[PDF] Application Of Nuclear Magnetic Resonance Spectroscopy To The Structure Determination Of The Integral Membrane Proteins Of The Mer Operon eBook
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Efforts at elucidating the structural biology of membrane proteins represent an ongoing challenge to conventional methods of structure determination. The emergence of new methods for the measurement and application of orientational restraints have offered new avenues of pursuing the determination of membrane protein structures. Presented in this thesis is the evolution of experimental and computation methods necessary to extend NMR based structure determination methods to the polytopic mercuric ion transport proteins of the mer operon. Primary structural efforts are focused upon the bi-spanning protein, MerF, using solution-state NMR methods on protein reconstituted into isotropic and weakly aligned micelles and solid-state NMR methods on protein reconstituted into statically aligned bicelles. The application of the methods developed for MerF are applied to a tri-spanning chimeric protein, MerTf, to extend the NMR based methodology toward the structure determination of the principal mercuric ion transporter, MerT.
In this Thesis, methodological developments to determine membrane protein structures using solution NMR spectroscopy and results on proteins with single and multiple hydrophobic trans-membrane domains and amphipathic in-plane helices are described. Measurements of weak anisotropic interactions provide unique structural information that can complement distance information. The potential of the developed methods for high-throughput applications to structural genomics of membrane proteins is tested.
The critically acclaimed laboratory standard, Methods in Enzymology, is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. The series contains much material still relevant today - truly an essential publication for researchers in all fields of life sciences. Nuclear Magnetic Resonance of Biological Macromolecules, Part C is written with a "hands-on" perspective. That is, practical applications with critical evaluations of methodologies and experimental considerations needed to design, execute, and interpret NMR experiments pertinent to biological molecules. * One of the most highly respected publications in the field of biochemistry since 1955 * Frequently consulted, and praised by researchers and reviewers alike * Truly an essential publication for anyone in any field of the life sciences
This volume and its companion, Volume 338, supplement Volumes 176, 177, 239, and 261. Chapters are written with a "hands-on" perspective. That is, practical applications with critical evaluations of methodologies and experimental considerations needed to design, execute, and interpret NMR experiments pertinent to biological molecules.
When I was asked to edit the second edition of Protein NMR Techniques, my first thought was that the time was ripe for a new edition. The past several years have seen a surge in the development of novel methods that are truly revolutionizing our ability to characterize biological macromolecules in terms of speed, accuracy, and size limitations. I was particularly excited at the prospect of making these techniques accessible to all NMR labs and for the opportunity to ask the experts to divulge their hints and tips and to write, practically, about the methods. I commissioned 19 chapters with wide scope for Protein NMR Techniques, and the volume has been organized with numerous themes in mind. Chapters 1 and 2 deal with recombinant protein expression using two organisms, E. coli and P. pastoris, that can produce high yields of isotopically labeled protein at a reasonable cost. Staying with the idea of isotopic labeling, Chapter 3 describes methods for perdeuteration and site-specific protonation and is the first of several chapters in the book that is relevant to studies of higher molecular weight systems. A different, but equally powerful, method that uses molecular biology to “edit” the spectrum of a large molecule using segmental labeling is presented in Chapter 4. Having successfully produced a high molecular weight target for study, the next logical step is data acquisition. Hence, the final chapter on this theme, Chapter 5, describes TROSY methods for stru- ural studies.
Volume 16 marks the beginning of a special topic series devoted to modern techniques in protein NMR, under the Biological Magnetic Resonance series. This volume is being followed by Volume 17 with the subtitle Structure Computation and Dynamics in Protein NMR. Volumes 16 and 17 present some of the recent, significant advances in biomolecular NMR field with emphasis on developments during the last five years. We are honored to have brought together in these volumes some of the world’s foremost experts who have provided broad leadership in advancing this field. Volume 16 contains advances in two broad categories: the first, Large Proteins, Complexes, and Membrane Proteins, and second, Pulse Methods. Volume 17, which will follow covers major advances in Computational Methods, and Structure and Dynamics. In the opening chapter of Volume 16, Marius Clore and Angela Gronenborn give a brief review of NMR strategies including the use of long range restraints in the structure determination of large proteins and protein complexes. In the next two chapters, Lewis Kay and Ron Venters and their collaborators describe state-of-t- art advances in the study of perdeuterated large proteins. They are followed by Stanley Opella and co-workers who present recent developments in the study of membrane proteins. (A related topic dealing with magnetic field induced residual dipolar couplings in proteins will appear in the section on Structure and Dynamics in Volume 17).
Volume 17 is the second in a special topic series devoted to modern techniques in protein NMR, under the Biological Magnetic Resonance series. Volume 16, with the subtitle Modern Techniques in Protein NMR , is the first in this series. These two volumes present some of the recent, significant advances in the biomolecular NMR field with emphasis on developments during the last five years. We are honored to have brought together in these volume some of the world s foremost experts who have provided broad leadership in advancing this field. Volume 16 contains - vances in two broad categories: I. Large Proteins, Complexes, and Membrane Proteins and II. Pulse Methods. Volume 17 contains major advances in: I. Com- tational Methods and II. Structure and Dynamics. The opening chapter of volume 17 starts with a consideration of some important aspects of modeling from spectroscopic and diffraction data by Wilfred van Gunsteren and his colleagues. The next two chapters deal with combined automated assignments and protein structure determination, an area of intense research in many laboratories since the traditional manual methods are often inadequate or laborious in handling large volumes of NMR data on large proteins. First, Werner Braun and his associates describe their experience with the NOAH/DIAMOD protocol developed in their laboratory.
