Insight into GFPmut2 pH Dependence by Single Crystal Microspectrophotometry and X-ray Crystallography.

The fluorescence of Green Fluorescent Protein (wtGFP) and variants has been exploited in distinct applications in cellular and analytical biology. GFPs emission depends on the population of the protonated (A-state) and deprotonated (B-state) forms of the chromophore. Whereas wtGFP is pH-independent, mutants in which Ser65 is replaced by either threonine or alanine (as in GFPmut2) are pH-dependent, with a p K around 6.

The STAS domain of mammalian SLC26A5 prestin harbours an anion-binding site.

Prestin is a unique ATP- and Ca(2+)-independent molecular motor with piezoelectric characteristics responsible for the electromotile properties of mammalian cochlear outer hair cells, i.e. the capacity of these cells to modify their length in response to electric stimuli.

Structure and single crystal spectroscopy of Green Fluorescent Proteins.

Usually, spectroscopic data on proteins in solution are interpreted at molecular level on the basis of the three-dimensional structures determined in the crystalline state. While it is widely recognized that the protein crystal structures are reliable models for the solution 3D structures, nevertheless it is also clear that sometimes the crystallization process can introduce some "artifacts" that can make difficult or even flaw the attempt to correlate the properties in solution with those in the crystalline state. In general, therefore, it would be desirable to identify some sort of control.

Structure of the cytosolic portion of the motor protein prestin and functional role of the STAS domain in SLC26/SulP anion transporters.

Prestin is the motor protein responsible for the somatic electromotility of cochlear outer hair cells and is essential for normal hearing sensitivity and frequency selectivity of mammals. Prestin is a member of mammalian solute-linked carrier 26 (SLC26) anion exchangers, a family of membrane proteins capable of transporting a wide variety of monovalent and divalent anions. SLC26 transporters play important roles in normal human physiology in different tissues, and many of them are involved in genetic diseases.

Expression, purification and characterisation of the C-terminal STAS domain of the SLC26 anion transporter prestin.

The membrane protein prestin is the voltage-sensitive molecular motor underlying somatic electromotility of outer hair cells. In order to produce adequate quantities to perform structural and functional studies, we cloned and expressed in bacterial systems three variants of the cytosolic C-terminal STAS domain of prestin from Rattus norvegicus. While the expression level of the longer form of the C-terminal domain (fragment [505-744]) was very low or absent, we succeeded in the overexpression of two shorter fragment of the STAS domain (fragments [529-744], PreCD(L), and [529-720], PreCD(S)).