Methods Development

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

We devote our efforts in this project to the development of improved NMR techniques and hardware, and to new approaches to protein engineering and isotopic labeling. We are continuing and expanding our efforts to develop new approaches to tagging macromolecules with paramagnetic centers and obtaining unique structural information about intermolecular interactions and structures of multi-component complexes. In addition, new approaches are developed when traditional methods require augmentation with new experiments or data. These methods include novel isotopic labeling schemes, pulse sequence development, automated methods and novel methods in relaxation NMR of proteins. This effort is continuing as a subset of the various structural biology projects described in other parts of the annual report. Specific developments have included the use of non-uniform sampling to speed up data acquisition of multi-dimensional NMR data, tagging with a range of molecules to facilitate paramagnetic relaxation enhancement to measure long-range distances in multi-component complexes. We have reported new and optimized approaches to engineering systems for deuteration, segmental labeling, lipidation, and construction of multi-domain systems that include intrinsically disordered regions. These tools enable the strength of NMR spectroscopy to be applied to more complex systems, much more similar to in vivo settings. Ongoing work in membrane mimetics is expected to impact both major projects in the lab in the coming year and beyond. New approaches to characterizing nano disk membrane mimetics have been developed and will be published in this year. Our recent work includes the development of new methods for examining fast conformational dynamics in proteins, and the application of this to E2:E3 interactions. A new aspect within this project is the development of NMR pulse sequence methods combined with novel isotopic labeling and the unique modification of NMR probe hardware to explore the use of triple resonance NMR to monitor sulfur sites in proteins. Through collaboration with the NHLBI Chemistry and Synthesis Center, we have developed double labeled 13C and 77Se compounds for incorporation into proteins. In our lab we re-engineered a conventional H/C/N triple resonance probe to perform H/C/Se triple resonance experiments. The first publication describing these efforts is submitted and under review.