Both liquid- and solid-state NMR have become major techniques in structural biology. Liquid-state NMR is a powerful, robust and noninvasive technique to investigate soluble proteins at atomic resolution (Wüthrich 1990). Compared to X-ray crystallography, solution NMR allows not only to investigate the protein structures in a nearly physiological environment, but is also to determine their dynamic properties.
Moreover, solution NMR technique allows the study protein-ligand and protein-protein interactions, protein folding, kinetics, catalysis and moreover structure determination.
1.7.1 Relevance of structure determination using NMR
Many biological activities are regulated by the interaction of proteins with nucleic acids and other biomacromolecules. Therefore it is quite essential to study these interactions both spatially and temporally. On top of that, it is important to investigate the mechanism involved in interactions including the specificity and selectivity.
Structural analysis at atomic-resolution will give deep insight in understanding these aspects. X-ray crystallography and NMR are the widely used techniques for structural analysis of biomacromolecules. X-ray crystallography is most suitable for high molecular weight systems where crystallization is possible. Whereas NMR is highly flexible and robust in terms of sample conditions and handling although there is a certain degree of size limitation.
It is becoming more relevant to study the protein-protein interactions that form even macromolecular complexes as many biological processes are mediated by interactions between proteins. The involvement of protein interactions with its partner proteins, peptides and drugs makes it important to study them at a molecular level which thereby helps in understanding the molecular basis of diseases.
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1.7.2 Application of NMR to structure determination of biomacromolecules
Structure determination of protein complexes using NMR has certain size limitations and is generally upto ~ 40kDa. The implementation of Transverse Relaxation Optimized SpectroscopY (TROSY) (Pervushin, Riek et al. 1997) in the traditional NMR methods and other NMR methodologies brought the size limitation near 100kDa (Pervushin, Riek et al. 1997, Riek, Wider et al. 1999, Riek, Fiaux et al. 2002). Isotope labeling and other methodological developments are the accelerating factors in NMR based structural calculations. NMR based 3D structure determination of biomolecules is usually performed by distance geometry calculations or simulated annealing (Güntert, Mumenthaler et al. 1997, GÜNTERT 1998, Schwieters, Kuszewski et al. 2003).
Structural constraints can be derived from NOE, J-coupling, Residual Dipolar Couplings (RDC), Paramagnetic Relaxation Enhancement (PRE) etc. Another feasible approach is to map the binding interface of large complexes if the exchange is in the NMR time scale is suitable for signal detection.
The strategy of structure determination by NMR depends on the timescales of chemical or conformational exchange processes, which may affect line shapes, relaxation rates, and chemical shifts of resonances. Depending on the strength of the interaction of a complex, it can be broadly classified into slow exchange, intermediate exchange and fast exchange. That is strong binding, weak binding and the intermediate exchange case, called coalescence.
1.7.3 Protein-ligand interactions by NMR spectroscopy
NMR spectroscopy is a useful technique in studying protein and protein-ligand interaction. The study of these interactions can be broadly classified into two classes on the basis of the target resonances in-study. (1) Focusing the protein resonances and (2) Ligand resonance studies (Carlomagno 2005). There are several factors that determine this selection. In the case of target resonance detected experiments it relies on the availability of the isotopically labeled target and the size of which is suitable to detect by NMR. Here mapping the binding interface of the target is possible with the help of
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two dimensional heteronuclear correlation experiments (eg: HSQC) (Bodenhausen and Ruben 1980). Whereas in the ligand detected methods such as transferred NOE (tr-NOE), saturation transfer difference (STD) (Mayer and Meyer 1999, Viegas, Manso et al. 2011), NOE pumping (Chen and Shapiro 1998), Water LOGSY (Dalvit, Pevarello et al. 2000) and INPHARMA (Sánchez-Pedregal, Reese et al. 2005, Orts, Griesinger et al.
2009), only the resonances of the ligand is observed and is exclusively applicable for the ligands in the low affinity range (millimolar range). Latter approach is highly desirable when the target is either too large to be detected by NMR or not available in the isotopically labeled form. Binding interface information can be obtained by following NMR spectral parameters:
(a) Line broadening
(b) Chemical shift perturbation (c) Change in NOE
(d) Intermolecular magnetization transfer
NMR chemical shifts are very sensitive to variations in the local electronic environment, for example, due to binding. Hence it is possible to map the binding interface of weakly binding ligand and protein receptor using the small changes in 1H and 15N shifts in a calibrated titration. The ligand can either be a protein, peptide or small molecule. No chemical shift changes will be observed for residues that do not participate in the interaction and those resonances that are shifted highlight the residues involved in the binding interface or are affected by minor conformational rearrangements caused by the interaction. Here, the intensity of the resonances will be varying according to the exchange rate and if it is near coalescence condition there will have severe line broadening that will again reflect the involvement of the respective regions of protein involved in binding.
Considering the Tau-tubulin/MT interaction, where tubulin is ~100kDa and MTs is in the range of several megadaltons it is beyond the limit of NMR detection. The application of sophisticated NMR methods is very much limited in the case of tubulin as it cannot be obtained in the isotopically labelled form. Till now the recombinant expression of tubulin in significant amount is unsuccessful because of their complexity in the folding (Clement, Savarin et al. 2010). However there are several studies involving
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tubulin and its binding partners as smaller drug molecules, peptides where the exchange transferred methods (Carlomagno 2005, Sánchez-Pedregal, Reese et al. 2005, Orts, Griesinger et al. 2009) have been employed. These include tr-NOE, STD-NMR, tr-CCR and INPHARMA and are detailed in the section methods.