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Fast NMR data acquisition from bicelles containing a membrane-associated peptide at natural-abundance.

ABSTRACT: In spite of recent technological advances in NMR spectroscopy, its low sensitivity continues to be a major limitation particularly for the structural studies of membrane proteins. The need for a large quantity of a membrane protein and acquisition of NMR data for a long duration are not desirable. Therefore, there is considerable interest in the development of methods to speed up the NMR data acquisition from model membrane samples. In this study, we demonstrate the feasibility of acquiring two-dimensional spectra of an antimicrobial peptide (MSI-78; also known as pexiganan) embedded in isotropic bicelles using natural-abundance (15)N nuclei. A copper-chelated lipid embedded in bicelles is used to speed-up the spin-lattice relaxation of protons without affecting the spectral resolution and thus enabling fast data acquisition. Our results suggest that even a 2D SOFAST-HMQC spectrum can be obtained four times faster using a very small amount (?3 mM) of a copper-chelated lipid. These results demonstrate that this approach will be useful in the structural studies of membrane-associated peptides and proteins without the need for isotopic enrichment for solution NMR studies.

SUBMITTER: Yamamoto K

PROVIDER: S-EPMC3203308 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Fast NMR data acquisition from bicelles containing a membrane-associated peptide at natural-abundance.

Yamamoto Kazutoshi K Vivekanandan Subramanian S Ramamoorthy Ayyalusamy A

The journal of physical chemistry. B 20111011 43

In spite of recent technological advances in NMR spectroscopy, its low sensitivity continues to be a major limitation particularly for the structural studies of membrane proteins. The need for a large quantity of a membrane protein and acquisition of NMR data for a long duration are not desirable. Therefore, there is considerable interest in the development of methods to speed up the NMR data acquisition from model membrane samples. In this study, we demonstrate the feasibility of acquiring two- ...[more]

PMID: 21939237

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Project description:Structural information about the coordination environment of calcium present in bone is highly valuable in understanding the role of calcium in bone formation, biomineralization, and bone diseases like osteoporosis. While a high-resolution structural study on bone has been considered to be extremely challenging, NMR studies on model compounds and bone minerals have provided valuable insight into the structure of bone. Particularly, the recent demonstration of (43)Ca solid-state NMR experiments on model compounds is an important advance in this field. However, application of (43)Ca NMR is hampered due to the low natural-abundance and poor sensitivity of (43)Ca. In this study, we report the first demonstration of natural-abundance (43)Ca magic angle spinning (MAS) NMR experiments on bone, using powdered bovine cortical bone samples. (43)Ca NMR spectra of bovine cortical bone are analyzed by comparing to the natural-abundance (43)Ca NMR spectra of model compounds including hydroxyapatite and carbonated apatite. While (43)Ca NMR spectra of hydroxyapatite and carbonated apatite are very similar, they significantly differ from those of cortical bone. Raman spectroscopy shows that the calcium environment in bone is more similar to carbonated apatite than hydroxyapatite. A close analysis of (43)Ca NMR spectra reveals that the chemical shift frequencies of cortical bone and 10% carbonated apatite are similar but the quadrupole coupling constant of cortical bone is larger than that measured for model compounds. In addition, our results suggest that an increase in the carbonate concentration decreases the observed (43)Ca chemical shift frequency. A comparison of experimentally obtained (43)Ca MAS spectra with simulations reveal a 3:4 mol ratio of Ca-I/Ca-II sites in carbonated apatite and a 2.3:3 mol ratio for hydroxyapatite. 2D triple-quantum (43)Ca MAS experiments performed on a mixture of carbonated apatite and the bone protein osteocalcin reveal the presence of protein-bound and free calcium sites, which is in agreement with a model developed from X-ray crystal structure of the protein.

Dataset Information

Fast NMR data acquisition from bicelles containing a membrane-associated peptide at natural-abundance.

Publications

Fast NMR data acquisition from bicelles containing a membrane-associated peptide at natural-abundance.

Similar Datasets

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