Correction: Dynein and Dynactin Leverage Their Bivalent Character to Form a High-Affinity Interaction.

[This corrects the article DOI: 10.1371/journal.pone.

Functional Expression and Characterization of Human Myristoylated-Arf1 in Nanodisc Membrane Mimetics.

Lipidated small GTP-binding proteins of the Arf family interact with multiple cellular partners and with membranes to regulate intracellular traffic and organelle structure. Here, we focus on the ADP-ribosylation factor 1 (Arf1), which interacts with numerous proteins in the Arf pathway, such as the ArfGAP ASAP1 that is highly expressed and activated in several cancer cell lines and associated with enhanced migration, invasiveness, and poor prognosis. Understanding the molecular and mechanistic details of Arf1 regulation at the membrane via structural and biophysical studies requires large quantities of fully functional protein bound to lipid bilayers.

Efficient and generalized processing of multidimensional NUS NMR data: the NESTA algorithm and comparison of regularization terms.

The advantages of non-uniform sampling (NUS) in offering time savings and resolution enhancement in NMR experiments have been increasingly recognized. The possibility of sensitivity gain by NUS has also been demonstrated. Application of NUS to multidimensional NMR experiments requires the selection of a sampling scheme and a reconstruction scheme to generate uniformly sampled time domain data.

Sensitivity gains, linearity, and spectral reproducibility in nonuniformly sampled multidimensional MAS NMR spectra of high dynamic range.

Recently, we have demonstrated that considerable inherent sensitivity gains are attained in MAS NMR spectra acquired by nonuniform sampling (NUS) and introduced maximum entropy interpolation (MINT) processing that assures the linearity of transformation between the time and frequency domains. In this report, we examine the utility of the NUS/MINT approach in multidimensional datasets possessing high dynamic range, such as homonuclear (13)C-(13)C correlation spectra. We demonstrate on model compounds and on 1-73-(U-(13)C,(15)N)/74-108-(U-(15)N) E.

Dynein and dynactin leverage their bivalent character to form a high-affinity interaction.

Cytoplasmic dynein and dynactin participate in retrograde transport of organelles, checkpoint signaling and cell division. The principal subunits that mediate this interaction are the dynein intermediate chain (IC) and the dynactin p150(Glued); however, the interface and mechanism that regulates this interaction remains poorly defined. Herein, we use multiple methods to show the N-terminus of mammalian dynein IC, residues 10-44, is sufficient for binding p150(Glued).

A time-saving strategy for MAS NMR spectroscopy by combining nonuniform sampling and paramagnetic relaxation assisted condensed data collection.

We present a time-saving strategy for acquiring 3D magic angle spinning NMR spectra for chemical shift assignments in proteins and protein assemblies in the solid state. By simultaneous application of nonuniform sampling (NUS) and paramagnetic-relaxation-assisted condensed data collection (PACC), we can attain 16-fold time reduction in the 3D experiments without sacrificing the signal-to-noise ratio or the resolution. We demonstrate that with appropriate concentration of paramagnetic dopant introduced into the sample the overwhelming majority of chemical shifts are not perturbed, with the exception of a limited number of shifts corresponding to residues located at the surface of the protein, which exhibit small perturbations.

Solid-state NMR spectroscopy of protein complexes.

Protein-protein interactions are vital for many biological processes. These interactions often result in the formation of protein assemblies that are large in size, insoluble, and difficult to crystallize, and therefore are challenging to study by structure biology techniques, such as single crystal X-ray diffraction and solution NMR spectroscopy. Solid-state NMR (SSNMR) spectroscopy is emerging as a promising technique for studies of such protein assemblies because it is not limited by molecular size, solubility, or lack of long-range order.

Spin diffusion driven by R-symmetry sequences: applications to homonuclear correlation spectroscopy in MAS NMR of biological and organic solids.

We present a family of homonuclear (13)C-(13)C magic angle spinning spin diffusion experiments, based on R2(n)(v) (n = 1 and 2, v = 1 and 2) symmetry sequences. These experiments are well suited for (13)C-(13)C correlation spectroscopy in biological and organic systems and are especially advantageous at very fast MAS conditions, where conventional PDSD and DARR experiments fail. At very fast MAS frequencies the R2(1)(1), R2(2)(1), and R2(2)(2) sequences result in excellent quality correlation spectra both in model compounds and in proteins.

Resonance Assignments and Secondary Structure Analysis of Dynein Light Chain 8 by Magic Angle Spinning NMR Spectroscopy.

Dynein light chain LC8 is the smallest subunit of the dynein motor complex and has been shown to play important roles in both dynein dependent and dynein independent physiological functions via its interaction with a number of its binding partners. It has also been linked to pathogenesis including roles in viral infections and tumorigenesis. Structural information for LC8-target proteins is critical to understanding the underlying function of LC8 in these complexes.

Disease-associated mutations in the p150(Glued) subunit destabilize the CAP-gly domain.

Point mutations within the CAP-gly domain of the p150(Glued) subunit of the dynactin complex have been identified in patients with distal spinal bulbar muscular atrophy (dSBMA) and Perry's syndrome. Herein, we show by CD and NMR experiments that each mutated CAP-gly domain is folded but less stable than the wild-type (WT) domain. We also demonstrate that the domains harboring these mutations bind to microtubules but fail to bind to EB1.

Solid-state and solution NMR studies of the CAP-Gly domain of mammalian dynactin and its interaction with microtubules.

Microtubules (MTs) and microtubule binding proteins (MTBPs) play fundamental physiological roles including vesicle and organelle transport, cell motility, and cell division. Despite the importance of the MT/MTBP assemblies, there remains virtually no structural or dynamic information about their interaction at the atomic level due to the inherent insolubility and lack of long-range order of MTs. In this study, we present a combined magic angle spinning solid-state and solution NMR study of the MTBP CAP-Gly domain of mammalian dynactin and its interaction with paclitaxel-stabilized microtubules.

Biochemical and structural characterization of the Pak1-LC8 interaction.

Pak1 (p21-activated kinase-1) and the dynein light chain, LC8, are overexpressed in breast cancer, and their direct interaction has been proposed to regulate tumor cell survival. These effects have been attributed in part to Pak1-mediated phosphorylation of LC8 at serine 88. However, LC8 is homodimeric, which renders Ser(88) inaccessible.

Dynamically driven protein allostery.

Allosteric interactions are typically considered to proceed through a series of discrete changes in bonding interactions that alter the protein conformation. Here we show that allostery can be mediated exclusively by transmitted changes in protein motions. We have characterized the negatively cooperative binding of cAMP to the dimeric catabolite activator protein (CAP) at discrete conformational states.