Isotope labeling strategies for the study of high-molecular-weight proteins by solution NMR spectroscopy

Tugarinov, Vitali; Kanelis, Voula; Kay, Lewis E.

doi:10.1038/nprot.2006.101

Cited by 491 publications

(435 citation statements)

References 27 publications

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“…BL21(DE3) codonplus were transformed with pET21b plasmids encoding histones or HMGN2 and subsequently grown in M9 minimal medium containing desired isotopes at 37°C. Methyl labeling of Ile-δ1-[ 13 CH 3 ] and Val/Leu-[ 13 CH 3 , 12 CD 3 ] (ILV labeling) followed the procedure of Tugarinov et al (32). Histones and HMGN2 were purified as described before (33,34).…”

Section: Methodsmentioning

confidence: 99%

Architecture of the high mobility group nucleosomal protein 2-nucleosome complex as revealed by methyl-based NMR

Kato

Ingen

Zhou

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

166

189

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Chromatin structure and function are regulated by numerous proteins through specific binding to nucleosomes. The structural basis of many of these interactions is unknown, as in the case of the high mobility group nucleosomal (HMGN) protein family that regulates various chromatin functions, including transcription. Here, we report the architecture of the HMGN2-nucleosome complex determined by a combination of methyl-transverse relaxation optimized nuclear magnetic resonance spectroscopy (methyl-TROSY) and mutational analysis. We found that HMGN2 binds to both the acidic patch in the H2A-H2B dimer and to nucleosomal DNA near the entry/exit point, "stapling" the histone core and the DNA. These results provide insight into how HMGNs regulate chromatin structure through interfering with the binding of linker histone H1 to the nucleosome as well as a structural basis of how phosphorylation induces dissociation of HMGNs from chromatin during mitosis. Importantly, our approach is generally applicable to the study of nucleosome-binding interactions in chromatin.

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Section: Methodsmentioning

confidence: 99%

Architecture of the high mobility group nucleosomal protein 2-nucleosome complex as revealed by methyl-based NMR

Kato

Ingen

Zhou

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

166

189

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“…T. elongatus KaiA, KaiB, KaiC, and SasA and Saccharomyces cerevisiae Ulp1 proteins were expressed in Escherichia coli BL21(DE3) (Novagen). The U-½ 15 N; 2 H -Ile-δ1-½ 13 C; 1 H -labeled proteins were generated by following a slightly modified version of a published protocol (53). For details about protein expression and purification, please see SI Appendix, Figs.…”

Section: Methodsmentioning

confidence: 99%

Flexibility of the C-terminal, or CII, ring of KaiC governs the rhythm of the circadian clock of cyanobacteria

Chang

Kuo

Tseng

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

144

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In the cyanobacterial circadian oscillator, KaiA and KaiB alternately stimulate autophosphorylation and autodephosphorylation of KaiC with a periodicity of approximately 24 h. KaiA activates autophosphorylation by selectively capturing the A loops of KaiC in their exposed positions. The A loops and sites of phosphorylation, residues S431 and T432, are located in the CII ring of KaiC. We find that the flexibility of the CII ring governs the rhythm of KaiC autophosphorylation and autodephosphorylation and is an example of dynamics-driven protein allostery. KaiA-induced autophosphorylation requires flexibility of the CII ring. In contrast, rigidity is required for KaiC-KaiB binding, which induces a conformational change in KaiB that enables it to sequester KaiA by binding to KaiA's linker. Autophosphorylation of the S431 residues around the CII ring stabilizes the CII ring, making it rigid. In contrast, autophosphorylation of the T432 residues offsets phospho-S431-induced rigidity to some extent. In the presence of KaiA and KaiB, the dynamic states of the CII ring of KaiC executes the following circadian rhythm: CII ST flexible → CII SpT flexible → CII pSpT rigid → CII pST very-rigid → CII ST flexible . Apparently, these dynamic states govern the pattern of phosphorylation, ST → SpT → pSpT → pST → ST. CII-CI ring-on-ring stacking is observed when the CII ring is rigid, suggesting a mechanism through which the ATPase activity of the CI ring is rhythmically controlled. SasA, a circadian clock-output protein, binds to the CI ring. Thus, rhythmic ring stacking may also control clock-output pathways.

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“…31 A 20 ml LB culture was inoculated from a dense overnight culture. After reaching an OD 600 of 0.5, the cells were spun down and washed once with PBS.…”

Section: Trosy Nmrmentioning

confidence: 99%

Structural basis of fluorescence quenching in caspase activatable‐GFP

Nicholls

Hardy

2013

Protein Science

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Apoptosis is critical for organismal homeostasis and a wide variety of diseases. Caspases are the ultimate executors of the apoptotic programmed cell death pathway. As caspases play such a central role in apoptosis, there is significant demand for technologies to monitor caspase function. We recently developed a caspase activatable-GFP (CA-GFP) reporter. CA-GFP is unique due to its ''dark'' state, where chromophore maturation of the GFP is inhibited by the presence of a C-terminal peptide. Here we show that chromophore maturation is prevented because CA-GFP does not fold into the robust b-barrel of GFP until the peptide has been cleaved by active caspase. Both CA-GFP and GFP 1-10 , a split form of GFP lacking the 11th strand, have similar secondary structure, different from mature GFP. A similar susceptibility to proteolytic digestion indicates that this shared structure is not the robust, fully formed GFP b-barrel. We have developed a model that suggests that as CA-GFP is translated in vivo it follows the same folding path as wild-type GFP; however, the presence of the appended peptide does not allow CA-GFP to form the barrel of the fully matured GFP. CA-GFP is therefore held in a ''pro-folding'' intermediate state until the peptide is released, allowing it to continue folding into the mature barrel geometry. This new understanding of the structural basis of the dark state of the CA-GFP reporter will enable manipulation of this mechanism in the development of reporter systems for any number of cellular processes involving proteases and potentially other enzymes.

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Isotope labeling strategies for the study of high-molecular-weight proteins by solution NMR spectroscopy

Cited by 491 publications

References 27 publications

Architecture of the high mobility group nucleosomal protein 2-nucleosome complex as revealed by methyl-based NMR

Architecture of the high mobility group nucleosomal protein 2-nucleosome complex as revealed by methyl-based NMR

Flexibility of the C-terminal, or CII, ring of KaiC governs the rhythm of the circadian clock of cyanobacteria

Structural basis of fluorescence quenching in caspase activatable‐GFP

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