Improving resolution in multidimensional NMR using random quadrature detection with compressed sensing reconstruction.

NMR spectroscopy is central to atomic resolution studies in biology and chemistry. Key to this approach are multidimensional experiments. Obtaining such experiments with sufficient resolution, however, is a slow process, in part since each time increment in every indirect dimension needs to be recorded twice, in quadrature. We introduce a modified compressed sensing (CS) algorithm enabling reconstruction of data acquired with random acquisition of quadrature components in gradient-selection NMR. We name this approach random quadrature detection (RQD). Gradient-selection experiments are essential to the success of modern NMR and with RQD, a 50 % reduction in the number of data points per indirect dimension is possible, by only acquiring one quadrature component per time point. Using our algorithm (CSRQD), high quality reconstructions are achieved. RQD is modular and combined with non-uniform sampling we show that this provides increased flexibility in designing sampling schedules leading to improved resolution with increasing benefits as dimensionality of experiments increases, with particular advantages for 4- and higher dimensional experiments.

Ca2+-independent binding of an EF-hand domain to a novel motif in the alpha-actinin-titin complex.

The interaction between alpha-actinin and titin, two modular muscle proteins, is essential for sarcomere assembly. We have solved the solution structure of a complex between the calcium-insensitive C-terminal EF-hand domain of alpha-actinin-2 and the seventh Z-repeat of titin. The structure of the complex is in a semi-open conformation and closely resembles that of myosin light chains in their complexes with heavy chain IQ motifs. However, no IQ motif is present in the Z-repeat, suggesting that the semi-open conformation is a general structural solution for calcium-independent recognition of EF-hand domains.

Structure of Cdc42 bound to the GTPase binding domain of PAK.

The Rho family GTPases, Cdc42, Rac and Rho, regulate signal transduction pathways via interactions with downstream effector proteins. We report here the solution structure of Cdc42 bound to the GTPase binding domain of alphaPAK, an effector of both Cdc42 and Rac. The structure is compared with those of Cdc42 bound to similar fragments of ACK and WASP, two effector proteins that bind only to Cdc42. The N-termini of all three effector fragments bind in an extended conformation to strand beta2 of Cdc42, and contact helices alpha1 and alpha5. The remaining residues bind to switches I and II of Cdc42, but in a significantly different manner. The structure, together with mutagenesis data, suggests reasons for the specificity of these interactions and provides insight into the mechanism of PAK activation.

The structure of mouse HP1 suggests a unique mode of single peptide recognition by the shadow chromo domain dimer.

The heterochromatin protein 1 (HP1) family of proteins is involved in gene silencing via the formation of heterochromatic structures. They are composed of two related domains: an N-terminal chromo domain and a C-terminal shadow chromo domain. Present results suggest that chromo domains may function as protein interaction motifs, bringing together different proteins in multi-protein complexes and locating them in heterochromatin. We have previously determined the structure of the chromo domain from the mouse HP1beta protein, MOD1. We show here that, in contrast to the chromo domain, the shadow chromo domain is a homodimer. The intact HP1beta protein is also dimeric, where the interaction is mediated by the shadow chromo domain, with the chromo domains moving independently of each other at the end of flexible linkers. Mapping studies, with fragments of the CAF1 and TIF1beta proteins, show that an intact, dimeric, shadow chromo domain structure is required for complex formation.

Structure of the small G protein Cdc42 bound to the GTPase-binding domain of ACK.

The proteins Cdc42 and Rac are members of the Rho family of small GTPases (G proteins), which control signal-transduction pathways that lead to rearrangements of the cell cytoskeleton, cell differentiation and cell proliferation. They do so by binding to downstream effector proteins. Some of these, known as CRIB (for Cdc42/Rac interactive-binding) proteins, bind to both Cdc42 and Rac, such as the PAK1-3 serine/threonine kinases, whereas others are specific for Cdc42, such as the ACK tyrosine kinases and the Wiscott-Aldrich-syndrome proteins (WASPs). The effector loop of Cdc42 and Rac (comprising residues 30-40, also called switch I), is one of two regions which change conformation on exchange of GDP for GTP. This region is almost identical in Cdc42 and Racs, indicating that it does not determine the specificity of these G proteins. Here we report the solution structure of the complex of Cdc42 with the GTPase-binding domain ofACK. Both proteins undergo significant conformational changes on binding, to form a new type of G-protein/effector complex. The interaction extends the beta-sheet in Cdc42 by binding an extended strand from ACK, as seen in Ras/effector interactions, but it also involves other regions of the G protein that are important for determining the specificity of effector binding.

An approach to global fold determination using limited NMR data from larger proteins selectively protonated at specific residue types.

A combination of calculation and experiment is used to demonstrate that the global fold of larger proteins can be rapidly determined using limited NMR data. The approach involves a combination of heteronuclear triple resonance NMR experiments with protonation of selected residue types in an otherwise completely deuterated protein. This method of labelling produces proteins with alpha-specific deuteration in the protonated residues, and the results suggest that this will improve the sensitivity of experiments involving correlation of side-chain ((1)H and (13)C) and backbone ((1)H and (15)N) amide resonances. It will allow the rapid assignment of backbone resonances with high sensitivity and the determination of a reasonable structural model of a protein based on limited NOE restraints, an application that is of increasing importance as data from the large number of genome sequencing projects accumulates. The method that we propose should also be of utility in extending the use of NMR spectroscopy to determine the structures of larger proteins.