Structural characterization of unfolded states of apomyoglobin using residual dipolar couplings

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 phi and psi dihedral angles favoring the beta 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 acid-denatured 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.     

Molecular features of the viral and cellular Src kinases involved in interactions with the GTPase-activating protein.

GTPase-activating protein (GAP) enhances the rate of GTP hydrolysis by cellular Ras proteins and is implicated in mitogenic signal transduction. GAP is phosphorylated on tyrosine in cells transformed by Rous sarcoma virus and serves as an in vitro substrate of the viral Src (v-Src) kinase. Our previous studies showed that GAP complexes stably with normal cellular Src (c-Src), although its association with v-Src is less stable. To further investigate the molecular basis for interactions between GAP and the Src kinases, we examined GAP association with and phosphorylation by a series of c-Src and v-Src mutants. Analysis of GAP association with c-Src/v-Src chimeric proteins demonstrates that GAP associates stably with Src proteins possessing low kinase activity and poorly with activated Src kinases, especially those that lack the carboxy-terminal segment of c-Src containing the regulatory amino acid Tyr-527. Phosphorylated Tyr-527 is a major determinant of c-Src association with GAP, as demonstrated by c-Src point mutants in which Tyr-527 is changed to Phe. While the isolated amino-terminal half of the c-Src protein is insufficient for stable GAP association, analysis of point substitutions of highly conserved amino acid residues in the c-Src SH2 region indicate that this region also influences Src-GAP complex formation. Therefore, our results suggest that both Tyr-527 phosphorylation and the SH2 region contribute to stable association of c-Src with GAP. Analysis of in vivo phosphorylation of GAP by v-Src mutants containing deletions encompassing the SH2, SH3, and unique regions suggests that the kinase domain of v-Src contains sufficient substrate specificity for GAP phosphorylation. Even though tyrosine phosphorylation of GAP correlates to certain extent with the transforming ability of various c-Src and v-Src mutants, our data suggest that other GAP-associated proteins may also have roles in Src-mediated oncogenic transformation. These findings provide additional evidence for the specificity of Src interactions with GAP and support the hypothesis that these interactions contribute to the biological functions of the Scr kinases.     

Reconstitution of interactions between the Src tyrosine kinases and Ras GTPase-activating protein using a baculovirus expression system.

Ras GTPase-activating protein (GAP) has been implicated in mitogenic signal transduction downstream of oncogenic and receptor tyrosine kinases. Previous studies have suggested that GAP is phosphorylated by oncogenic viral Src (v-Src) and that GAP is associated with a complex containing normal cellular Src (c-Src) in vertebrate fibroblasts. To investigate molecular interactions between the Src kinases and GAP, we developed an in vitro system for reconstituting Src-GAP complexes. For this purpose, we constructed recombinant baculovirus vectors that direct expression of Rous sarcoma virus v-Src, chicken c-Src, and bovine GAP in infected Sf9 insect cells. In vitro reconstitution experiments using baculovirus-expressed proteins demonstrate that both v-Src and c-Src associate in complexes with GAP. In addition, in vitro and in vivo phosphorylation analyses indicate that GAP serves as a substrate for both the v-Src and c-Src tyrosine kinases. To determine which structural features of GAP are involved in interactions with the Src kinases, we constructed recombinant baculoviruses that encode deletion mutants of bovine GAP. Deletion of the GAP amino-terminal portion containing Src homology 2 regions, which are highly conserved structural motifs postulated to mediate interactions among proteins, diminishes GAP phosphorylation and association with Src. This reconstitution system should facilitate further studies of molecular interactions between the Src kinases and GAP.     

Structure and Disorder in an Unfolded State under Nondenaturing Conditions from Ensemble Models Consistent with a Large Number of Experimental Restraints

Obtaining detailed structural models of disordered states of proteins under nondenaturing conditions is important for a better understanding of both functional intrinsically disordered proteins and unfolded states of folded proteins. Extensive experimental characterization of the drk N-terminal SH3 domain unfolded state has shown that, although it appears to be highly disordered, it possesses significant nonrandom secondary and tertiary structure. In our previous attempts to generate structural models of the unfolded state using the program ENSEMBLE, we were limited by insufficient experimental restraints and conformational sampling. In this study, we have vastly expanded our experimental restraint set to include ¹H-¹⁵N residual dipolar couplings, small-angle X-ray scattering measurements, nitroxide paramagnetic relaxation enhancements, O₂-induced ¹³C paramagnetic shifts, hydrogen-exchange protection factors, and ¹⁵N R₂ data, in addition to the previously used nuclear Overhauser effects, amino terminal Cu²⁺-Ni²⁺ binding paramagnetic relaxation enhancements, J-couplings, chemical shifts, hydrodynamic radius, and solvent accessibility restraints. We have also implemented a new ensemble calculation methodology that uses iterative conformational sampling and seeks to calculate the simplest possible ensemble models. As a result, we can now generate ensembles that are consistent with much larger experimental data sets than was previously possible. Although highly heterogeneous and having broad molecular size distributions, the calculated drk N-terminal SH3 domain unfolded-state ensembles have very different properties than expected for random or statistical coils and possess significant nonnative α-helical structure and both native-like and nonnative tertiary structure.     

