scholarly journals Conformational Flexibility of p150Glued(1-191) Subunit of Dynactin Assembled with Microtubules

2019 ◽  
Author(s):  
C. Guo ◽  
J. C. Williams ◽  
T. Polenova
Keyword(s):  
Magic Angle Spinning ◽  
Magic Angle ◽  
Binding Interactions ◽  
Microtubule Binding ◽  
Basic Domain ◽  
Intrinsically Disordered ◽  
Rich Domain ◽  
Associated Proteins ◽  
Angle Spinning ◽  
High Degree

ABSTRACTMicrotubule-associated proteins (MAPs) perform diverse functions in cells. These functions are dependent on their interactions with microtubules. Dynactin, a cofactor of dynein motor, assists the binding of dynein to various organelles and is crucial to the long-distance processivity of dynein-based complexes. The largest subunit of dynactin, the p150glued, contains a N-terminus segment that is responsible for the microtubule-binding interactions and long-range processivity of dynactin. We employed solution and magic angle spinning NMR spectroscopy to characterize the structure and dynamics of the p150glued N-terminal region, both free and in complex with polymerized microtubules. This 191-residue region encompasses the CAP-Gly domain, the basic domain and serine-proline-rich (SP-rich) domain. We demonstrate that the basic and SP-rich domains are intrinsically disordered in solution and significantly enhance the binding affinity to microtubules as these regions contain the second microtubule-binding site on the p150Glued subunit. The majority of the basic and SP-rich domains are predicted to be random-coil, while the segments S111–I116, A124–R132 and K144–T146 in the basic domain contain short α-helical or β-sheet structures. These three segments possibly encompass the microtubule binding site. Surprisingly, the protein retains high degree of flexibility upon binding to microtubules except for the regions that are directly involved in the binding interactions with microtubules. This conformational flexibility may be essential for the biological functions of the p150Glued subunit.STATEMENT OF SIGNIFICANCEMicrotubule-associated proteins (MAPs) perform diverse functions in cells. Many of them comprises intrinsically disordered regions, whose structural flexibility are central to microtubule-based cellular functions of MAPs. We employed solution and magic angle spinning NMR spectroscopy to characterize the structure and dynamics of the p150glued N-terminal region encompassing the CAP-Gly domain, the basic domain and serine-proline-rich (SP-rich) domain, both free and in complex with polymerized microtubules. The results reveal that the basic and SP-rich domains are largely unstructured and retains high degree of flexibility upon binding to microtubules except for the regions that are possibly involved in the binding interactions with microtubules. This approach is informative for dynamics studies of intrinsically disordered MAPs and other disordered proteins in large biological assemblies.

scholarly journals Atomic-resolution structure of the CAP-Gly domain of dynactin on polymeric microtubules determined by magic angle spinning NMR spectroscopy

2015 ◽  
Vol 112 (47) ◽  
pp. 14611-14616 ◽  
Author(s):  
Si Yan ◽  
Changmiao Guo ◽  
Guangjin Hou ◽  
Huilan Zhang ◽  
Xingyu Lu ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Cell Biology ◽  
Double Resonance ◽  
Magic Angle Spinning ◽  
Atomic Resolution ◽  
Organelle Transport ◽  
Magic Angle ◽  
Associated Proteins ◽  
Angle Spinning ◽  
Resolution Structure

Microtubules and their associated proteins perform a broad array of essential physiological functions, including mitosis, polarization and differentiation, cell migration, and vesicle and organelle transport. As such, they have been extensively studied at multiple levels of resolution (e.g., from structural biology to cell biology). Despite these efforts, there remain significant gaps in our knowledge concerning how microtubule-binding proteins bind to microtubules, how dynamics connect different conformational states, and how these interactions and dynamics affect cellular processes. Structures of microtubule-associated proteins assembled on polymeric microtubules are not known at atomic resolution. Here, we report a structure of the cytoskeleton-associated protein glycine-rich (CAP-Gly) domain of dynactin motor on polymeric microtubules, solved by magic angle spinning NMR spectroscopy. We present the intermolecular interface of CAP-Gly with microtubules, derived by recording direct dipolar contacts between CAP-Gly and tubulin using double rotational echo double resonance (dREDOR)-filtered experiments. Our results indicate that the structure adopted by CAP-Gly varies, particularly around its loop regions, permitting its interaction with multiple binding partners and with the microtubules. To our knowledge, this study reports the first atomic-resolution structure of a microtubule-associated protein on polymeric microtubules. Our approach lays the foundation for atomic-resolution structural analysis of other microtubule-associated motors.

