Labeling backbone-labeled proteins

An advantage of NMR spectroscopy is the analysis of protein dynamics. Measurement and analysis of the relaxation parameters R1 R2, and the 15N NOE of 15N-labeled proteins leads to an order parameter (S2) that can describe the relative mobility of the backbone of the protein. Both collagenase-1 and stromelysin-1 have been studied either as inhibited complexes or the free protein [19, 52], Stromleysin-1 was studied with inhibitors binding to prime or nonprime subsites. Presence or absence of inhibitors in the nonprime sites had minor effects on the highly ordered structure of residues in these subsites, which are in contact with the... 

Boucher W, Laue ED, Campbell SL, Domaille PJ. Improved 4D NMR experiments for the assignment of backbone nuclei in 13C/15N labelled proteins. J Biomol NMR 1992 2 631-637. 

Backbone dynamics are most commonly investigated by measurement of 15N T and T% relaxation times and the fyH -15N NOE in uniformly 15N-labeled protein. To circumvent problems associated with the limited dispersion of the NMR spectra of unfolded proteins, the relaxation and NOE data are generally measured using 2D HSQC-based methods (Farrow et al., 1994 Palmer et al., 1991). 

The first experiment to be recorded on isotope-labeled proteins is the [ N/HJ-HSQC experiment. Inspection of the [ N. HJ-correlalion map and simple counting of cross peaks reveals whether multiple conformers exist, whether some parts of the backbone signals are broadened, possibly because of slow conformational exchange, or whether parts of the sequence are not visible at all. As mentioned above, the spectrum will also show whether the protein is well folded or not. 

Strip plots can only be constructed when the crosspeaks have already been assigned in the 2D HSQC spectrum. In a 15N-labeled protein, sequence-specific assignments come from sequential NOE (a,N, /3,N and N,N) crosspeaks located in the 3D HSQC-NOESY spectrum. The walk through the protein backbone is done in the same way as with unlabeled proteins, except that overlap in NOESY spectra is greatly reduced by spreading the crosspeaks out in the 15N dimension of a 3D spectrum. 

Most recently, Peti et al.225 have demonstrated the potential of pCoil for the chemical shift assignment of aromatic side-chain protons of proteins. Using the conserved hypothetical protein TM0979 from Thermotoga maritima, the authors demonstrated the sequence-specific backbone resonance assignments with < 500 pg of 13C15N-doubly labeled protein. 

The backbone and side-chain and N signals of a solid 62-residue (U- C, N)-labelled protein containing the a-spectrin SH3 domain were assigned by 2D MAS N- C and dipolar correlation spectroscopy at... 

Morelle, N., et al.. Computer Assignment of the Backbone Resonances of Labeled Proteins Using Two-Dimensional Correlation Experiments, J. Biomol. NMR, 5, 154,... 

It is necessary to assign at least the NMR resonances of atoms comprising the protein backbone prior to further site-specific NMR studies. This can be accomplished routinely by using a suite of triple-resonance experiments and uniformly 2H/13C/15N-labeled protein samples [35], However, the relative high concentrations (100-600 gM) necessary for assignment require a careful choice of measuring conditions. Only in some cases, the published... 

Logan TM, Olejniczak ET, Xu RX, Fesik SW (1992) Side chain and backbone assignments in isotopically labeled proteins from two heteronuclear triple resonance experiments. FEES Lett 314 413 18... 

Szyperski T, Wider G, Bushweller JH, Wiithrich K (1993) 3D 13 C-15 N-heteronuclear two-spin coherence spectroscopy for polypeptide backbone assignments in 13 C-15 N-double-labeled proteins. J Biomol NMR 3 127-132... 

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