Structure and disorder in the ribonuclease S-peptide probed by NMR residual dipolar couplings

NMR residual dipolar couplings for the S-peptide of ribonuclease A aligned in C8E5/n-octanol liquid crystals are consistent with the presence of a native-like alpha-helix structure undergoing dynamic fraying. Residues 3-13, which correspond to the first alpha-helix of ribonuclease A, show couplings that become more negative at low temperature and in the presence of salt, conditions which stabilize alpha-helical structure in the S-peptide. By contrast, dipolar couplings from the N and C termini of the peptide are close to zero and remain nearly invariant with changes in solution conditions. Torsion angle dynamics simulations using a gradient of dihedral restraint bounds that increase from the center to the ends of the peptide reproduce the experimentally observed sequence dependence of dipolar couplings. The magnitudes of residual dipolar couplings depend on the anisotropy of the solute. Native proteins often achieve nearly spherical shapes due to the hydrophobic effect. Embryonic partially folded structures such as the S-peptide alpha-helix have an intrinsically greater potential for anisotropy that can result in sizable residual dipolar couplings in the absence of long-range structure.     

Mercuric chloride mediates a protein sulfhydryl modification-based pathway of signal transduction for activating Src kinase which is independent of the phosphorylation / dephosphorylation of a carboxyl terminal tyrosine

Little is known about the regulatory mechanism of c-Src kinase in cells except the suggested regulation through phosphorylation and dephosphorylation of its carboxyl terminal tyrosine residue (Y527). We here demonstrated that exposure of NIH3T3 cells to mercuric chloride (HgCl₂) induces both aggregation and activation of Src kinase protein through a redox-linked mechanism. The aggregation of Src proteins was suggested to be induced by the sulfhydryl groups-to-Hg²⁺ reaction-mediated polymerization of cell membrane proteins to which the Src proteins associate noncovalently. The possibility was ruled out that the aggregation occurred secondarily to the promotion of protein tyrosine phosphorylation. Further study revealed that the Src kinase was activated by HgCl₂ at least in part independent of the known Csk kinase-linked or Y527-phosphorylation/dephosphorylation-mediated control. Correspondingly, CNBr cleavage mapping of phosphopeptides for autophosphorylated c-Src protein demonstrated selective promotion of phosphorylation at Y416 in HgCl₂-treated cells without obvious change in the phosphorylation level at Y527. These results suggest a unique protein sulfhydryl modification-based pathway of signal transduction for activating Src kinase in NIH3T3 cells. © 1996 Wiley-Liss, Inc.     

Structural characterization of the intrinsically unfolded protein beta-synuclein, a natural negative regulator of alpha-synuclein aggregation

The synuclein family of intrinsically unfolded proteins is composed of three highly homologous members, alpha-synuclein (alphaS), beta-synuclein (betaS) and gamma-synuclein (gammaS), which are linked to neurodegenerative disorders and cancer. alphaS has been studied intensively after its identification as the major protein component of amyloid-like deposits in Parkinson's disease and dementia with Lewy bodies. betaS, on the other hand, was found to act as a potent inhibitor of alphaS amyloid formation, and it is proposed as a natural regulator of its neurotoxicity. It is then of particular interest to elucidate the structural and dynamic features of the soluble state of betaS as a first step to understand the molecular basis of its anti-amyloidogenic effect on alphaS. We present here the characterization of natively unstructured betaS by high resolution heteronuclear NMR techniques. A combination of pulse-field gradient, three-dimensional heteronuclear correlation, residual dipolar couplings, paramagnetic relaxation enhancement and backbone relaxation experiments were employed to characterize the ensemble of conformations populated by the protein. The results indicate that betaS adopts extended conformations in its native state, characterized by the lack of the long-range contacts as previously reported for alphaS. Despite the lack of defined secondary structure, we found evidence for transient polyproline II conformations clustered at the C-terminal region. The structuring of the backbone at the C terminus is locally encoded, stabilized by the presence of eight proline residues embedded in a polypeptide stretch rich in hydrophilic and negatively charged amino acids. The structural and functional implications of these findings are analyzed via a thorough comparison with its neurotoxic homolog alphaS.     

