Optimization and stabilization of Rho small GTPase proteins for solution NMR studies

Cao, Shufen; Buck, Matthias

doi:10.4161/sgtp.19257

Cited by 3 publications

(1 citation statement)

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“…Cdc42 (Feltham et al 1997;Oswald 2006, 2007;Buck et al 2004). Rac1 (Thapar et al 2003;Bouguet-Bonnet and Buck, 2006;Modha et al 2008;Lian et al 2000) and RhoA Mazhab-Jafari et al 2010;Cierpicki et al 2009) have been extensively studied by solution NMR, direct NMR studies of other Rnd1 have only been very recently reported (Cao and Buck 2012). Although optimization made the protein more suitable for solution NMR study, it appears the Rnd1 protein is still more unstable and more dynamic than other GTPases, allowing us to only obtain partial assignments.…”

Section: Biological Contextmentioning

confidence: 97%

Backbone assignment and secondary structure of Rnd1, an unusual Rho family small GTPase

et al. 2012

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Rho GTPases have attracted considerable interest as signaling molecules due to their variety of functional roles in cells. Rnd1 is a relatively recently discovered Rho GTPase with no enzymatic activity against its bound GTP nucleotide, setting it apart from other family members. Research has revealed a critical role for Rnd1 not only in neurite outgrowth, dendrite development, axon guidance, but also in gastric cancer and in endothelial cells during inflammation. Structural information is crucial for understanding the mechanism that forms the basis for protein-protein interactions and functions, but until recently there were no reports of NMR studies directly on the Rnd1 protein. In this paper we report assignments for the majority of Rnd1 NMR resonances based on 2D and 3D NMR spectra. Rnd1 assignment was a challenging task, however, despite optimization strategies that have facilitated NMR studies of the protein (Cao and Buck in Small GTPase 2:295-304, 2012). Besides common triple-resonance experiments, 3D HNCA, 3D HN(CO)CA, 3D HNCO which are usually employed for sequence assignment, 3D NOESY experiments and specific labeling of 13 kinds of amino acids were also utilized to gain as many (1)H(N), (13)C, and (15)N resonances assignments as possible. For 170 cross peaks observed out of 183 possible mainchain N-H correlations in the (1)H-(15)N TROSY spectrum, backbone assignment was finally completed for 127 resonances. The secondary structure was then defined by chemical shifts and TALOS+ based on the assignments. The overall structure in solution compares well with that of Rnd1 in a crystal, except for two short segments, residues 77-83 and residues 127-131. Given that some features are shared among Rho GTPases, Rnd1 assignments are also compared with two other family members, Cdc42 and Rac1. The overall level of Rnd1 assignment is lower than for Cdc42 and Rac1, consistent with its lower stability and possibly increased internal dynamics. However, while the Rnd1 switch II region remained un-assigned, the switch I region could be more fully assigned compared to Cdc42 and Rac1. The NMR assignment and structure analysis reported here provides a robust basis for future study of the binding between Rnd1 and other proteins, as well as for further studies of the molecular function of this unusual GTPase.

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Section: Biological Contextmentioning

confidence: 97%

Backbone assignment and secondary structure of Rnd1, an unusual Rho family small GTPase

et al. 2012

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Characterizing Plexin GTPase Interactions Using Gel Filtration, Surface Plasmon Resonance Spectrometry, and Isothermal Titration Calorimetry

Muller‐Greven

Kim

Hota

et al. 2016

Methods in Molecular Biology

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Plexins are unique, as they are the first example of a transmembrane receptor that interacts directly with small GTPases, a family of proteins that are essential for cell motility and proliferation/survival. We and other laboratories have determined the structure of the Rho GTPase-binding domain (RBD) of several plexins and also of the entire intracellular region of plexin-B1. Structures of plexin complexes with Rho GTPases, Rac1 and Rnd1, and a structure with a Ras GTPase, Rap1b, have also been solved. The relationship between plexin-Rho and plexin-Ras interactions is still unclear and in vitro biophysical experiments that characterize the protein interactions of purified components play an important role in advancing our understanding of the molecular mechanisms that underlie the function of plexin. This chapter describes the use of gel filtration (also known as size-exclusion chromatography or SEC), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) in studies of plexin-small GTPase interactions with plexin-B1:Rac1 as an example. Together with other assays and manipulations (e.g., by mutagenesis or protein domain truncation/deletion), these in vitro measurements provide an important reference for the role and extent of the interactions.

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STI1 antagonizes cytoskeleton collapse mediated by small GTPase Rnd1 and regulates neurite growth

Souza

Costa

Bilek

et al. 2014

Experimental Cell Research

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Optimization and stabilization of Rho small GTPase proteins for solution NMR studies

Cited by 3 publications

References 57 publications

Backbone assignment and secondary structure of Rnd1, an unusual Rho family small GTPase

Backbone assignment and secondary structure of Rnd1, an unusual Rho family small GTPase

Characterizing Plexin GTPase Interactions Using Gel Filtration, Surface Plasmon Resonance Spectrometry, and Isothermal Titration Calorimetry

STI1 antagonizes cytoskeleton collapse mediated by small GTPase Rnd1 and regulates neurite growth

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