have used metabolic profiling to discover combinations of metabolites that predict freezing tolerance and its heterosis in Arabidopsis thaliana. We identified compatible solutes and, in particular, the pathway leading to raffinose as crucial statistical predictors for freezing tolerance and its heterosis, while some TCA cycle intermediates contribute only to predicting the heterotic phenotype. This indicates coordinate links between heterosis and metabolic pathways, suggesting that a limited number of regulatory genes may determine the extent of heterosis in this complex trait. In addition, several unidentified metabolites strongly contributed to the prediction of both freezing tolerance and its heterosis and we present an exemplary analysis of one of these, identifying it as a hexose conjugate

The structure of a PII signaling protein from a halophilic archaeon reveals novel traits and high-salt adaptations

To obtain insights into archaeal nitrogen signaling and haloadaptation of the nitrogen/carbon/energy-signaling protein PII, we determined crystal structures of recombinantly produced GlnK2 from the extreme halophilic archaeon Haloferax mediterranei, complexed with AMP or with the PII effectors ADP or ATP, at respective resolutions of 1.49 Å, 1.45 Å, and 2.60 Å. A unique trait of these structures was a three-tongued crown protruding from the trimer body convex side, formed by an 11-residue, N-terminal, highly acidic extension that is absent from structurally studied PII proteins. This extension substantially contributed to the very low pI value, which is a haloadaptive trait of H. mediterranei GlnK2, and participated in hexamer-forming contacts in one crystal. Similar acidic N-extensions are shown here to be common among PII proteins from halophilic organisms. Additional haloadaptive traits prominently represented in H. mediterranei GlnK2 are a very high ratio of small residues to large hydrophobic aliphatic residues, and the highest ratio of polar to nonpolar exposed surface for any structurally characterized PII protein. The presence of a dense hydration layer in the region between the three T-loops might also be a haloadaptation. Other unique findings revealed by the GlnK2 structure that might have functional relevance are: the adoption by its T-loop of a three-turn α-helical conformation, perhaps related to the ability of GlnK2 to directly interact with glutamine synthetase; and the firm binding of AMP, confirmed by biochemical binding studies with ATP, ADP, and AMP, raising the possibility that AMP could be an important PII effector, at least in archaea.This work was supported by grants from the Spanish government (BFU2011-30407 and BIO2008_00082, to V. Rubio and M. J. Bonete, respectively) and from the Valencian government (Prometeo 2009/51 to V. Rubio). C. Palanca is a JAE-Predoc fellow of the CSIC and, during this work, L. Pedro-Roig was a FPU fellow of the Spanish Ministry of Education. The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007–2013) under BioStruct-X (grant agreement No. 283570)

The Effect of Osmolytes on Protein Fibrillation

Osmolytes are small molecules that are exploited by cells as a protective system against stress conditions. They favour compact protein states which makes them stabilize globular proteins in vitro and promote folding. Conversely, this preference for compact states promotes aggregation of unstructured proteins. Here we combine a brief review of the effect of osmolytes on protein fibrillation with a report of the effect of osmolytes on the unstructured peptide hormone glucagon. Our results show that osmolytes either accelerate the fibrillation kinetics or leave them unaffected, with the exception of the osmolyte taurine. Furthermore, the osmolytes that affected the shape of the fibrillation time profile led to fibrils with different structure as revealed by CD. The structural changes induced by Pro, Ser and choline-O-sulfate could be due to specific osmolytes binding to the peptides, stabilizing an otherwise labile fibrillation intermediate