The Tyrosine Kinase Csk Dimerizes through Its SH3 Domain

The Src family kinases possess two sites of tyrosine phosphorylation that are critical to the regulation of kinase activity. Autophosphorylation on an activation loop tyrosine residue (Tyr 416 in commonly used chicken c-Src numbering) increases catalytic activity, while phosphorylation of a C-terminal tyrosine (Tyr 527 in c-Src) inhibits activity. The latter modification is achieved by the tyrosine kinase Csk (C-terminal Src Kinase), but the complete inactivation of the Src family kinases also requires the dephosphorylation of the activation loop tyrosine. The SH3 domain of Csk recruits the tyrosine phosphatase PEP, allowing for the coordinated inhibition of Src family kinase activity. We have discovered that Csk forms homodimers through interactions mediated by the SH3 domain in a manner that buries the recognition surface for SH3 ligands. The formation of this dimer would therefore block the recruitment of tyrosine phosphatases and may have important implications for the regulation of Src kinase activity.     

Neuron-specific Cdk5 kinase is responsible for mitosis-independent phosphorylation of c-Src at Ser75 in human Y79 retinoblastoma cells.

c-Src is phosphorylated at specific serine and threonine residues during mitosis in fibroblastic and epithelial cells. These sites are phosphorylated in vitro by the mitotic kinase Cdk1 (p34(cdc2)). In contrast, c-Src in Y79 human retinoblastoma cells, which are of neuronal origin, is phosphorylated at one of the mitotic sites, Ser75, throughout the cell cycle. The identity of the serine kinase that nonmitotically phosphorylates c-Src on Ser75 remains unknown. We now are able to show for the first time that Cdk5 kinase, which has the same consensus sequence as the Cdk1 and Cdk2 kinases, is required for the phosphorylation in asynchronous Y79 cells. The Ser75 phosphorylation was inhibited in a dose-dependent manner by butyrolactone I, a specific inhibitor of Cdk5-type kinases. Three stable subclones that have almost no kinase activity were selected by transfection of an antisense Cdk5-specific activator p35 construct into Y79 cells. The loss of the kinase activity caused an approximately 85% inhibition of the Ser75 phosphorylation. These results present compelling evidence that Cdk5/p35 kinase is responsible for the novel phosphorylation of c-Src at Ser75 in neuronal cells, raising the intriguing possibility that c-Src acts as an effector of Cdk5/p35 kinase during neuronal development.     

Csk inhibition of c-Src activity requires both the SH2 and SH3 domains of Src.

The protein tyrosine kinase c-Src is negatively regulated by phosphorylation of Tyr527 in its carboxy-terminal tail. A kinase that phosphorylates Tyr527, called Csk, has recently been identified. We expressed c-Src in yeast to test the role of the SH2 and SH3 domains of Src in the negative regulation exerted by Tyr527 phosphorylation. Inducible expression of c-Src in Schizosaccharomyces pombe caused cell death. Co-expression of Csk counteracted this effect. Src proteins mutated in either the SH2 or SH3 domain were as lethal as wild type c-Src, but were insensitive to Csk, even though they were substrates for Csk in vivo. Peptide binding experiments revealed that Src proteins with mutant SH3 domains adopted a conformation in which the SH2 domain was not interacting with the tail. These data support the model of an SH2 domain-phosphorylated tail interaction repressing c-Src activity, but expand it to include a role for the SH3 domain. We propose that the SH3 domain contributes to the maintenance of the folded, inactive configuration of the Src molecule by stabilizing the SH2 domain-phosphorylated tail interaction. Moreover, the system we describe here allows for further study of the regulation of tyrosine kinases in a neutral background and in an organism amenable to genetic analysis.     

Src kinase regulation by phosphorylation and dephosphorylation

Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTPalpha, PTPepsilon, and PTPlambda. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined.     

Role of intrinsic flexibility in signal transduction mediated by the cell cycle regulator, p27 Kip1

p27^Kip1 (p27), which controls eukaryotic cell division through interactions with cyclin-dependent kinases (Cdks), integrates and transduces promitogenic signals from various nonreceptor tyrosine kinases by orchestrating its own phosphorylation, ubiquitination and degradation. Intrinsic flexibility allows p27 to act as a “conduit” for sequential signaling mediated by tyrosine and threonine phosphorylation and ubiquitination. While the structural features of the Cdk/cyclin-binding domain of p27 are understood, how the C-terminal regulatory domain coordinates multistep signaling leading to p27 degradation is poorly understood. We show that the 100-residue p27 C-terminal domain is extended and flexible when p27 is bound to Cdk2/cyclin A. We propose that the intrinsic flexibility of p27 provides a molecular basis for the sequential signal transduction conduit that regulates p27 degradation and cell division. Other intrinsically unstructured proteins possessing multiple sites of posttranslational modification may participate in similar signaling conduits.     

