Development of Nuclear Magnetic Resonance Pulse Sequences and Probes to Study Biomacromolecules

Cosman, M ; Krishnan, V V ; Maxwell, R

Lawrence Livermore National Laboratory

关键词: Lawrence Livermore National Laboratory;    Membrane Proteins;    Magnetic Fields;    X-Ray Diffraction;    Enzymes;   

DOI  :  10.2172/15005390
RP-ID  :  UCRL-ID-143261
RP-ID  :  W-7405-ENG-48
RP-ID  :  15005390

美国|英语

来源: UNT Digital Library

【 摘 要 】

The determination of the three dimensional structures at high resolution of biomolecules, such as proteins and nucleic acids, enables us to understand their function at the molecular level. At the present time, there are only two methods available for determining such structures, nuclear magnetic resonance (NMR) spectroscopy and X-ray diffraction. Compared to well-established X-ray diffraction techniques, NMR methodology is relatively new and has many areas in which improvement can still be attained. In this project, we focused on the development of new NMR probes and pulse sequences that were tailored to tackle specific problems that are not adequately addressed by current technology. Probes are the hardware that contain the radio frequency (RF) circuitry used to both excite and detect the NMR signals. Pulse sequences are composed of a series of RF pulses and delays, which are applied to the sample held within the magnetic field by the probe, so as to manipulate the nuclear spins. Typically, a probe is developed for a specific set of nuclei and types of experiments and the pulse sequences are then written to use the probe in an optimal manner. In addition, the inter-development of instrumentation and methods are determined by the specific biological question to be examined. Thus our efforts focused on addressing an area of importance in NMR Structural Biology namely more effective ways to use the phosphorus ({sup 31}P) nucleus. Phosphorus is a very important biological element that is strategically located in nucleic acids, where it imparts negative charge and flexibility to RNA and DNA. It is also a component of the cellular membrane and thus interacts with membrane proteins. It is used in mechanisms to signal, activate or deactivate enzymes; and participates in energy storage and release. However, the phosphorus nucleus exhibits certain properties, such as poor spectral dispersion, low sensitivity of detection, and fast relaxation, which limit its effective use in NMR studies of biomolecules. Our unique combination of expertise at LLNL allowed us to tackle each of the negative features of {sup 31}P-NMR in a three-pronged, concerted effort. The nature of our work necessitated an interdependent, multidisciplinary approach that required knowledge of spin physics (pulse sequences), engineering (probes), and structural biology (sample preparation and structure determination).