Protein NMR for the Millennium is the third volume in a special thematic series devoted to the latest developments in protein NMR under the Biological Magnetic Resonance umbrella. This book is divided into three major sections dealing with significant recent advances in the study of large proteins in solution and solid state, structure refinement, and screening of bioactive ligands. Key Features: TROSY, Segmental isotope labeling of proteins, Hydrogen bond scalar couplings, Structure refinement based on residual dipolar couplings, Written by the world's foremost experts who have provided broad leadership in advancing the protein NMR field.
The cell membrane is critical for life. The functionality of this membrane is dependent on the interactions between the many different molecules found within and connected to the cell membrane. Proteins and peptides are responsible for a variety of biological activities including signal transmission and ion movement. Membrane proteins can be associated very closely with the membrane (known as integral membrane proteins) or loosely with the membrane (peripheral proteins). The functionality of these membrane proteins depends on their structure, which can be studied using nuclear magnetic resonance (NMR). In structural studies, protons present themselves as desirable nuclei for detection, owing to their high abundance in biological systems. However, strong homonuclear dipolar interactions often lead to broad signals and obscured details. This thesis describes the study and development of NMR pulse sequences that can be used to investigate model membrane systems that include small molecules. Beginning with a leucine-rich peptide P0813, proton suppression techniques were developed using a unique gradient probe assembly. The addition of pulsed field gradients within the pulse sequences resulted in significant proton coherence suppression in a sample composed of protonated lipid and peptide. Gramicidin-A was used in the development of proton-detected correlation experiments. These experiments were further studied with Conolysin-Mt1, a natural cytolytic peptide found in sea snails. Cholesterol was employed in the study of how the nuclear Overhauser effect can be used to probe molecular structure and dynamics. Finally, the majority of the modified experiments were tested on a synthetic peptide ALGA, which was originally designed to produce well-resolved proton spectra. Throughout the studies performed on the five different small molecules, new proton detection techniques were investigated, with varying degrees of success. In addition, different sample preparations were explored to help determine how changes in sample composition can affect sample stability.
Atomic-resolution membrane protein structures can be determined by solid-state Nuclear Magnetic Resonance (NMR) spectroscopy, and the unique advantage of the approach is that membrane proteins reside in near-native lipid bilayer environment at physiological pH and temperature, which minimizes the potential distortions of the protein structure caused by the environment. Here, the full-length mercury transporter protein, MerF, is the focus of the structural studies, and the protein is an essential part of the bacterial mercury detoxification system that has been exploited as a potential engineering target for mercury bioremediation strategies. The backbone structures of the full-length MerF are determined in two environments, (i) magnetically aligned bicelles by oriented-sample (OS) solid-state NMR and (ii) proteoliposome by rotationally aligned (RA) solid-state NMR; and notably, both environments provide the planar lipid bilayer environment for the protein. The structural study of MerF in aligned bicelle has initially been challenging for the OS solid-state NMR, and consequently, methods have been developed to tackle the two major obstacles, the spectral resolution and resonance assignments. New pulse sequence, MSHOT-Pi4/Pi, has demonstrated a reduction of the 1H resonance line width by more than a factor of two, a significant improvement in spectral resolution. New resonance assignment method, Dipolar Coupling Correlated Isotropic Chemical Shift (DCCICS) Analysis, has been developed that is able to transfer resonance assignment from isotropic NMR methods to OS solid-state NMR spectra. The combined usage of several resonance assignment strategies and special tactics, such as applying DCCICS to the new high-resolution proton-evolved local field experiments for terminal and loop residues, has resulted in the complete assignment of all backbone immobile residues of the full-length MerF protein in magnetically aligned bicelle. Meanwhile, RA solid-state NMR is developed in the lab as a new method that combines the strength of magic-angle-spinning (MAS) solid-state NMR in obtaining resonance assignment and the concept of molecular alignment from OS solid-state NMR in obtaining angular restraints. In applying to the structural study of MerF, the method is further incorporated with multi-contact cross polarization and sequential backbone "walk" with three three-dimensional experiments, and the first structure of full-length MerF is determined with the method. In comparison to the previously determined structure of the truncated MerF (MerFt), the full-length structure reveals that the protein truncation has caused large conformational rearrangement at a place more than ten residues away from the truncation site, which serves as an example to demonstrate the importance of studying the full-length unmodified proteins by structural biologists. Additionally, the structure reveals that both mercury-binding sites are located at the intracellular side of the membrane, hinting at the observation of a conformation that allows intramolecular transfer of mercury ions. Subsequently after the complete assignment of MerF in OS solid-state NMR, the MerF structure determined by RA solid-state NMR is further improved by incorporating additional angular restraints from OS solid-state NMR and by the new treatment of dihedral restraints derived from the experimental study of C-terminal dynamics. Lastly, as a side project, the theoretical foundation of MSHOT-Pi4 pulse sequence is further explored. The observation that the pulse sequence selectively improves the resolution of membrane protein samples but not of standard single crystal sample has been analytically generalized as the principle of "motion-adapted" pulse sequence, where it is found that the interference between sample's spatial rotational motion and the radio-frequency pulse rotation in the quantum spin space is the cause of the selectivity. As a related endeavor, the mechanisms of dilute spin exchange and the magic-angle 1H spin-lock pulses have been analyzed theoretically and demonstrated in standard and biological samples. Mixed-order proton-relay mechanism is proposed to be the main contributor to dilute spin exchange in stationary aligned sample, and once more, the difference of pulse performance between standard and biological samples is observed that may be a consequence of several causes including sample motion. In conclusion, the development of various methods in OS and RA solid-state NMR are likely to find their usage in future structural studies of membrane proteins; the theoretical principle of motion-adapted property opens up new avenue to develop pulse sequences for membrane protein samples; and the atomic-resolution backbone structures of MerF contribute information for structural biologist and for the mechanistic study of mercury transportation.