January 13, 2007
Characterization of the ABC AF1982 using NMR Spectroscopy
Rayshonda Williams, Arthur Palmer, Tatyana Igumenova
A long-term objective of this research project is the characterization of structural and dynamic properties of the bacterial ATP Binding Cassette (ABC) transporter, AF1982. ATP-Binding Cassette transporters are a family of proteins that catalyze transmembrane transport of solutes against a concentration gradient. Forty- eight ABC transporters have been identified in humans and are linked to diseases such as cystic fibrosis, multidrug resistance, age-related macular degeneration, and adreno-leukodystrophy. AF1982 represents a tractable bacterial model system for studying structural and dynamic changes that occur in ABCs as they progress through the steps of nucleotide binding, hydrolysis, and release. We seek to identify conformational changes distal to the nucleotide binding sites that are essential for allosteric coupling between nucleotide binding and hydrolysis/transport, as well as for the nucleotide release. To achieve this objective, nuclear magnetic resonance (NMR) relaxation techniques will be used to probe conformational dynamics of apo-, ADP-, and ATP-bound AF1982. To facilitate NMR relaxation studies, isotopically labeled species of AF1982 with uniform [15N, 2H] and [15N, 13C, 2H] enrichment will be prepared and characterized by heteronuclear solution NMR spectroscopy. As initial objectives, the aggregation states of the AF1982, as a function of ligation, will be determined using 15N relaxation NMR experiments. From the current NMR relaxation and mass spectroscopy data, it is known that the protein is aggregating. A trial of various buffers has been conducted to determine the optimal conditions for the protein.