NMR analysis of methyl groups at 100-500 kDa: Model systems and Arp2/3 complex

Mara Kreishman-Deitrick, Coumaran Egile, David W. Hoyt, Joseph J. Ford, Rong Li, Michael K. Rosen

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Abstract

Large macromolecular machines are among the most important and challenging targets for structural and mechanistic analyses. Consequently, there is great interest in development of NMR methods for the study of multicomponent systems in the 50-500 kDa range. Biochemical methods also must be developed in concert to produce such systems in selectively labeled form. Here, we present 1H/13C-HSQC spectra of protonated methyl groups in a model system that mimics molecular weights up to ∼560 kDa. Signals from side chain methyl groups of Ile, Leu, and Val residues are clearly detectable at correlation times up to ∼330 ns. We have also developed a biochemical procedure to produce the 240 kDa, heteroheptameric Arp2/3 actin nucleation complex selectively labeled at one subunit and obtained 1H/13C-HSQC spectra of this assembly. Sensitivity in spectra of both the Arp2/3 complex and the model system indicate that methyl groups will be useful sources of information in nonsymmetric systems with molecular weights greater than 600 kDa at concentrations less than 100 μM. Methyl analyses will complement TROSY and CRINEPT analyses of amides in NMR studies of structure and molecular interactions of extremely large macromolecules and assemblies.

Original languageEnglish (US)
Pages (from-to)8579-8586
Number of pages8
JournalBiochemistry
Volume42
Issue number28
DOIs
StatePublished - Jul 22 2003

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Actin-Related Protein 2-3 Complex
Molecular weight
Nuclear magnetic resonance
Molecular interactions
Molecular Weight
Macromolecules
Amides
Actins
Nucleation
Molecular Structure

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kreishman-Deitrick, M., Egile, C., Hoyt, D. W., Ford, J. J., Li, R., & Rosen, M. K. (2003). NMR analysis of methyl groups at 100-500 kDa: Model systems and Arp2/3 complex. Biochemistry, 42(28), 8579-8586. https://doi.org/10.1021/bi034536j

NMR analysis of methyl groups at 100-500 kDa : Model systems and Arp2/3 complex. / Kreishman-Deitrick, Mara; Egile, Coumaran; Hoyt, David W.; Ford, Joseph J.; Li, Rong; Rosen, Michael K.

In: Biochemistry, Vol. 42, No. 28, 22.07.2003, p. 8579-8586.

Research output: Contribution to journalArticle

Kreishman-Deitrick, M, Egile, C, Hoyt, DW, Ford, JJ, Li, R & Rosen, MK 2003, 'NMR analysis of methyl groups at 100-500 kDa: Model systems and Arp2/3 complex', Biochemistry, vol. 42, no. 28, pp. 8579-8586. https://doi.org/10.1021/bi034536j
Kreishman-Deitrick M, Egile C, Hoyt DW, Ford JJ, Li R, Rosen MK. NMR analysis of methyl groups at 100-500 kDa: Model systems and Arp2/3 complex. Biochemistry. 2003 Jul 22;42(28):8579-8586. https://doi.org/10.1021/bi034536j
Kreishman-Deitrick, Mara ; Egile, Coumaran ; Hoyt, David W. ; Ford, Joseph J. ; Li, Rong ; Rosen, Michael K. / NMR analysis of methyl groups at 100-500 kDa : Model systems and Arp2/3 complex. In: Biochemistry. 2003 ; Vol. 42, No. 28. pp. 8579-8586.
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