The rate of movement of fatty acids (FA) across phospholipid bilayers is an important consideration for their mechanism of transport across cell membranes but has not yet been measured. When FA move undirectionally across phospholipid bilayers, the rapid movement of un-ionized FA compared to ionized FA results in transport of protons. We have previously used this property to show that FA move spontaneously ("flip-flop") across the bilayer of small unilamellar vesicles within approximately 1 s (Kamp & Hamilton, 1992, 1993). This work extends the time resolution of this assay into the millisecond time range by use of stopped flow fluorometry. In small unilamellar vesicles (diameter, approximately 25 nm) at neutral pH, flip-flop of all fatty acids studied (lauric, myristic, palmitic, oleic, and stearic) was > or = 80% complete within 5-10 ms. In large unilamellar vesicles (diameter, approximately 100 nm), the same fatty acids exhibited fast flip-flop but with a measureable rate (t 1/2 = 23 +/- 12 ms). The calculated pseudounimolecular rate constant of the un-ionized FA (kFAH) approximately 15 s-1. There was no dependence of the flip-flop rate on the fatty acid chain length or structure. We also monitored the rate of desorption and transbilayer movement of (anthroyloxy)stearic acid in small unilamellar vesicles. Whereas previous studies suggested slow flip-flop of this FA analogue, the present studies suggest that (anthroyloxy)stearic acid flip-flops rapidly and that earlier studies did not truly measure the transbilayer movement step. These findings further support the view that proteins are not required for translocation of FA across cell membranes.

The oncoprotein MDM2 regulates the activity and stability of the tumor suppressor p53 through protein-protein interaction involving their N-terminal domains. The N-terminal lid of MDM2 has been implicated in p53 regulation; however, due to its flexible nature, limited data are available concerning its role in ligand binding. The quantitative dynamics study using NMR reported here shows, for the first time, that the lid in apo-MDM2 slowly interconverts between a "closed" state that is associated with the p53-binding cleft and an "open" state that is highly flexible. Our results reveal that apo-MDM2 predominantly populates the closed state, whereas the p53-bound MDM2 exclusively populates the open state. Unlike p53 binding, the small molecule MDM2 antagonist nutlin-3 binds to the cleft essentially without perturbing the closed lid state. The lid dynamics thereby represents a signature for the experimental and virtual screening of therapeutic antagonists that target the p53-MDM2 interaction.

Covariance nuclear magnetic resonance (NMR) spectroscopy is introduced, which is a new scheme for establishing nuclear spin correlations from NMR experiments. In this method correlated spin dynamics is directly displayed in terms of a covariance matrix of a series of one-dimensional (1D) spectra. In contrast to two-dimensional (2D) Fourier transform NMR, in a covariance spectrum the spectral resolution along the indirect dimension is determined by the favorable spectral resolution obtainable along the detection dimension, thereby reducing the time-consuming sampling requirement along the indirect dimension. The covariance method neither involves a second Fourier transformation nor does it require separate phase correction or apodization along the indirect dimension. The new scheme is demonstrated for cross-relaxation (NOESY) and J-coupling based magnetization transfer (TOCSY) experiments.

A novel NMR scheme is presented that establishes homonuclear spin correlations without requiring direct detection of the spin species. This covariance NMR method is experimentally demonstrated for a mixture of amino acids and for the uniformly 13C-labeled cyclic decapeptide antamanide using a 13C-edited TOCSY experiment. The method opens up new avenues for the experimental analysis of molecules containing insensitive spins encountered in biomolecular NMR and analytical chemistry including metabolomics.