Chapter 9
The Ubiquitin NMR Resource 1
Downloaded by STONY BROOK UNIV SUNY on December 14, 2016 | http://pubs.acs.org Publication Date: August 16, 2007 | doi: 10.1021/bk-2007-0969.ch009
Richard Harris and Paul C. Driscoll
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Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, United Kingdom National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom 2
We describe the ubiquitin NMR resource (http://www.biochem.ucl.ac.uk/bsm/nmr/ubq), which evolved from the need to teach postgraduate students, within our laboratory, the process of obtaining sequence specific resonance assignments. Ubiquitin was chosen as a model protein, because it is commercially available and high quality NMR spectra can be acquired in a relatively short period of time. The data in this resource can be used as a practical demonstration of obtaining sequence specific resonance assignments, which provides an introduction to triple resonance NMR experiments and how the structures of the individual amino acid types give rise to different ranges of Cα and Οβ chemical shifts. From the assignment of the backbone resonances a comparison of the chemical shifts of the Cα, Cβ and CO resonances to random coil values allows for a straightforward prediction of the secondary structure content. All the software used in the resource for processing and spectral analysis isfreelyavailablefromthe Internet.
Introduction Ubiquitin is a small protein (76 amino acid residues) that derives its name from its occurrence throughout the plant and animal kingdoms (1,2). The threedimensional structure of ubiquitin has been extensively characterized by both X-ray crystallography and NMR spectroscopy (3, 4) and comprises a 114
© 2007 American Chemical Society
Rovnyak and Stockland; Modern NMR Spectroscopy in Education ACS Symposium Series; American Chemical Society: Washington, DC, 2007.
115 combination of alpha helices and beta strands in a ββαββαβ motif, with a chain topology now known as the ubiquitin fold. Over-expression of N- and C/ Nisotope labeled ubiquitin in Escherichia coli regularly yields tens of milligrams per litre of media and, as such, represents an economic method to provide a stable, low molecular weight, non-aggregating, highly soluble protein that is ideal for testing new NMR pulse sequences and other aspects of the application of NMR methodology. The Ubiquitin NMR Resource, described here, comprises a series of NMR spectra acquired on commercially available samples of N-labeled and doublelabeled C/ N ubiquitin (5mg in ca. 300uL 20mM potassium phosphate, pH 5.8, VLI Research Inc.). The spectra are divided into three archives: (i) triple resonance experiments for assignment of the backbone resonances; (ii) experiments to obtain side chain assignments; and (iii) spectra for assignment of inter-proton distance restraints (Table 1). Each archive contains the raw Varian FID data, made available to be processed according to taste, an NMRpipe (5) script for referencing information and for outputting the data into a format for the user's preferred NMR analysis software, and pre-processedfilesready to be analyzed using the program ANSIG (6). A discussion of NMR data processing is beyond the scope of this chapter, however most general NMR texts review this area (for example Rule and Hitchens (7)); for a more in-depth treatise on data processing see Hoch and Stern (8). 15
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Table 1. Experiments acquired on N or C/ N ubiquitin at 25°C (mixing times given in parenthesis) Archive 1 ,5
Archive 2
Archive 3
2D 'Η, Ν HSQC
2D ^ " C HSQC
2D *Η, Ν HSQC
3DHNCA
2D (HC)CH-TOCSY
3D N TOCSY-HSQC (80ms)
3D HN(CO)CA
3D H(C)CH-TOCSY (7ms)
3D NNOESY-HSQC (100ms)
3D HNCACB
3D H(C)CH-TOCSY (21ms) 2D H, C CT-HSQC
3D HN(COCA)CB
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3D C NGESY-HSQC (100ms)
3DHNCO 3D HN(CA)CO For an explanation of the nomenclature see (9, 10) The NMR study of any protein, whether aimed at the investigation of ligand binding, dynamics or determination of the three-dimensional solution structure, will require the cross peaks in the 2D ^^N-heteronuclear single quantum correlation spectroscopy (HSQC) spectrum to be assigned to their individual amino acid residues. The 'H^N-HSQC is often described as a "fingerprint" of
Rovnyak and Stockland; Modern NMR Spectroscopy in Education ACS Symposium Series; American Chemical Society: Washington, DC, 2007.
116 the protein because every amino acid (except proline) contains a backbone amide group that should give rise to a single cross peak that correlates the amide proton and amide nitrogen. Figure 1 shows the (already-assigned) H, N-HSQC spectrum of human ubiquitin acquired on a Varian UnityPlus spectrometer operating at a nominal Ήfrequencyof 600MHz. For small proteins, such as ubiquitin, the assignment process is relatively straightforward and may by achieved with good accuracy by "traditional" analysis of three-dimensional Nedited TOCSY-HSQC and NOESY-HSQC spectra (10). !
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