Magic angle spinning NMR spectroscopy of membrane proteins

1996 ◽  
Vol 29 (4) ◽  
pp. 395-449 ◽  
Author(s):  
Steven O. Smith ◽  
Kathryn. Aschheim ◽  
Michel Groesbeek
Keyword(s):  
Membrane Proteins ◽  
Electrostatic Interactions ◽  
Membrane Surface ◽  
Fatty Acyl ◽  
Magic Angle Spinning ◽  
Magic Angle ◽  
Associated Proteins ◽  
Acyl Chains ◽  
Membrane Spanning ◽  
Angle Spinning

The passage of molecules and information across cell membranes is mediated largely by membrane-spanning proteins acting as channels, pumps, receptors and enzymes. These proteins perform many tasks: they control electrochemical gradients across the membrane, receive signals from the environment or from other cells, convert light energy into chemical signals, transport small molecules into and out of cells, and harness proton gradients to generate the energy consumed in metabolism. Indeed, of the estimated 50000–100000 genes in the human genome, fully 20–40 % are thought to encode integral membrane proteins. If one also includes membrane-associated proteins, which are attached to the membrane surface through fatty acyl chains or electrostatic interactions, this percentage is likely to be much higher.

Preparation of Cellulose Succinates with High Degree of Substitution in Solvent System [C4mim]Cl/DMSO

2012 ◽  
Vol 554-556 ◽  
pp. 244-249 ◽  
Author(s):  
Deng Shan Bao ◽  
Chuan Fu Liu ◽  
Run Cang Sun
Keyword(s):  
Ionic Liquid ◽  
Succinic Anhydride ◽  
Solvent System ◽  
Magic Angle Spinning ◽  
Degree Of Substitution ◽  
Magic Angle ◽  
Cross Polarization ◽  
Angle Spinning ◽  
High Degree ◽  
Modified Cellulose

Homogeneous derivatization of cellulose was investigated with succinic anhydride (SA) in a solvent system containing 1-butyl-3-methylimidazolium chloride ionic liquid ([C4mim]Cl) and dimethylsulfoxide (DMSO) using iodine, N-bromosuccinimide (NBS), and 4-dimethylaminopyridine (DMAP) as a catalyst. The results showed that the high degree of substitution (DS) of modified cellulose significantly increased from 0.24 without any catalysts to 0.84, 2.31, and 2.34 under same conditions except with I2, NBS, and DMAP, respectively, as a catalyst. The possible mechanism of succinoylation catalysed with different catalysts was discussed. Fourier transform infrared and solid-state cross-polarization/magic angle spinning 13C NMR spectroscopies also provided evidences of catalyzed homogeneous succinoylation reaction. The results indicated that iodine, NBS, and DMAP could effectively improve the succinoylation efficiency of cellulose in [C4mim]Cl/DMSO.

Pulse Shaped Dynamic Nuclear Polarization Under Magic Angle Spinning

2019 ◽  
Author(s):  
ASIF EQUBAL ◽  
Kan Tagami ◽  
Songi Han
Keyword(s):  
Electron Spin ◽  
Lattice Relaxation ◽  
Magic Angle Spinning ◽  
Spin Lattice Relaxation ◽  
Magic Angle ◽  
Total Electron ◽  
Spin Lattice ◽  
Maximum Benefit ◽  
Selective Saturation ◽  
Angle Spinning

In this paper, we report on an entirely novel way of improving the MAS-DNP efficiency by shaped μw pulse train irradiation for fast and broad-banded (FAB) saturation of the electron spin resonance. FAB-DNP achieved with Arbitrary Wave Generated shaped μw pulse trains facilitates effective and selective saturation of a defined fraction of the total electron spins, and provides superior control over the DNP efficiency under MAS. Experimental and quantum-mechanics based numerically simulated results together demonstrate that FAB-DNP significantly outperforms CW-DNP when the EPR-line of PAs is broadened by conformational distribution and exchange coupling. We demonstrate that the maximum benefit of FAB DNP is achieved when the electron spin-lattice relaxation is fast relative to the MAS frequency, i.e. at higher temperatures and/or when employing metals as PAs. Calculations predict that under short T<sub>1e </sub>conditions AWG-DNP can achieve as much as ~4-fold greater enhancement compared to CW-DNP.

High Resolution Magic Angle Spinning NMR in Combinatorial Chemistry

2001 ◽  
Vol 4 (4) ◽  
pp. 333-351 ◽  
Author(s):  
G. Lippens ◽  
R. Warrass ◽  
J. Wieruszeski ◽  
P. Rousselot-Pailley ◽  
G. Chessari
Keyword(s):  
High Resolution ◽  
Combinatorial Chemistry ◽  
Magic Angle Spinning ◽  
Magic Angle ◽  
Angle Spinning

Wideline 2H -NMR Spectroscopy and Imaging of Solids

1994 ◽  
Vol 49 (1-2) ◽  
pp. 19-26 ◽  
Author(s):  
B. Blümich
Keyword(s):  
Nmr Spectroscopy ◽  
Excitation Power ◽  
Magic Angle Spinning ◽  
Magic Angle ◽  
2H Nmr ◽  
Stochastic Excitation ◽  
2H Nmr Spectroscopy ◽  
Spatially Inhomogeneous ◽  
Recent Developments ◽  
Angle Spinning

Abstract Recent developments, focussing on reduction of the rf excitation power by stochastic excitation, on improvements in sensitivity and excitation bandwidth by magic angle spinning, and on combining wideline spectroscopy with spatial resolution for investigations o f spatially inhomogeneous objects are reviewed.

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