Incorporating residual dipolar couplings into the NMR solution structure determination of nucleic acids

NMR solution structures of nucleic acids are generally less well defined than similar-sized proteins. Most NMR structures of nucleic acids are defined only by short-range interactions, such as intrabase-pair or sequential nuclear Overhauser effects (NOEs), and J-coupling constants, and there are no long-range structural data on the tertiary structure. Residual dipolar couplings represent an extremely valuable source of distance and angle information for macromolecules but they average to zero in isotropic solutions. With the recent advent of general methods for partial alignment of macromolecules in solution, residual dipolar couplings are rapidly becoming indispensable constraints for solution NMR structural studies. These residual dipolar couplings give long-range global structural information and thus complement the strictly local structural data obtained from standard NOE and torsion angle constraints. Such global structural data are especially important in nucleic acids due to the more elongated, less-globular structure of many DNAs and RNAs. Here we review recent progress in application of residual dipolar couplings to structural studies of nucleic acids. We also present results showing how refinement procedures affect the final solution structures of nucleic acids.Copyright 2001 John Wiley & Sons, Inc.     

Temperature sensitivities of metazoan and pre-metazoan Src kinases

Homologous enzymes from different species display functional characteristics that correlate with the physiological and environmental temperatures encountered by the organisms. In this study, we have investigated the temperature sensitivity of the nonreceptor tyrosine kinase Src. We compared the temperature dependencies of c-Src and two Src kinases from single-celled eukaryotes, the choanoflagellate Monosiga brevicollis and the filasterean Capsaspora owczarzaki. Metazoan c-Src exhibits temperature sensitivity, with high activity at 30 °C and 37 °C. This sensitivity is driven by changes in substrate binding as well as maximal velocity, and it is dependent on the amino acid sequence surrounding tyrosine in the substrate. When tested with a peptide that displays temperature-dependent phosphorylation by c-Src, the enzymatic rates for the unicellular Src kinases show much less variation over the temperatures tested. The data demonstrate that unicellular Src kinases are temperature compensated relative to metazoan c-Src, consistent with an evolutionary adaptation to their environments.     

Src-Tyrosine kinases are major agents in mitochondrial tyrosine phosphorylation

Mitochondrial tyrosine phosphorylation is emerging as an important mechanism in regulating mitochondrial function. This article, aimed at identifying which kinases are the major agents in mitochondrial tyrosine phosphorylation, shows that this role should be attributed to Src family members. Indeed, various members of this family, for example, Fgr, Fyn, Lyn, c-Src, are constitutively present in the internal structure of mitochondria as well as Csk, a key enzyme in the regulation of the activity of this family. By means of different approaches, biochemical fractioning, Western blotting and immunogold analysis "in situ" of phosphotyrosine signaling, evidence is reported on the existence of a signal transduction pathway from plasma membrane to mitochondria, resulting in increasing Src-dependent mitochondrial tyrosine phosphorylation. The activation of Src kinases at mitochondrial level is associated with the proliferative status where several mitochondrial proteins are specifically tyrosine-phosphorylated.     

Structure of the Na,K-ATPase regulatory protein FXYD1 in micelles

FXYD1 is a major regulatory subunit of the Na,K-ATPase and the principal substrate of hormone-regulated phosphorylation by c-AMP dependent protein kinases A and C in heart and skeletal muscle sarcolemma. It is a member of an evolutionarily conserved family of membrane proteins that regulate the function of the enzyme complex in a tissue-specific and physiological-state-specific manner. Here, we present the three-dimensional structure of FXYD1 determined in micelles by NMR spectroscopy. Structure determination was made possible by measuring residual dipolar couplings in weakly oriented micelle samples of the protein. This allowed us to obtain the relative orientations of the helical segments and information about the protein dynamics. The structural analysis was further facilitated by the inclusion of distance restraints, obtained from paramagnetic spin label relaxation enhancements, and by refinement with a micelle depth restraint, derived from paramagnetic Mn line broadening effects. The structure of FXYD1 provides the foundation for understanding its intra-membrane association with the Na,K-ATPase alpha subunit and suggests a mechanism whereby the phosphorylation of conserved Ser residues, by protein kinases A and C, could induce a conformational change in the cytoplasmic domain of the protein to modulate its interaction with the alpha subunit.     

