A selective protonation strategy is described that uses [3-2H] 13C alpha-ketoisovalerate to introduce (1H-delta methyl)-leucine and (1H-gamma methyl)-valine into 15N-, 13C-, 2H-labeled proteins. A minimum level of 90% incorporation of label into both leucine and valine methyl groups is obtained by inclusion of approximately 100 mg/L alpha-ketoisovalerate in the bacterial growth medium. Addition of [3,3-2H2] alpha-ketobutyrate to the expression media (D2O solvent) results in the production of proteins with (1H-delta1 methyl)-isoleucine (> 90% incorporation). 1H-13C HSQC correlation spectroscopy establishes that CH2D and CHD2 isotopomers are not produced with this method. This approach offers enhanced labeling of Leu methyl groups over previous methods that utilize Val as the labeling agent and is more cost effective.

No abstract

Proline is established as a potent breaker of both et-helical and 13-sheet structures in soluble (globular) proteins.

The conformational propensities of unfolded states of apomyoglobin have been investigated by measurement of residual dipolar couplings between 15 N and 1 H in backbone amide groups. Weak alignment of apomyoglobin in acid and urea-unfolded states was induced with both stretched and compressed polyacrylamide gels. In 8 M urea solution at pH 2.3, conditions under which apomyoglobin contains no detectable secondary or tertiary structure, significant residual dipolar couplings of uniform sign were observed for all residues. At pH 2.3 in the absence of urea, a change in the magnitude and/or sign of the residual dipolar couplings occurs in local regions of the polypeptide where there is a high propensity for helical secondary structure. These results are interpreted on the basis of the statistical properties of the unfolded polypeptide chain, viewed as a polymer of statistical segments. For a folded protein, the magnitude and sign of the residual dipolar couplings depend on the orientation of each bond vector relative to the alignment tensor of the entire molecule, which reorients as a single entity. For unfolded proteins, there is no global alignment tensor; instead, residual dipolar couplings are attributed to alignment of the statistical segments or of transient elements of secondary structure. For apomyoglobin in 8 M urea, the backbone is highly extended, with f and c dihedral angles favoring the b or P II regions. Each statistical segment has a highly anisotropic shape, with the N -H bond vectors approximately perpendicular to the long axis, and becomes weakly aligned in the anisotropic environment of the strained acrylamide gels. Local regions of enhanced flexibility or chain compaction are characterized by a decrease in the magnitude of the residual dipolar couplings. The formation of a small population of helical structure in the aciddenatured state of apomyoglobin leads to a change in sign of the residual dipolar couplings in local regions of the polypeptide; the population of helix estimated from the residual dipolar couplings is in excellent agreement with that determined from chemical shifts. The alignment model described here for apomyoglobin can also explain the pattern of residual dipolar couplings reported previously for denatured states of staphylococcal nuclease and other proteins. In conjunction with other NMR experiments, residual dipolar couplings can provide valuable insights into the dynamic conformational propensities of unfolded and partly folded states of proteins and thereby help to chart the upper reaches of the folding landscape.

Transcriptional activation is mediated by large protein complexes assembled on target gene promoter regions. These complexes contain activators and coactivators of transcription as well as elements of the basal transcription machinery (1). The specificity, timing, and degree of transcriptional activation depend not only on which proteins form this complex but also on how they interact with each other. At the simplest level, direct interactions involved in complex formation provide a mechanism for recruiting specific functionalities to a promoter sequence. However, in some cases, binding events also give rise to allosteric effects on binding or enzymatic activity, providing another means by which transcriptional activity can be modulated.A transcriptional coactivator that may be allosterically regulated by simultaneous interactions with more than one target protein is the cAMP-response element (CREB) 1 -binding protein (CBP), as well as its paralog p300. In addition to its catalytic histone acetyltransferase domain, CBP is composed of several protein-binding modules that serve to bridge genespecific transcription factors with components of the basal transcription complex (2). CBP domains such as KIX, CH1, and CH3 recognize a diverse range of protein sequences that in some cases appear to share a general structural motif in the bound state. For example, the KIX domain binds an amphipathic helix formed by either the phosphorylated kinaseinducible domain (pKID) of CREB or the transactivation domain of c-Myb (3, 4). Although both of these ligands bind to the shallow hydrophobic groove formed between KIX helices ␣ 1 and ␣ 3 , the high affinity of CREB for CBP is mediated by critical interactions involving the pKID phosphoryl group and is therefore inducible, whereas the c-Myb activation domain binds constitutively with somewhat lower affinity (5, 6).It has been proposed that CBP-mediated transcriptional activation can be enhanced by cooperative interactions between CBP and more than one DNA-bound transcriptional activator (7-9). Since the nuclear concentration of CBP is thought to be limiting (10 -12), the presence of multiple protein-binding domains on CBP, together with the high affinity of CBP for the activators involved, should sequester a larger proportion of CBP to specific promoter sites and thereby provide a mechanism for transcriptional synergy by multiple activators. There are a number of cases where synergistic increases in transcription have been shown to occur when multiple CBP-binding activators bind to the same gene promoter. Examples include cooperative transcriptional activation by CREB and the serum response factor (13, 14) as well as enhancement of c-Myb transcriptional activation by core binding factor (15, 16), CCAAT/