ABSTRACT
GFP mutants are known to display fluorescence flickering, a process that occurs in a wide time range. Because serine 65, threonine 203, glutamate 222, and histidine 148 have been indicated as key residues in determining the GFP fluorescence photodynamics, we have focused here on the role of histidine 148 and glutamate 222 by studying the fluorescence dynamics of GFPmut2 (S65A, V68L, and S72A GFP) and its H148G (Mut2G) and E222Q (Mut2Q) mutants. Two relaxation components are found in the fluorescence autocorrelation functions of GFPmut2: a 10-100 micros pH-dependent component and a 100-500 micros laser-power-dependent component. The comparison of these three mutants shows that the mutation of histidine 148 to glycine induces a 3-fold increase in the protonation rate, thereby indicating that the protonation-deprotonation of the chromophore occurs via a proton exchange with the solution mediated by the histidine 148 residue. The power-dependent but pH-independent relaxation mode, which is not affected by the E222Q and H148G mutations, is due to an excited-state process that is probably related to conformational rearrangements of the chromophore after the photoexcitation, more than to the chromophore excited-state proton transfer.
Subject(s)
Green Fluorescent Proteins/chemistry , Luminescent Agents/chemistry , Photons , Protons , Computer Simulation , Glutamic Acid/chemistry , Histidine/chemistry , Hydrogen-Ion Concentration , Mutation , Protein Conformation , Serine/chemistry , Spectrometry, Fluorescence , Threonine/chemistryABSTRACT
Proteins belonging to the superfamily of pyridoxal 5'-phosphate-dependent enzymes are currently classified into three functional groups and five distinct structural fold types. The variation within this enzyme group creates an ideal system to investigate the relationships among amino acid sequences, folding pathways, and enzymatic functions. The number of known three-dimensional structures of pyridoxal 5'-phosphate-dependent enzymes is rapidly increasing, but only for relatively few have the folding mechanisms been characterized in detail. The dimeric O-acetylserine sulfhydrylase from Salmonella typhimurium belongs to the beta-family and fold type II group. Here we report the guanidine hydrochloride-induced unfolding of the apo- and holoprotein, investigated using a variety of spectroscopic techniques. Data from absorption, fluorescence, circular dichroism, (31)P nuclear magnetic resonance, time-resolved fluorescence anisotropy, and photon correlation spectroscopy indicate that the O-acetylserine sulfhydrylase undergoes extensive disruption of native secondary and tertiary structure before monomerization. Also, we have observed that the holo-O-acetylserine sulfhydrylase exhibits a greater conformational stability than the apoenzyme form. The data are discussed in light of the fact that the role of the coenzyme in structural stabilization varies among the pyridoxal 5'-phosphate-dependent enzymes and does not seem to be linked to the particular enzyme fold type.
Subject(s)
Cysteine Synthase/chemistry , Pyridoxal Phosphate/chemistry , Guanidine/chemistry , Light , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Protein Denaturation , Scattering, Radiation , Spectrometry, Fluorescence , Spectrophotometry, UltravioletABSTRACT
Two cases of oculomotor nerves involvement as uncommon initial manifestation of a parasellar extramedullary plasmacytoma are reported. The first patient presented with an isolated left VI cranial nerve palsy; the second one had an incomplete left III cranial nerve palsy. In both cases clinical investigation and computed tomography revealed an intracranial plasmacytoma associated with multiple myeloma. It is important to underline the difference between an intracranial extramedullary plasmacytoma and a plasmacytoma associated with multiple myeloma. In fact the absence of a general associated illness should show a most favourable course.
