(1). A major response is to halt cell cycle progression with corresponding activation of genes needed for DNA repair. A second kind of response is apoptosis (2). Genotoxic stresses activate many cellular kinases that phosphorylate various serine and threonine residues in p53, as well as enzymes that cause lysine acetylations (3). Presently, at least 12 serine/threonines and 2 lysines are known to be modified in p53 and there may be more modification sites. This signaling system and the code of post-translational modification are still incompletely understood (1). An understanding of the structural basis of such signal integration and transduction remains a distant dream.p53 is a multidomain protein. The N-terminal domain 1-73 is responsible for transactivation function and binding of such proteins as Mdm2/Hdm2, which down-regulate the levels of p53 (4). The N-terminal domain of p53 is emerging as one of the most important regions in p53 function. One of the major pathways of transactivation is through interaction with p300/ CBP 1 and recruitment of this protein to the required promoter region. p300/CBP has well known histone acetyltransferase activity, which results in histone acetylation and remodeling of the chromosome in that region leading to enhanced transcription (5). Clearly, interactions of the N-terminal domain of p53 with Mdm2 and p300/CBP and possibly with other regulatory proteins are of crucial importance in understanding the various roles played by p53 as the guardian of the genome.Although crystal or NMR structures of other domains have been reported (6, 7), very little is known about the structure of this important domain. Only very recently, an NMR study of this domain has shown this to be disordered, although the authors concluded that secondary structural elements are present (8). It now appears that there are at least eight phosphorylation sites within the N-terminal subdomain: serines 6, 9, 15, 20, 33, 37, and 46 and threonine 18 (1). Functions of several of these phosphorylations are not clear. However, it appears that the Ser-15 phosphorylation plays a crucial role in the transactivation process (9 -11). Phosphorylation of Ser-15 occurs very soon after initial stressing of the cells (12). Among * This work was supported in part by the Nissan Science Foundation and a grant-in-aid for scientific research (to K. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.§ 1 The abbreviations used are: CBP, cAMP-responsive element-binding protein-binding protein; Pep53, peptide corresponding to residues 17-28 of human p53; Bpep53, same peptide as Pep53 except five ␣-aminoisobutyric acid residues incorporated in designated positions; Aib, ␣-aminoisobutyric acid; Ser15P, p53-(1-39) domain phosphorylated at serine 15; Thr18P, p53-(1-39) domain phosphorylated at threonine 18; Ser20P, p53-(1-39) domain phosphorylated at serine ...

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...

The nature of solvent molecules around proteins in native and different non-native states is crucial for understanding the protein folding problem. We have characterized two compact denatured states of glutaminyl-tRNA synthetase (GlnRS) under equilibrium conditions in the presence of a naturally occurring osmolyte, l-glutamate. The solvation dynamics of the compact denatured states and the fully unfolded state has been studied using a covalently attached probe, acrylodan, near the active site. The solvation dynamics progressively becomes faster as the protein goes from the native to the molten globule to the pre molten globule to the fully unfolded state. Anisotropy decay measurements suggest that the pre-molten-globule intermediate is more flexible than the molten globule although the secondary structure is largely similar. Dynamic light scattering studies reveal that both the compact denatured states are aggregated under the measurement conditions. The implications of solvation dynamics in aggregated compact denatured states have been discussed.