Miya Kamihira

CONFORMATION AND DYNAMICS OF MEMBRANE PROTEINS AND BIOLOGICALLY ACTIVE PEPTIDES AS STUDIED BY HIGH-RESOLUTION SOLID-STATE 13C NMR

We have explored an empirical approach to clarify the three-dimensional structure and dynamics of membrane proteins such as bacteriorhodopsin (bR) based on 13C NMR measurements, utilizing the concept of the conformation-dependent displacements of 13C chemical shifts as determined by cross polarization-magic angle spinning (CP-MAS) and dipolar decoupled-magic angle spinning (DD-MAS) methods. This is possible because 13C chemical shifts of the amino acid residues under consideration are appreciably displaced, up to 8 ppm, depending upon particular conformations from several portions of membrane proteins such as the transmembrane α-helices, loops, N- or C-terminus, etc. as referred to the data accumulated to date of an appropriate model system. It is also possible to distinguish a region characterized by a variety of backbone motions with at least three different time scales from NMR data: rapid motions with correlation times shorter than 10−8 s, intermediate motions with correlation times of 10−4 to 10−5 s, and slow motions with a time scale of 10−2 s. In addition, we also explored a non-empirical approach to reveal the three-dimensional structure of a smaller molecular system such as biologically active peptides as a messenger molecule in signal transduction, based on accurately determined appropriate sets of interatomic distances as determined by rotational echo double resonance (REDOR). Examination of 13C or 15N chemical shifts before and after REDOR experiments proved to be an indispensable means to examine whether or not conformations of several kinds of 13C, 15N-doubly labeled samples at different positions are not changed all the time. Here, we summarize some illustrative examples to this end, selected from our recent studies on [3-13C]Ala-labeled bR and biologically active peptides such as enkephalins.

Kinetics of Amyloid Fibril Formation of Human Calcitonin

Amyloid fibril formation is one of the common phenomena associated with many serious diseases such as Alzheimer’s disease, Parkinson’s, bovine spongiform encephalopathy (BSE), scrapie, and so on. Independent of the constituent polypeptides, the amyloid fibrils exhibit highly organized filamentous structures which are typically ∼100 A in diameter, as revealed by electron microscopy and atomic force microscopy. Mechanism of the amyloid fibril formation has been extensively studied by various spectroscopic techniques, related to misfolding of proteins. Especially, solid-state NMR spectroscopy has made a great contribution to determine the structures of the fibrils from several peptides/proteins at the molecular level. For example, Alzheimer’s β-amyloid peptides, which consist of 40–42 amino acid residues, have gained insights into the three-dimensional (3D) structures in the fibrils as a double-layered cross-β structure with parallel β-sheets by accumulating the local and spatial conformational restraints [1–3]. Also, an 11-residue fragment of human transthyretin (TTR) in its fibrillar form which in vivo is allied with familial amyloid polyneuropathy and senile systemic amyloidosis, was revealed the complete 3D structures of the extended β-strand conformation, by establishing dihedral angles of the backbone and 13C– 15N distances [4,5]. These results indicate that solid-state NMR spectroscopy is a powerful tool to determine the non-crystal, non-soluble, fibrillar structures. In this chapter, a solid-state NMR application on the kinetics analyses of the amyloid fibril formation is described. Human calcitonin (hCT) is a thyroid hormone which regulates the mineral metabolism in the bones [6– 8]. hCT contains 32 amino acid residues and its sequence is CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAPNH2 with a disulfide bond between Cys1 and Cys7 and a C-terminus amide. In a high concentrated solution, however, it is known to form the amyloid fibrils, which are typically 80 A in diameter [9,10].

Solid state NMR of biomolecules

Publisher Summary Nuclear magnetic resonance (NMR) spectroscopy is recognized as one of the most powerful means to elucidate structures and dynamics of a wide variety of molecules. In biological sciences, it is now possible to determine the three-dimensional structure of proteins in solution. Since high-resolution solid-state NMR spectroscopy was developed, it has been useful for obtaining information on the structure and dynamics of biologically important molecules, such as membrane proteins and insoluble fibril proteins. The latter technique combines high-power proton decoupling with magic angle spinning (MAS) and cross polarization (CP) techniques. This allows the observation of high-resolution NMR signals of solid biomolecules. In this technique, anisotropic interactions are eliminated to achieve high-resolution signals, although these interactions contain important geometric information for elucidating the structure of biomolecules. To reintroduce the anisotropic interaction under a high-resolution condition, a number of useful techniques have been developed. Solid state NMR of anisotropic media such as lipid bilayer systems serves as a magnetically anisotropic interaction to explore the molecular orientation in the membrane.