Abstract:
: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.
journal_name
J Mol Bioljournal_title
Journal of molecular biologyauthors
Simard JR,Zunszain PA,Hamilton JA,Curry Sdoi
10.1016/j.jmb.2006.06.028subject
Has Abstractpub_date
2006-08-11 00:00:00pages
336-51issue
2eissn
0022-2836issn
1089-8638pii
S0022-2836(06)00750-9journal_volume
361pub_type
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