Improving two-dimensional proton NMR NOESY spectra of a large protein by selective deuteriation

Reisman, Joseph M.; Jariel-Encontre, Isabelle; Hsu, Victor L.; Parello, Joseph; Geiduschek, E. Peter; Kearns, David R.

doi:10.1021/ja00007a083

Cited by 23 publications

(16 citation statements)

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“…The 'Hlabeled variants are needed to establish these dipolar links between the aromatic protons and the corresponding aliphatic protons of each residue. As previously reported (Reisman et al, 1991), many of these through-space correlations are not observed in the TFl-'H NOESY spectra. Upon selective 'Hlabeling, however, these aromatic-to-aliphatic correlations were clearly observed.…”

Section: Assignment and Analytical Strategiessupporting

confidence: 78%

“…For this reason, we have employed a selective 'H-labeling approach (Torchia et al, 1988;Arrowsmith et al, 1991;Reisman et al, 1991) toward solving the solution structure of TF1.…”

Section: Hu Sequences (A)mentioning

confidence: 99%

“…For example, if the selected unlabeled amino acids are Phe, Gly, Ile and Tyr, and the remaining amino acids are 'H-labeled, the variant protein is denoted by TFl-'H[F, G, I, Y]. Selectively 'H-labeled isotopic variants of TF1 yield simplified spectra and allow for improved analysis of two-dimensional NOESY data (Reisman et al, 1991 ;Pachter et al, 1992).…”

Section: Hu Sequences (A)mentioning

confidence: 99%

“…A selective 'H-labeling approach has been employed with the objective of obtaining a solution structure for the 22-kDa transcription factor 1 (TF1) encoded by Bacillus subtilis phage SPOl by high-resolution 'H-NMR (Reisman et al, 1991). TF1 is a homodimeric type I1 DNA-binding protein (DBP 11); proteins of this family are highly basic, dimeric and found in all bacteria (Pettijohn, 1988).…”

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confidence: 99%

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A ¹H‐NMR study of the transcription factor 1 from Bacillus subtilis phage SPO1 by selective ²H‐labeling

Reisman¹,

Hsu²,

Jariel-Encontre³

et al. 1993

European Journal of Biochemistry

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'H-NMR experiments have been performed on transcription factor 1 (TF1) encoded by Bacillus subtilis phage SPOl. To study this 22-kDa homodimeric DNA-binding protein, a selective 'Hlabeling strategy has been employed. Complete sequence-specific assignments of all the resonances from the five aromatic residues were determined by a modified standard sequential-assignment procedure. The reduced contribution of spin diffusion upon the long-mixing-time nuclear-overhauser-enhancement spectroscopy for the selectively 'H-labeled variants, as opposed to the fully 'Hcontaining protein, has allowed for the identification of the spin systems and of the long-range dipolar contacts between Phe28 and Phe47 protons in the protein core and between Phe61 and Phe97 protons. The latter suggests an interaction between the proposed P-ribbon DNA-binding arm and the carboxy terminus of the paired monomer. A previously proposed TF1 structural model [Geiduschek, E. P., Schneider, G. J. & Sayre, M. H. (1990) J. Struct. Biol. 104,[84][85][86][87][88][89][90]] has been modified using constrained-energy-minimization calculations incorporating the experimentally determined set of aromatic-to-aromatic contacts. This new model has been analyzed with regard to the relative mobility and the relative solvent accessibility of the aromatic residues which have been measured by the nonselective T, relaxation times of the aromatic resonances for the fully 'Hcontaining protein and the relaxation time enhancements upon selective 2H-labeling, respectively.

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Section: Assignment and Analytical Strategiessupporting

confidence: 78%

“…For this reason, we have employed a selective 'H-labeling approach (Torchia et al, 1988;Arrowsmith et al, 1991;Reisman et al, 1991) toward solving the solution structure of TF1.…”

Section: Hu Sequences (A)mentioning

confidence: 99%

Section: Hu Sequences (A)mentioning

confidence: 99%

mentioning

confidence: 99%

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A ¹H‐NMR study of the transcription factor 1 from Bacillus subtilis phage SPO1 by selective ²H‐labeling

Reisman¹,

Hsu²,

Jariel-Encontre³

et al. 1993

European Journal of Biochemistry

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“…An attractive approach is the replacement of protons by deuterium in order to decrease the efficiency of 13 C relaxation due to their attached 1 H. The application of deuteration for protein assignment and structure determination has been explored in several publications 50 - 54 . Results proposed that, while 100% deuteration gives maximum sensitivity for backbone assignment experiments HNCOCA and HNCA, 53 , 54 maximum sensitivity for side chain assignment experiments were achieved with 50% deuteration 52 . For NOESY experiments, deuteration affects HN-HN, HN-HC and HC-HC cross peaks in different ways, 55 but 50% is a useful compromise.…”

Section: Resultsmentioning

confidence: 99%

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

Cao

Buck²

2011

Small GTPases

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Rho GTPases of the Ras superfamily have important roles in regulating the organization of the actin filament system, morphogenesis and migration of cells. Structural details for these proteins are still emerging, and information on their dynamics in solution is much needed to understand the mechanisms underlying their signaling functions. This report reviews conditions for solution NMR studies of Rho GTPases and describes our optimization and stabilization of Rnd1 for such experiments. Rnd1 belongs to the Rnd protein subfamily branch of Rho small GTPases and functions in neurite outgrowth, dendrite development and in axon guidance. However, as we report here, solution NMR studies of this protein are challenging. Multiple methods have been employed to enhance the stability of Rnd1, including by cleavage of an N-terminal His expression tag and by addition of non-hydrolysable GMPPNP (β: γ-imidoguanosine 5'-triphosphate) nucleotide. Further stabilization of Rnd1 against aggregation was achieved through a structure informed point mutation while maintaining its conformation and binding affinity for a partner protein. The NMR spectrum of the optimized protein reveals significant improvement in NMR signal dispersion and intensity. This work paves the way for structural and protein-protein/protein-ligand interaction studies of Rnd1 by solution NMR and also provides a guide for optimization and stabilization of other Rho GTPases.

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Multidimensional 2H-Based NMR Methods for Resonance Assignment, Structure Determination, and The Study of Protein Dynamics

Gardner

Kay

2002

Modern Techniques in Protein NMR

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Improving two-dimensional proton NMR NOESY spectra of a large protein by selective deuteriation

Cited by 23 publications

References 1 publication

A ¹H‐NMR study of the transcription factor 1 from Bacillus subtilis phage SPO1 by selective ²H‐labeling

A ¹H‐NMR study of the transcription factor 1 from Bacillus subtilis phage SPO1 by selective ²H‐labeling

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

Multidimensional 2H-Based NMR Methods for Resonance Assignment, Structure Determination, and The Study of Protein Dynamics

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Improving two-dimensional proton NMR NOESY spectra of a large protein by selective deuteriation

Cited by 23 publications

References 1 publication

A 1H‐NMR study of the transcription factor 1 from Bacillus subtilis phage SPO1 by selective 2H‐labeling

A 1H‐NMR study of the transcription factor 1 from Bacillus subtilis phage SPO1 by selective 2H‐labeling

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

Multidimensional 2H-Based NMR Methods for Resonance Assignment, Structure Determination, and The Study of Protein Dynamics

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Product

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A ¹H‐NMR study of the transcription factor 1 from Bacillus subtilis phage SPO1 by selective ²H‐labeling

A ¹H‐NMR study of the transcription factor 1 from Bacillus subtilis phage SPO1 by selective ²H‐labeling