This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Many spectroscopic methods in Structural Biology are based on the measurement of distances providing constraints used to resolve structures and thus shed light on the mechanisms underlying the related functions. In protein research, these constraints are used to reduce the number of degrees of freedom available to the polypeptide chain, to orient and dock proteins that participate in supramolecular assemblies, and to characterize the time evolution of these structures during their functionality. The approach to structural studies, based on site directed mutagenesis, spin labeling, and modern pulsed ESR techniques, e.g. those based on pulse ESR distance measurements, particularly DQC and DEER, is well-timed in the context of structural genomics. The method yields the distances over a range of at least 11 to 75 with high throughput and requires less than nanomole amounts of protein, including those that are difficult to produce in amounts needed for other structural methods. It is well-suited for resolving structures of ?difficult systems' such as membrane proteins, large protein complexes, and RNA using a small number of long-distance constrains. In many cases knowing just a single large distance is all that is needed. Distance distributions could help gain insight into the static structure of membrane proteins and conformationally heterogeneous water soluble proteins and the fluctuations in these structures. In our published work on T4-Lysozyme we proposed ?triangulation? method as the most suitable in context of nitroxides. Now, for the first time, we realized the idea with CheA/CheW serving as a testing ground. We used metric matrix distance geometry to solve for the structure, based on 21 constraints. Clearly, not all distances may be measurable and there is an uncertainty in distances. Also, the position of backbone is not immediately known. Therefore, more sophisticated algorithms need be developed. We have chosen CNS as a platform for using the constraints from pulse ESR.
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