Identification of Specific Effect of Chloride on the Spectral Properties and Structural Stability of Multiple Extracellular Glutamic Acid Mutants of Bacteriorhodopsin.

In the present work we combine spectroscopic, DSC and computational approaches to examine the multiple extracellular Glu mutants E204Q/E194Q, E204Q/E194Q/E9Q and E204Q/E194Q/E9Q/E74Q of bacteriorhodopsin by varying solvent ionic strength and composition. Absorption spectroscopy data reveal that the absorption maxima of multiple EC Glu mutants can be tuned by the chloride concentration in the solution. Visible Circular dichroism spectra imply that the specific binding of Cl- can modulate weakened exciton chromophore coupling and reestablish wild type-like bilobe spectral features of the mutants.

Analysis of gene expression for studying tumor progression: the case of glucocorticoid administration.

Background: Glucocorticoids are commonly used as adjuvant treatment for side-effects and have anti-proliferative activity in several tumors but, on the other hand, their proliferative effect has been reported in several studies, some of them involving the spread of cancer. We shall attempt to reconcile these incongruities from the genomic and tissue-physiology perspectives with our findings.

Methods: An accurate phenotype analysis of microarray data can help to solve multiple paradoxes derived from tumor-progression models.

MGDB: crossing the marker genes of a user microarray with a database of public-microarrays marker genes.

Summary: The microarrays performed by scientific teams grow exponentially. These microarray data could be useful for researchers around the world, but unfortunately they are underused. To fully exploit these data, it is necessary (i) to extract these data from a repository of the high-throughput gene expression data like Gene Expression Omnibus (GEO) and (ii) to make the data from different microarrays comparable with tools easy to use for scientists.

The effect of triple glutamic mutations E9Q/E194Q/E204Q on the structural stability of bacteriorhodopsin.

In the present study, we report on the structural features of the bacteriorhodopsin triple mutant E9Q/E194Q/E204Q (3Glu) of bacteriorhodopsin by combining experimental and molecular dynamics (MD) approaches. In 3Glu mutant, Glu9, Glu194 and Glu204 residues located at the extracellular side of the protein were mutated altogether to glutamines. UV-visible and differential scanning calorimetry experiments served as diagnostic tools for monitoring the resistance against thermal stress of the active site and the tertiary structures of the 3Glu.

Study on the feasibility of bacteriorhodopsin as bio-photosensitizer in excitonic solar cell: a first report.

Bacteriorhodopsin (bR) is a membrane protein found in the archae Halobacterium salinarum. Here, we studied wild type bR and especially the triple mutant bR, 3Glu [E9Q/E194Q/E204Q], in combination with wide gap semiconductor TiO2 for their suitability as efficient light harvester in solar cell. Our differential scanning calorimetry data show thermal robustness of bR wild type and 3Glu mutant, which make them good candidates as photosensitizer in solar cells.

Coupling between the retinal thermal isomerization and the Glu194 residue of bacteriorhodopsin.

Glu194 is a residue located at the end of F helix on the extracellular side of the light-induced proton pump bacteriorhodopsin (BR). Currently, it is well recognized that Glu194 and Glu204 residues, along with water clusters, constitute the proton release group of BR. Here we report that the replacement of Glu194 for Gln affects not only the photocycle of the protein but also has tremendous effect on the all-trans to 13-cis thermal isomerization.

Influence of proline on the thermostability of the active site and membrane arrangement of transmembrane proteins.

Proline residues play a fundamental and subtle role in the dynamics, structure, and function in many membrane proteins. Temperature derivative spectroscopy and differential scanning calorimetry have been used to determine the effect of proline substitution in the structural stability of the active site and transmembrane arrangement of bacteriorhodopsin. We have analyzed the Pro-to-Ala mutation for the helix-embedded prolines Pro50, Pro91, and Pro186 in the native membrane environment.

Inter-helical hydrogen bonds are essential elements for intra-protein signal transduction: the role of Asp115 in bacteriorhodopsin transport function.

The behavior of the D115A mutant was analyzed by time-resolved UV-Vis and Fourier transformed infrared (FTIR) spectroscopies, aiming to clarify the role of Asp115 in the intra-protein signal transductions occurring during the bacteriorhodopsin photocycle. UV-Vis data on the D115A mutant show severely desynchronized photocycle kinetics. FTIR data show a poor transmission of the retinal isomerization to the chromoprotein, evidenced by strongly attenuated helical changes (amide I), the remarkable absence of environment alterations and protonation/deprotonation events related to Asp96 and direct Schiff base (SB) protonation form the bulk.

X-ray absorption and molecular dynamics study of cation binding sites in the purple membrane.

The present work describes the results of a study aimed at identifying candidate cation binding sites on the extracellular region of bacteriorhodopsin, including a site near the retinal pocket. The approach used is a combined effort involving computational chemistry methods (computation of cation affinity maps and molecular dynamics) together with the Extended X-Ray Absorption Fine Structure (EXAFS) technique to obtain relevant information about the local structure of the protein in the neighborhood of Mn(2+) ions in different affinity binding sites. The results permit the identification of a high-affinity binding site where the ion is coordinated simultaneously to Asp212(-) and Asp85(-).

The role of proline residues in the dynamics of transmembrane helices: the case of bacteriorhodopsin.

Proline residues in transmembrane helices have been found to have important roles in the functioning of membrane proteins. Moreover, Pro residues occur with high frequency in transmembrane alpha-helices, as compared to alpha-helices for soluble proteins. Here, we report several properties of the bacteriorhodopsin mutants P50A (helix B), P91A (helix C) and P186A (helix F).

Thr-90 plays a vital role in the structure and function of bacteriorhodopsin.

The role of Thr-90 in the bacteriorhodopsin structure and function was investigated by its replacement with Ala and Val. The mutant D115A was also studied because Asp-115 in helix D forms a hydrogen bond with Thr-90 in helix C. Differential scanning calorimetry showed a decreased thermal stability of all three mutants, with T90A being the least stable.

Specific effects of chloride on the photocycle of E194Q and E204Q mutants of bacteriorhodopsin as measured by FTIR spectroscopy.

Low-temperature Fourier transform infrared spectroscopy has been used to study mutants of Glu194 and Glu204, two amino acids that are involved in proton release to the extracellular side of bacteriorhodopsin. Difference spectra of films of E194Q, E204Q, E194Q/E204Q, E9Q/E194Q/E204Q, and E9Q/E74Q/E194Q/E204Q at 243, 277, and 293 K and several pH values were obtained by continuous illumination. A specific effect of Cl(-) ions was found for the mutants, promoting a N-like intermediate at alkaline pH and an O' intermediate at neutral or acid pH.