The impact of window functions on NMR-based paramagnetic relaxation enhancement measurements in membrane proteins.
ABSTRACT Though challenging, solution NMR spectroscopy allows fundamental interrogation of the structure and dynamics of membrane proteins. One major technical hurdle in studies of helical membrane proteins by NMR is the difficulty of obtaining sufficient long range NOEs to determine tertiary structure. For this reason, long range distance information is sometimes sought through measurement of paramagnetic relaxation enhancements (PRE) of NMR nuclei as a function of distance from an introduced paramagnetic probe. Current PRE interpretation is based on the assumption of Lorentzian resonance lineshapes. However, in order to optimize spectral resolution, modern multidimensional NMR spectra are almost always subjected to resolution-enhancement, leading to distortions in the Lorentizian peak shape. Here it is shown that when PREs are derived using peak intensities (i.e., peak height) and linewidths from both real and simulated spectra that were produced using a wide range of apodization/window functions, that there is little variation in the distances determined (<1 A at the extremes). This indicates that the high degree of resolution enhancement required to obtain well-resolved spectra from helical membrane proteins is compatible with the use of PRE data as a source of distance restraints. While these conclusions are particularly important for helical membrane proteins, they are generally applicable to all PRE measurements made using resolution-enhanced data.
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ABSTRACT: Apodizing functions are used in Fourier transform spectroscopy (FTS) to reduce the magnitude of the sidelobes in the instrumental line shape (ILS), which are a direct result of the finite maximum optical path difference in the measured interferogram. Three apodizing functions, which are considered optimal in the sense of producing the smallest loss in spectral resolution for a given reduction in the magnitude of the largest sidelobe, find frequent use in FTS [J. Opt. Soc. Am.66, 259 (1976)]. We extend this series to include optimal apodizing functions corresponding to increases in the width of the ILS ranging from factors of 1.1 to 2.0 compared with its unapodized value, and we compare the results with other commonly used apodizing functions.Journal of the Optical Society of America A 12/2007; 24(11):3644-8. · 1.56 Impact Factor
Article: Structural studies of the transmembrane C-terminal domain of the amyloid precursor protein (APP): does APP function as a cholesterol sensor?[show abstract] [hide abstract]
ABSTRACT: The amyloid precursor protein (APP) is subject to alternative pathways of proteolytic processing, leading either to production of the amyloid-beta (Abeta) peptides or to non-amyloidogenic fragments. Here, we report the first structural study of C99, the 99-residue transmembrane C-terminal domain of APP liberated by beta-secretase cleavage. We also show that cholesterol, an agent that promotes the amyloidogenic pathway, specifically binds to this protein. C99 was purified into model membranes where it was observed to homodimerize. NMR data show that the transmembrane domain of C99 is an alpha-helix that is flanked on both sides by mostly disordered extramembrane domains, with two exceptions. First, there is a short extracellular surface-associated helix located just after the site of alpha-secretase cleavage that helps to organize the connecting loop to the transmembrane domain, which is known to be essential for Abeta production. Second, there is a surface-associated helix located at the cytosolic C-terminus, adjacent to the YENPTY motif that plays critical roles in APP trafficking and protein-protein interactions. Cholesterol was seen to participate in saturable interactions with C99 that are centered at the critical loop connecting the extracellular helix to the transmembrane domain. Binding of cholesterol to C99 and, most likely, to APP may be critical for the trafficking of these proteins to cholesterol-rich membrane domains, which leads to cleavage by beta- and gamma-secretase and resulting amyloid-beta production. It is proposed that APP may serve as a cellular cholesterol sensor that is linked to mechanisms for suppressing cellular cholesterol uptake.Biochemistry 10/2008; 47(36):9428-46. · 3.42 Impact Factor
Article: Using multiple quantum coherence to increase the 15N resolution in a three-dimensional TROSY HNCO experiment for accurate PRE and RDC measurements.[show abstract] [hide abstract]
ABSTRACT: We present a new version of the 3D TROSY HNCO pulse scheme, referred to as HR-TROSY HNCO, with comparable resolution in the (15)N dimension to a 2D (1)H-(15)N HSQC experiment. In the conventional 3D TROSY HNCO, the constant time period (1/2J(NC) approximately 32 ms) severely limits the maximum resolution in the (15)N dimension. In the HR-TROSY HNCO experiment presented here, both constant time periods (approximately 32 ms each) for coherence forward and backward transfer between (15)N and (13)C' are utilized to double the (15)N evolution time. This leads to a dramatic enhancement in peak separation along the (15)N dimension, making the HR-TROSY HNCO an ideal pulse scheme for accurate paramagnetic relaxation enhancement and residual dipolar coupling measurements.Journal of Magnetic Resonance 07/2009; 200(2):173-7. · 2.14 Impact Factor