Complexes of gamma-tubulin with nonreceptor protein tyrosine kinases Src and Fyn in differentiating P19 embryonal carcinoma cells

Nonreceptor protein tyrosine kinases of the Src family have been shown to play an important role in signal transduction as well as in regulation of microtubule protein interactions. Here we show that gamma-tubulin (gamma-Tb) in P19 embryonal carcinoma cells undergoing neuronal differentiation is phosphorylated and forms complexes with protein tyrosine kinases of the Src family, Src and Fyn. Elevated expression of both kinases during differentiation corresponded with increased level of proteins phosphorylated on tyrosine. Immunoprecipitation experiments with antibodies against Src, Fyn, gamma-tubulin, and with anti-phosphotyrosine antibody revealed that gamma-tubulin appeared in complexes with these kinases. In vitro kinase assays showed tyrosine phosphorylation of proteins in gamma-tubulin complexes isolated from differentiated cells. Pretreatment of cells with Src family selective tyrosine kinase inhibitor PP2 reduced the amount of phosphorylated gamma-tubulin in the complexes. Binding experiments with recombinant SH2 and SH3 domains of Src and Fyn kinases revealed that protein complexes containing gamma-tubulin bound to SH2 domains and that these interactions were of SH2-phosphotyrosine type. The combined data suggest that Src family kinases might have an important role in the regulation of gamma-tubulin interaction with tubulin dimers or other proteins during neurogenesis.     

TRANCE, a TNF family member, activates Akt/PKB through a signaling complex involving TRAF6 and c-Src

TRANCE, a TNF family member, and its receptor, TRANCE-R, are critical regulators of dendritic cell and osteoclast function. Here, we demonstrate that TRANCE activates the antiapoptotic serine/threonine kinase Akt/PKB through a signaling complex involving c-Src and TRAF6. A deficiency in c-Src or addition of Src family kinase inhibitors blocks TRANCE-mediated PKB activation in osteoclasts. c-Src and TRAF6 interact with each other and with TRANCE-R upon receptor engagement. TRAF6, in turn, enhances the kinase activity of c-Src leading to tyrosine phosphorylation of downstream signaling molecules such as c-Cbl. These results define a mechanism by which TRANCE activates Src family kinases and PKB and provide evidence of cross-talk between TRAF proteins and Src family kinases.     

Structures of p38alpha active mutants reveal conformational changes in L16 loop that induce autophosphorylation and activation

p38 mitogen-activated protein (MAP) kinases function in numerous signaling processes and are crucial for normal functions of cells and organisms. Abnormal p38 activity is associated with inflammatory diseases and cancers making the understanding of its activation mechanisms highly important. p38s are commonly activated by phosphorylation, catalyzed by MAP kinase kinases (MKKs). Moreover, it was recently revealed that the p38alpha is also activated via alternative pathways, which are MKK independent. The structural basis of p38 activation, especially in the alternative pathways, is mostly unknown. This lack of structural data hinders the study of p38's biology as well as the development of novel strategies for p38 inhibition. We have recently discovered and optimized a novel set of intrinsically active p38 mutants whose activities are independent of any upstream activation. The high-resolution crystal structures of the intrinsically active p38alpha mutants reveal that local alterations in the L16 loop region promote kinase activation. The L16 loop can be thus regarded as a molecular switch that upon conformational changes promotes activation. We suggest that similar conformational changes in L16 loop also occur in natural activation mechanisms of p38alpha in T-cells. Our biochemical studies reveal novel mechanistic insights into the activation process of p38. In this regard, the results indicate that the activation mechanism of the mutants involves dimerization and subsequent trans autophosphorylation on Thr180 (on the phosphorylation lip). Finally, we suggest a model of in vivo p38alpha activation induced by the L16 switch with auto regulatory characteristics.     

Line narrowing in spectra of proteins dissolved in a dilute liquid crystalline phase by band-selective adiabatic decoupling: Application to 1HN-15N residual dipolar coupling measurements

Residual heteronuclear dipolar couplings obtained from partially oriented protein samples can provide unique NMR constraints for protein structure determination. However, partial orientation of protein samples also causes severe ¹ H line broadening resulting from residual ¹ H-¹H dipolar couplings. In this communication we show that band-selective ¹H homonuclear decoupling during data acquisition is an efficient way to suppress residual ¹H-¹H dipolar couplings, resulting in spectra that are still amenable to solution NMR analysis, even with high degrees of alignment. As an example, we present a novel experiment with improved sensitivity for the measurement of one-bond ¹ H^N-¹⁵N residual dipolar couplings in a protein sample dissolved in magnetically aligned liquid crystalline bicelles.