Interaction between Escherichia coli glutaminyl-tRNA synthetase (GlnRS) and its substrates have been studied by fluorescence quenching. In the absence of other substrates, glutamine, tRNAG'" and ATP bind with dissociation constants of 460, 0.22 and 180 pM, respectively. The presence of other substrates has either no effect or, at best a weak effect, on binding of ligands. Attempts to isolate enzyme-bound aminoacyl adenylate did not succeed. Binding of the phosphodiester, 5'-(methy1)adenosine monophosphate (MeAMP), to GlnRS was studied by fluorescence quenching and radioactive-ligand binding. tRNA also only has a weak effect on phosphodiester binding.Selectively pyrene-labeled GlnRS was used to obtain shape and size information for free GlnRS. A comparison with the GlnRS shape in the GlnRS/tRNAG1" crystal structure indicates that no major change in shape and size occurs upon tRNAG'" binding to GlnRS. S,S'-Bis(8-anilino-l-naphthalene sulfonate) (bis-ANS), a non-covalent fluorescent probe, was also used to probe for conformational changes in GlnRS. This probe also indicated that no major conformational change occurs upon tRNAG'" binding.We conclude that lack of tRNA-independent pyrophosphate-exchange activity in this enzyme is not a result of either lack of glutamine or ATP binding in the absence of tRNA, or formation of aminoacyl adenylate and slow release of pyrophosphate. A conformational change is implied upon tRNA binding, which promotes pyrophosphate exchange. Fluorescence studies indicate that this conformational change must be limited and local in nature.

The urea induced equilibrium denaturation behavior of glutaminyl-tRNA synthetase from Escherichia coli (GlnRS) in 0.25 M potassium L-glutamate, a naturally occurring osmolyte in E. coli, has been studied. Both the native to molten globule and molten globule to unfolded state transitions are shifted significantly toward higher urea concentrations in the presence of L-glutamate, suggesting that L-glutamate has the ability to counteract the denaturing effect of urea. D-Glutamate has a similar effect on the equilibrium denaturation of glutaminyltRNA synthetase, indicating that the effect of L-glutamate may not be due to substrate-like binding to the native state. The activation energy of unfolding is not significantly affected in the presence of 0.25 M potassium L-glutamate, indicating that the native state is not preferentially stabilized by the osmolyte. Dramatic increase of coefficient of urea concentration dependence (m) values of both the transitions in the presence of glutamate suggests destabilization and increased solvent exposure of the denatured states. Four other osmolytes, sorbitol, trimethylamine oxide, inositol, and triethylene glycol, show either a modest effect or no effect on native to molten globule transition of glutaminyl-tRNA synthetase. However, glycine betaine significantly shifts the transition to higher urea concentrations. The effect of these osmolytes on other proteins is mixed. For example, glycine betaine counteracts urea denaturation of tubulin but promotes denaturation of S228N -repressor and carbonic anhydrase. Osmolyte counteraction of urea denaturation depends on osmolyte-protein pair.Folding of a polypeptide chain into a precise three-dimensional structure has been a subject of intense study over the past several decades (1, 2). Despite much progress, a complete understanding still eludes us. Much attention is now devoted to protein folding in vivo, where the cellular environment profoundly influences folding (3, 4). This has led to the discovery of the chaperones. Another aspect of in vivo environments that differ significantly from in vitro environments normally used for protein folding studies is the presence of osmolytes. Osmolytes are small molecules that accumulate inside the cell at relatively high concentrations and protect the intracellular proteins against environmental stress. Thus, they play a crucial role in protein stabilization.Although some efforts have been directed toward understanding the effects of osmolytes on protein stability, little is known about their effect on folding intermediates and partially unfolded states of proteins. Partially unfolded states are not only of crucial importance in understanding the folding processes but may play a crucial role in many diseases that involve extracellular protein aggregation and amyloid fibril formation, such as Alzheimer's disease and Scrapie (5, 6). Similar intracellular protein aggregates (Lewy bodies) are also known to play an important role in other neurodegenerative diseases, such as Parkinson's disease (7). Ver...

Despite emergence of bis-ANS as a major fluorescence probe of proteins structure, conformational and spectroscopic properties of protein/bis-ANS complexes remains largely unexplored. We have shown that fluorescence polarization of both ANS and bis-ANS is excitation wavelength dependent and this is a property of all protein-ANS/bis-ANS complexes studied. Bis-ANS excitation maximum is always more red shifted than the corresponding ANS complex. Even when corrected for the red shift, the bis-ANS complexes in some, but not all, cases show only a little lowering of polarization, suggesting modest additional depolarization in bis-ANS compared to ANS. Calculation of energy migration rate between the two rings suggests that energy migration rate should be high at all values of the naphthyl-naphthyl dihedral angle. Although, Molecular mechanics and dynamics calculations show that the lowest energy conformation of bis-ANS is when the two naphthalene rings are roughly perpendicular to each other, due to rapid energy migration this conformation should lead to dramatic lowering of emission anisotropy, unlike what is observed. Salt and temperature dependence of bis-ANS/protein interaction suggests little ionic interaction and pre-dominant interaction through hydrophobic aromatic rings. We conclude that bis-ANS binds to proteins through interaction with the aromatic rings and with two rings nearly parallel to each other.

The utility of collisional quenching of energy donors in fluorescence energy transfer is described. In multi-donor single acceptor systems, which contain different classes of donors (as distinguished by their accessibility towards a collisional quencher), donor quenching may be used to assess the fraction of energy transfer from each class of donor. The tubulin-colchicine complex was used as a donor-acceptor system to show that two inaccessible tryptophans are at or near the colchicine binding site.Tubulin (a heterodimer ap>, the major component of microtubules, binds the plant alkaloid colchicine, specifically and quasi-irreversibly at a single site and inhibits tubulin polymerization [l]. In spite of the intensive study of colchicine-tubulin interaction, there is no consensus about the location of the colchicine binding site on tubulin. Some studies have placed the colchicine binding site on the a subunit [2], some on the p subunit [3] and some at the interface [4].Luduena and co-workers [5] have used cross linkers to crosslink two sulfhydryl groups which are at the colchicine binding site. They have shown that these two sulfhydryl groups are protected from chemical modification by colchicine and its analogs and identified them as Cys239 and Cys354 of the p subunit [6, 71.One approach for localizing a binding site on a protein is to measure distances from fixed points in proteins by fluorescence energy transfer. Colchicine, which is non-fluorescent in solution, fluoresces upon binding to tubulin [8]. Its excitation spectrum overlaps well with the emission spectrum of tryptophan, thus forming a good donor-acceptor pair. The presence of eight tryptophans in tubulin, however, makes it difficult to have even a qualitative assessment of the involvement of various tryptophan residues in energy transfer to bound colchicine. Donor quenching of fluorescence energy transfer has previously been used to estimate distance distribution between a donor-acceptor pair [9]. In this experiment, a collisional quencher is used which quenches donor fluorescence causing a reduction in R , and hence energy transfer efficiency. A study with model peptide and protein with single indole and dansyl moieties has firmly established the feasibility of this approach [9]. In a multi-tryptophan protein, where classes of tryptophan residues differ in accessibility towards a collisional quencher, donor quenching may be used to assess the degree of energy transfer from different classes. We have explored this concept in the colchicine-tubulin complex using acrylamide as the collisional quencher.