Human serum albumin (HSA), the most abundant protein in human blood plasma, serves as a transporting agent for various endogenous compounds and drug molecules. [1,2] Its capability to bind and transport multiple fatty acids (FA), in particular, has been studied extensively in the past. [3,4] Research on HSA was severely hampered by the complexity of the protein and has benefitted tremendously from crystallographic high-resolution structures. Nearly 20 years ago, He and Carter reported the first crystal structure of HSA. [5] To date, a multitude of HSA crystal structures have been deposited in the Protein Data Bank.
Even more important for understanding the binding properties of the protein are the structures of complexes of HSA and transported molecules. Thanks to the pioneering work of Curry et al., crystal structures of various HSA/fatty acid complexes have become accessible. [6][7][8] They found that fatty acids are distributed highly asymmetrically in the protein crystal although HSA itself exhibits a symmetric primary and secondary structure. Up to seven distinct binding sites were found for long-chain fatty acids, most of which comprised ionic anchoring units and long, hydrophobic pockets. [8,9] The location of two to three high-affinity binding sites [3,10] was assigned by correlation of the X-ray structure with NMR studies on competitive binding of drugs that replaced 13 Clabeled fatty acids. [11,12] Sites 2, 4, and 5 bind fatty acids with a high affinity, while sites 1, 3, 6, and 7 exhibit a somewhat lower affinity to fatty acids (see Figure 1 a).
More generally, there is a long-standing debate as to what extent protein crystal structures reflect the dynamic and functional structures of proteins in solution. This debate is often fueled by apparent discrepancies between X-ray crystallographic data and results from solution-state-based techniques (e.g. NMR and other types of spectroscopy as well as neutron scattering) or from molecular dynamics simulations. Moreover, there is an increasing awareness that protein dynamics in solution is connected to biological function.
Recent NMR studies revealed that many proteins exhibit [*] M.