Protein Dynamics and Adiabatic Fast Passage NMR Experiments

The functional properties of proteins are intimately linked to their dynamical features (e.g conformational plasticity). Most striking examples for the relevance of protein dynamics are protein folding and the exchange between conformational substates involved in enzyme allostery. NMR spectroscopy is a unique experimental technique for studying protein dynamics in solution as it provides dynamic information with atomic resolution (e.g. residue-specific motions). Measurement of heteronuclear relaxation rates probes molecular dynamics over a wide time range and provides important insight into the motional behaviour of protein in solution. Dynamic processes on microsecond to millisecond time scales are probed by a variety of different experimental schemes (e.g. Carr- Purcell-Meiboom-Gill, CPMG and/or offset T1 -dispersion measurements). Despite tremendous achievements in the past, experimental limitations, such as, for example, inevitable RF inhomogeniety can lead to pulse imperfections and may impair the extraction of reliable motional parameters. In this project it is planned to apply adiabatic fast passage techniques to studies of protein dynamics. Adiabatic inversion pulses (RF pulses with a linear frequency sweep) were introduced in NMR spectroscopy to overcome frequency bandwidth limitations and to ensure spin inversion over a very broad frequency range. Here we investigate adiabatic fast passage inversion schemes as an extension (and/or alternative) to existing NMR techniques for studying s-ms time-scale protein dynamics. At a given sweep rate, different net amounts of transverse magnetization during the time course of the adiabatic inversion pulse can be created by a variation of the RF amplitude. The interpretation of spin relaxation in the adiabatic spin lock frame is straightforward (e.g. linear relationship between relaxation rate and effective tilt angle) and the presence of conformational exchange processes leads to a pronounced deviation from linearity. In the project it is planned to establish this novel methodology for measurements of protein backbone 15N, 13Ca and 13C` as well as side-chain methyl groups. Additionally, we will also explore the possibility to analyze multiple-quantum coherences. Preliminary data obtained in our laboratory indicate that relaxation measurements in the adiabatic spin-lock frame offer better sensitivity and may extend the methodology to faster motions which are not detectable by conventional NMR technology. Given the fact that adiabatic fast passage pulse schemes are less sensitive to experimental imperfections it is anticipated that these robust pulse schemes may extend the realm of biomolecular NMR spectroscopy to analyze biologically important protein dynamics.

The functional properties of proteins are intimately linked to their dynamical features (e.g conformational plasticity). Most striking examples for the relevance of protein dynamics are protein folding and the exchange between conformational substrates involved in enzyme allostery. NMR spectroscopy is a unique experimental technique for studying protein dynamics in solution as it provides dynamic information with atomic resolution (e.g. residue-specific motions). Measurement of heteronuclear relaxation rates probes molecular dynamics over a wide time range and provides important insight into the motional behaviour of protein in solution. Despite tremendous achievements in the past experimental and conceptual limitations impair the extraction of reliable motional parameters. In this project we developed an adiabatic fast passage (AFP) technique to study protein dynamics. Adiabatic inversion pulses were introduced in NMR spectroscopy to overcome frequency bandwidth limitations and to ensure spin inversion over a very broad frequency range. Since the presence of ms-ms time scale motions leads to a pronounced deviation from linearity that complicates the quantitative analysis we developed a numerical procedure to extract motional parameters from 15N AFP data. Based on the observed results and methodological developments during the course of the project we developed novel applications to analyze 1H-1H spin pairs in protein-ligand systems and probe subtle transient structural changes in intrinsically disordered proteins (IDPs). IDPs have recently attracted great scientific interest as they challenge conventional structural biology concepts and because of their involvement in fundamental biological processes. The findings and relevance of the project can be summarized as follows: 1) the novel numerical analysis tool for AFP experiments offers unique possibilities for the extraction of reliable motional parameters of protein systems undergoing conformational exchange processes; 2) the developed 1H-1H AFP-NOESY experiment turned out to provide very valuable information about binding modes and can be used for pharmacophore mapping and thus constitutes a novel starting point for rational drug (lead) discovery. Most importantly, this strategy doesnt require the a priori knowledge of the targets 3D structure and we therefore anticipate widespread application in drug discovery programs; 3) finally, the application to BASP1, a protein involved in neuronal development, illustrated the potential of the technique to provide unprecedented insight into the structural plasticities of intrinsically disordered proteins.