Ed S. Mooberry

PRACTICAL INTRODUCTION TO THEORY AND IMPLEMENTATION OF MULTINUCLEAR, MULTIDIMENSIONAL NUCLEAR MAGNETIC RESONANCE EXPERIMENTS

Publisher Summary The objectives of this chapter are 2-fold. First, it presents basic unifying features of pulse sequences so that the underlying mechanics of even complicated sequences become more transparent. Second, a step-by-step guide to present the practical implementation and processing of multidimensional experiments is illustrated. Much progress has resulted from generalization of heteronuclear twodimensional (2D) NMR experiments with 13 C- and 15 N-labeled biomolecules to higher dimensions. The ultimate goal of NMR investigations of biomolecules is to obtain structural and dynamic information. To this end, many specialized experimental techniques similar to those described above have been developed, which allow the measurement of parameters that provide distance and dihedral angle constraints. In conjunction with the methods described in this chapter, computer-automated resonance assignments and spectral analysis techniques should facilitate efficient studies of larger biomolecules and are expected to accelerate the pace of NMR contributions to structural biochemistry.

High Pressure Effects on Protein Structure

The protein stability problem is one of the most intriguing questions in protein chemistry. The central conundrum is why the folded (native) form of a protein, with its specific three-dimensional structure, is stabilized relative to the unfolded (denatured) form, which is conformationally disperse. The related problems of protein dynamics, stability, and design are all related ultimately to protein energetics. Our knowledge of the microscopic characteristics of proteins is constantly growing as more three-dimensional structures are determined by X-ray crystallography and nuclear magnetic resonance spectroscopy. To fully understand the energetic details of proteins, it is necessary to have information not only about their structures but also of their macroscopic behavior. A fundamental approach to protein energetics is the study of thermodynamics, i.e., the equilibrium position of the system (Callen, 1985). In the context of protein stability, this equilibrium is between the active, folded form, of a protein and the inactive, conformationally disperse, unfolded form. Not surprisingly, under physiological conditions this equilibrium usually favors the native form. By examining the response of this equilibrium process to different perturbants, we can infer a remarkable amount of information regarding the energetics of protein folding. In this chapter, we focus on the use of pressure as a variable for perturbing equilibria connecting protein structural states. As detailed below, the standard volume change for a reaction is the central component of pressure studies, although higher order terms, such as the standard compressibility change can also be important. Volume changes associated with reactions can be determined from investigations of the pressure dependence of equilibria; activation volumes for reactions can be obtained from measurements of pressure effects on reaction rates (Markley et al., 1996).

Mixing apparatus for preparing NMR samples under pressure.

The size limit for protein NMR spectroscopy in solution arises in large part from line broadening caused by slow molecular tumbling. One way to alleviate this problem is to increase the effective tumbling rate by reducing the viscosity of the solvent. Because proteins generally require an aqueous environment to remain folded, one approach has been to encapsulate hydrated proteins in reverse micelles formed by a detergent and to dissolve the encapsulated protein in a low-viscosity fluid. The high volatility of suitable low-viscosity fluids requires that the samples be prepared and maintained under pressure. We describe a novel apparatus used for the preparation of such samples. The apparatus includes a chamber for mixing the detergent with the low-viscosity solvent, a second chamber for mixing this with hydrated protein, and a 5-mm (o.d.) zirconium oxide NMR sample tube with shut-off valves designed to contain pressures on the order of 10 bar, sufficient for liquid propane. Liquids are moved from one location to another by introducing minor pressure differentials between two pressurization vessels. We discuss the operation of this apparatus and illustrate this with data on a 30-kDa protein complex (chymotrypsin:turkey ovomucoid third domain) encapsulated in reverse micelles of the detergent, sodium bis (2-ethylhexyl) sulfosuccinate, aerosol-ot (AOT), dissolved in liquid propane.

MODIFICATIONS OF OLDER MODEL NUCLEAR MAGNETIC RESONANCE CONSOLE FOR COLLECTION OF MULTINUCLEAR, MULTIDIMENSIONAL SPECTRAL DATA

Publisher Summary Pursuant to the original description by Kay, Ikura, Tschudin, and Bax, a growing variety of double- and triple-resonance, three-dimensional (3D) and four-dimensional (4D) nuclear magnetic resonance (NMR) experiments have been developed. Although fully equipped commercial NMR spectrometer consoles produced after 1990 have been capable of performing these demanding experiments, earlier consoles lack the necessary decoupling capabilities and requisite number of transmitter channels. Thus, although the original two-dimensional (2D) triple-resonance experiments have been able to be carried out some of with a standard 1985 vintage Bruker Instruments AM-400 NMR spectrometer and BSV-3 X-nucleus decoupler, it was needed to make extensive modifications in order to perform multinuclear 3D and 4D experiments. Kay et al. described the adaptation of a Bruker AM console for early 3D and 4D triple-resonance experiments. With more extensive modifications, most of the current multinuclear 3D and 4D experiments have been able to be performed with older model Bruker Instruments AM-500 and AM-600 consoles and BSV-3 X-nucleus decouplers.

Computational Aspects of Multinuclear NMR Spectroscopy of Proteins at NMRFAM

In recent studies, we have explored various strategies for using isotope labeling in conjunction with 1D, 2D, and 3D NMR spectroscopy to obtain information about how proteins work. With larger proteins, a concerted approach to the assignment of protein spectra that makes use of all available information about through-bond and through-space connectivities appears to provide the most reliable results. We summarize here various ways in which we have been using computers to process and analyze data and to derive information about protein structure and dynamics. We also describe in brief our protein NMR database project.