During the past two decades, advances in NMR and its related technologies have tremendously increased the sensitivity of NMR relaxation experiments. NMR-derived parameters now have sufficient sensitivity to allow comparison with parameters obtained by other methods, such as molecular dynamics simulations and thermodynamics experiments. However, for meaningful results to emerge and for reasonable comparison of NMR-derived dynamics with those obtained by other methods, accurate evaluation of uncertainties in the NMR-derived parameters is critical. The objective of this project is to provide a comprehensive assessment of the relaxation errors for a variety of experiments. Based on this assessment, uncertainty estimations will be incorporated into software packages to allow accurate extraction of protein dynamics. Such analyses will significantly impact the field and are crucial at the present time when NMR dynamics studies are increasingly used to interpret unique features in enzymatic mechanisms and other biological processes.
The results of this research will be widely distributed via journal publications and websites, promoting their broad application in the NMR community and facilitating the development of new and existing analysis tools in the field. Removing impediments caused by improper error estimation, this work will allow the merging and integration of NMR results with those derived by other biophysical methods. Such seamless integration is important for future progress in Biophysics and Structural Biology. This project will also provide interdisciplinary training for students. An advanced NMR course comprising theory and experiments will be integrated into the Molecular Biophysics and Structural Biology Graduate Curriculum and increased opportunities for summer undergraduate research will be created. Also, science exposure in the local communities will be increased and Structural Biology will be brought to K-12 schools.
The overall objective of the award was to thoroughly evaluate experimental uncertainties in Nuclear Magnetic Resonance (NMR) relaxation data of proteins. To accurately compare NMR-derived parameters with other biophysical parameters, the PI evaluated the uncertainties associated with the NMR-derived parameters, taking into account systematic errors as well as experimental noise error. In the intellectual merit, the PI obtained important results, mainly related to error analysis in transverse (R2) relaxation experiments with a Carr Purcell Meiboom Gill (CPMG) sequence and also cross correlation effects in longitudinal relaxation (to be submitted). (1) Uncertainties in 15N CPMG R2, a commonly used parameter to elucidate protein dynamics, were compared using different 1H decoupling schemes, and using a 600 MHz NMR instrument equipped with an ambient temperature probe and an 800 MHz instrument with a cryogenic probe. More recently, the PI performed simulation and NMR experiments to elucidate R2 accuracy and the uncertainty at practical experimental parameters using an alternative phase scheme. (2) Computational simulation of magnetization decay of the CPMG R2 dispersion was performed by taking into account relaxation effect during 15N CPMG pulsing and during the evolution time. (3) A better fitting algorithm by adding lower and upper boundaries of the fit parameters for the analysis of the CPMG R2 dispersion data was proposed and, a useful dispersion presentation by plotting R2 RMSD of each dataset against the residue number was demonstrated. (4) Cross correlation effects in the longitudinal relaxation was assessed. (5) The PI wrote a review article that describes the nuts and bolts of current NMR relaxation experiments to detect protein backbone dynamics. The award resulted in four peer-reviewed articles and one review article, all directly supported by the NSF, and an additional six articles that indirectly benefited from the results of the NSF-supported studies. In the broader impact, the PI accomplished (1) broad dissemination of the NSF-sponsored research on her website and through numerous invited seminars and lectures. Proper data analysis is a fundamentally important issue in any basic science. (2) To enhance strength of the local NMR community, we actively attended local NMR symposiums. (3) The PI involved students in the proposed research project. (4) The PI newly prepared and instructed an advanced NMR course that has a hand-on for NMR software as well as theory. (5) The PI accomplished activities for the Summer Undergraduate Research Program (SURP) in the University of Pittsburgh School of Medicine. (6) The PI organized and/or helped several tours of the NMR and X-ray facilities in the Department of Structural Biology, with support and participation from other colleagues (Figure attached) (7) The PI had out-reach activities through the Spectroscopy Society in Pittsburgh.
