A. Joshua Wand

Improved labeling strategy for 13C relaxation measurements of methyl groups in proteins

Selective incorporation of 13C into the methyl groupsof protein side chains is described as a means for simplifying themeasurement and interpretation of 13C relaxation parameters.High incorporation (>90%) is accomplished by using pyruvate(3-13C, 99%) as the sole carbon source in the growthmedia for protein overexpression in E. coli. This improved labeling schemeincreases the sensitivity of the relaxation experiments by approximatelyfivefold when compared to randomly fractionally 13C-labeledprotein, allowing high-quality measurements on relatively dilute (<1 mM)protein samples at a relatively low cost.

Protein complexes studied by NMR spectroscopy.

Recent advances in NMR methods now allow protein complexes to be studied in great detail in a wide range of solution conditions. Isotope-enrichment strategies, resonance-assignment approaches and structural-determination methods have evolved to the point where almost any type of complex involving proteins of reasonable size may be studied in a straightforward way. A variety of isotope editing and filtering strategies underlie these powerful methodologies. Approaches to the characterization of the dynamics of protein complexes have also matured to the point where detailed studies of the effects of complexation on dynamics can be studied over a wide range of timescales.

A Structural Biologist’s View of Precision and Accuracy of Structural Models of Proteins Based on NMR Data

Advances in the field of nuclear magnetic resonance (NMR) since its first observation 50 years ago have been staggering and have revolutionized many areas of physics, chemistry and biology. As the technology as a whole has developed there has been an explosion in the number of applications of NMR spectroscopy to studies of proteins and other biopolymers. The dramatic increase in the power, flexibility and efficiency of modern multidimensional and multinuclear NMR spectroscopy now provide the structural biologist with a previously unavailable avenue to high resolution structural information in solution (Clore and Gronenborn, 1991b; Bax and Grzesiek, 1993; Leopold et al., 1994). At the center of the studies of the structure and dynamics of proteins by NMR is the solution of the general problem of assigning individual resonances to specific nuclei within the macromolecule. Recent developments of conceptually new experimental frameworks coupled with technical advancements have had a swift and profound effect on the solution of the resonance assignment problem. The issue of resolving and mapping individual resonances of a protein to specific atomic sites within the molecule has now largely been solved for proteins ranging up to 25 kDa in size. Parallel to these recent advances in resonance assignment methodologies has been an ever increasing level of sophistication in the marshaling of NMR-based structural parameters for the determination of the solution structures of proteins.

Solution structures of horse ferro- and ferricytochrome c using 2D and 3D 1H NMR and restrained simulated annealing

Publisher Summary Cytochrome c has become a paradigm for protein folding and electron transfer studies, because of its stability, solubility, and ease of preparation. This chapter describes the high-resolution solution structures for ferro- and ferricytochrome c, using 1H 2D and 3D NMR spectroscopy and hybrid distance geometry, simulated annealing calculations. The solution structures of horse ferro- and ferricytochrome c to high resolution are determined using a comprehensive battery of 1H-based NMR experiments, including three dimensional homonuclear spectra, and the key to this approach is the use of advanced statistical methods to compensate for the lack of extensive torsion angle constraints in the identification of prochiral hydrogens. The use of three dimensional spectroscopy in conjunction with good working models of the structures has allowed the number of NOE-based restraints that could be determined to approach the density required for high resolution structures to be obtained. The chapter suggests that these detailed structural studies will provide the basis for a comprehensive re-evaluation of hypotheses, concerning the fundamental nature of the electron transfer processes in proteins and also serves to illustrate that highly defined molecular models of proteins of moderate size can be determined using structural restraints derived solely from 1H NMR experiments.