The long term goals of this project are to understand the structure and function of physiologically and clinically important membrane transporters through a combination of X-ray crystallography and biochemical-biophysical approaches. Membrane proteins play crucial roles in many aspects of cell function;some are targets for pharmacologically and toxicologically active substances. The human facilitated glucose transporters (Gluts), members of the Major Facilitator Superfamily (MFS), comprise a relatively large family of structurally related proteins (GLUT1-12). Glut1, which is abundant in the human red blood cells (RBCs) and the blood-brain barrier, is an extensively-studied representative of these facilitated transporters. Glut4 is responsible for insulin-regulated glucose disposal. Several diseases have been identified with the mutation and malfunction of Gluts, such as GLUT1 deficiency syndrome, a hereditary neurological syndrome, and type II diabetes, one of major threats to human health that is becoming alarmingly epidemic in scale. Therefore, it is important to obtain a high-resolution structure, as well as characterize the transport mechanism at a molecular level. So far, there is no 3-D crystal structure available for any glucose facilitator, although many attempts have been made, particularly with Glut1. This proposal will focus on the crystallization of eukaryotic facilitated Gluts, particularly Glut1 from human RBCs. Isolation and purification of Glut1 from human RBCs was achieved more than two decades ago. Recently, the PI of this proposal observed that phospholipids (PL) play an important role in the crystallization of lactose permease of Escherichia coli, a member of MFS. Manipulating PL has dramatically improved our success rate for crystallization related membrane proteins. Therefore, the use of PL in crystallization should yield a promising approach for obtaining crystal structures of eukaryotic members of the MFS. Glut1, a human protein of MFS, will be purified from RBC membranes;over-expression systems of eukaryotic Glut1-4 in different systems will be simultaneously tested. Manipulation of PL content will be introduced in addition to conventional crystallization methods, to crystallize human Glut1 and/or the eukaryotic Gluts, which will lay the foundation for solving X-ray crystal structures of these important eukaryotic transporters in the future. Successful crystallization of eukaryotic Gluts is imperative to obtaining X-ray crystal structures;the expected structures will substantially improve our understanding of facilitated glucose transport and provide important clues for therapeutic intervention, which will have significant impact in biology and medicine.
