We have used a nanosecond pH-jump technique, coupled with simultaneous transient absorption and fluorescence emission detection, to characterize the dynamics of the acid-induced spectral changes in the GFPmut2 chromophore. Disappearance of the absorbance at 488 nm and the green fluorescence emission occurs with a thermally activated, double exponential relaxation. To understand the source of the two transients we have introduced mutations in amino acid residues that interact with the chromophore (H148G, T203V, and E222Q). Results indicate that the faster transient is associated with proton binding from the solution, while the second process, smaller in amplitude, is attributed to structural rearrangement of the amino acids surrounding the chromophore. The protonation rate shows a 3-fold increase for the H148G mutant, demonstrating that His148 plays a key role in protecting the chromophore from the solvent. The deprotonation rate for T203V is an order of magnitude smaller, showing that the hydrogen bond with the hydroxyl of Thr203 is important in stabilizing the deprotonated form of the chromophore. A kinetic model suggests that, in addition to protecting the chromophore from the solvent, His148 may act as the primary acceptor for the protons on the way to the chromophore.

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The transglutaminase (TGase) from Streptomyces mobaraensis catalyzes transamidation reactions in a protein substrate leading to the modification of the side chains of Gln and Lys residues according to the A-CONH(2) + H(2)N-B → A-CONH-B + NH(3) reaction, where both A and B can be a protein or a ligand. A noteworthy property of TGase is its susbstrate specificity, so that often only a few specific Gln or Lys residues can be modified in a globular protein. The molecular features of a globular protein dictating the site-specific reactions mediated by TGase are yet poorly understood. Here, we have analyzed the reactivity toward TGase of apomyoglobin (apoMb), α-lactalbumin (α-LA), and fragment 205-316 of thermolysin. These proteins are models of protein structure and folding that have been studied previously using the limited proteolysis technique to unravel regions of local unfolding in their amino acid sequences. The three proteins were modified by TGase at the level of Gln or Lys residues with dansylcadaverine or carbobenzoxy-l-glutaminylglycine, respectively. Despite these model proteins containing several Gln and Lys residues, the sites of TGase derivatization occur over restricted chain regions of the protein substrates. In particular, the TGase-mediated modifications occur in the "helix F" region in apoMb, in the β-domain in apo-α-LA in its molten globule state, and in the N-terminal region in fragment 205-316 of thermolysin. Interestingly, the sites of limited proteolysis are located in the same chain regions of these proteins, thus providing a clear-cut demonstration that chain flexibility or local unfolding overwhelmingly dictates the site-specific modification by both TGase and a protease.

Tryptophan synthase is a pyridoxal 5'-phosphate-dependent alpha(2)beta(2) complex catalyzing the last two steps of tryptophan biosynthesis in bacteria, plants and fungi. Structural, dynamic and functional studies, carried out over more than 40 years, have unveiled that: (1) alpha- and beta-active sites are separated by about 20 A and communicate via the selective stabilization of distinct conformational states, triggered by the chemical nature of individual catalytic intermediates and by allosteric ligands; (2) indole, formed at alpha-active site, is intramolecularly channeled to the beta-active site; and (3) naturally occurring as well as genetically generated mutants have allowed to pinpoint functional and regulatory roles for several individual amino acids. These key features have made tryptophan synthase a text-book case for the understanding of the interplay between chemistry and conformational energy landscapes.

The last step of cysteine biosynthesis in bacteria and plants is catalyzed by O-acetylserine sulfhydrylase. In bacteria, two isozymes, O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, have been identified that share similar binding sites, although the respective specific functions are still debated. O-acetylserine sulfhydrylase plays a key role in the adaptation of bacteria to the host environment, in the defense mechanisms to oxidative stress and in antibiotic resistance. Because mammals synthesize cysteine from methionine and lack O-acetylserine sulfhydrylase, the enzyme is a potential target for antimicrobials. With this aim, we first identified potential inhibitors of the two isozymes via a ligand- and structure-based in silico screening of a subset of the ZINC library using FLAP. The binding affinities of the most promising candidates were measured in vitro on purified O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B from Salmonella typhimurium by a direct method that exploits the change in the cofactor fluorescence. Two molecules were identified with dissociation constants of 3.7 and 33 µM for O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, respectively. Because GRID analysis of the two isoenzymes indicates the presence of a few common pharmacophoric features, cross binding titrations were carried out. It was found that the best binder for O-acetylserine sulfhydrylase-B exhibits a dissociation constant of 29 µM for O-acetylserine sulfhydrylase-A, thus displaying a limited selectivity, whereas the best binder for O-acetylserine sulfhydrylase-A exhibits a dissociation constant of 50 µM for O-acetylserine sulfhydrylase-B and is thus 8-fold selective towards the former isozyme. Therefore, isoform-specific and isoform-independent ligands allow to either selectively target the isozyme that predominantly supports bacteria during infection and long-term survival or to completely block bacterial cysteine biosynthesis.

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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.