Abstract
Solving high-resolution structures for membrane proteins continues to be a daunting challenge in the structural biology community. In this study we report our high-resolution NMR results for a transmembrane protein, outer envelope protein of molar mass 16 kDa (OEP16), an amino acid transporter from the outer membrane of chloroplasts. Three-dimensional, high-resolution NMR experiments on the 13C, 15N, 2H-triply-labeled protein were used to assign protein backbone resonances and to obtain secondary structure information. The results yield over 95% assignment of N, HN, CO, Cα, and Cβ chemical shifts, which is essential for obtaining a high resolution structure from NMR data. Chemical shift analysis from the assignment data reveals experimental evidence for the first time on the location of the secondary structure elements on a per residue basis. In addition T 1Z and T2 relaxation experiments were performed in order to better understand the protein dynamics. Arginine titration experiments yield an insight into the amino acid residues responsible for protein transporter function. The results provide the necessary basis for high-resolution structural determination of this important plant membrane protein.
Highlights
Integral membrane proteins are a rapidly growing field of interest in structural biology and biochemistry
The residues show significant chemical shift perturbations which are nonlinear compared to the other residues, such as D128 and A139, which suggests that any binding that occurs at these sites is strictly nonspecific
In this work we have addressed three important properties of the membrane protein OEP16: the formation of transmembrane helices of OEP16 protein, its likely monomeric state in sodium dodecyl sulfate (SDS) detergent micelles, and the ligand binding properties of this protein using results obtained from various high-resolution nuclear magnetic resonance (NMR)
Summary
Integral membrane proteins are a rapidly growing field of interest in structural biology and biochemistry. Nuclear magnetic resonance (NMR) spectroscopy shows great promise in the field of structure determination, it remains a challenge for membrane proteins for several reasons One such hurdle involves obtaining high yields of isotope-enriched proteins that are structurally stable in detergent micelles at concentrations high enough to produce adequate signal to noise. This narrow 1H dispersion, combined with the increased number of residues present in larger membrane proteins, yields a problem with regard to peak overlap in high-resolution NMR spectra Another obstacle to consider is the necessity of solubilizing the protein in detergent micelles, which are used to maintain protein structural integrity. This increase in size of the complex results in slower rotational averaging, and decreased transverse relaxation times [5]
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