Location of high and low affinity fatty acid binding sites on human serum albumin revealed by NMR drug-competition analysis.

Human serum albumin (HSA) is an abundant plasma protein that transports a wide variety of drugs and endogenous compounds. The complex binding capacity of HSA has made it a challenging system to study in detail but in order to develop our understanding of the interactions between ligands for HSA, the locations and relative affinities of different ligand binding sites must be determined. Albumin possesses multiple binding sites for its primary physiological ligand, non-esterified fatty acids (FA). Previously, titration of BSA with (13)C-labeled FA revealed multiple chemical shifts and allowed identification of a subset of three chemical shifts that were associated with high affinity FA binding. Recent crystallographic studies of HSA have mapped at least seven FA binding sites for long-chain FA and delineated the overlap with binding sites for drugs and other endogenous compounds. We aim to correlate NMR and structural data for FA to provide a more complete description of the binding capacity of HSA. Our recent mutagenesis studies allowed us to identify two high affinity binding sites in domain III of HSA. Here, we use NMR to study the binding of (13)C-carboxyl labeled palmitate to HSA in the presence and absence of competitor ligands to complete the correlation of NMR chemical shifts with specific structural binding sites. We carefully selected ligands with specific binding sites identified by crystallography and used them, either singly or in combination, to compete with [(13)C]palmitate for binding to HSA. We show that FA sites 2, 4 and 5 bind FA with high affinity, while sites 1, 3, 6 and 7 exhibit low affinity for FA, thus providing the first complete determination of relative affinities of all seven long-chain FA sites on HSA. Our results also yield direct insights into the interactions between FA and other ligands.

Visualization and tracking of single protein molecules in the cell nucleus.

A recently developed laser fluorescence videomicroscopy method was used to determine for the first time the intranuclear trajectories of single protein molecules. Using the recombinant Escherichia coli beta-galactosidase protein P4K, labeled with an average of 4.6 ALEXA 488 chromophores per tetramer, single P4K molecules could be localized and tracked in the nuclei of permeabilized 3T3 cells at a spatial accuracy of approximately 30 nm and a time resolution of 18 ms. Our previous photobleaching measurements indicated that P4K had two fractions inside the nucleus, a larger mobile and a smaller immobile fraction. The present study supported this observation but revealed a much larger variety of mobility classes. Thus, a fraction of P4K molecules appeared to be truly immobile while another fraction was mobile but confined to very small areas. In addition, a large fraction of the P4K molecules appeared to be mobile and to move over extended distances by diffusion. However, a quantitative analysis showed that at least two subpopulations were present differing widely in diffusion coefficients. Importantly, both the diffusion coefficients and the fractions of these subpopulations were time-dependent. Our results suggest that proteins can move inside the nucleus over extended distances by diffusion. However, intranuclear protein diffusion is severely restricted, most likely by multiple association-dissociation events and/or impermeable obstacles.