This project was performed in collaboration with Professor Rod MacKinnon at Rockefeller who succeeded at determining the structure of KcsA. KcsA is a potassium channel from the bacteria Streptomyces lividens. Until the study by MacKinnon, no voltage-dependent potassium ion channel had been structurally characterized by high-resolution methods. KcsA (160 residues wild-type) forms a tetrameric integral membrane structure within the lipid bilayer. The structure is believed to be highly conserved in all cellular organisms. In the crystallization study, a detergent micelle was used in lieu of phospholipid membrane to stabilize the tetrameric KcsA structure. Initially, MacKinnon found that full length recombinant KcsA failed to crystallize. Unstructured regions of the protein were suspected to be causing poor contacts between the KcsA tetramers, leading to sub-optimal crystallization conditions. MALDI mass spectrometry was of great assistance at this point in the project because it quickly provided information concerning the compact folding domain of KcsA. A C-terminal His-tag construct of the full-length protein was subjected to various rounds of proteolysis using trypsin, chymotrypsin and subtilisin. Time-course digestions and MALDI-MS peptide mapping revealed that the C-terminal of the protein (35 residues) was rapidly cleaved (~1 hr) leaving a highly protease-resistant compact domain (residues 1-125). The N-terminal of the protein (~23 residues) underwent a very slow cleavage (many days) at a single site by subtilisin. A new construct of KcsA spanning the compact domain ultimately yielded diffraction-grade crystals that showed electron density from residues 23 to 119. We speculated that the N-terminal 23 residues were outside of the micelle making non-specific contact with the detergent micelle in a random fashion. Because of its random orientation, the N-terminal does not appear in the electron density map but is protected from enzymatic cleavage through interactions with the bulky micelle. Since membrane proteins are notoriously difficult to analyze by mass spectrometric methods, conditions for analyzing KcsA by mass spectrometry needed to be explored. Various long alkyl-chain detergents were examined and found to be compatible with both the crystallization conditions and MALDI-MS analysis. Mass spectrometry was continually used to monitor the purity and stability of the KcsA protein. The compact construct of KcsA used for crystallization was prepared by cleaving off a C-terminal His-tag with chymotrypsin. MALDI-MS revealed that the chymotrypsin was contaminated with trypsin activity, resulting in degradation of the protein; inclusion of a trypsin inhibitor ensured protein stability. Finally, mass spectrometry was used in the selection of suitable heavy-atom derivatization reagents for X-ray phase determination. Different mercurating reagents were screened against a wide variety of positional cysteine KcsA mutants. The presence of the large detergent micelle embedding the KcsA tetramer meant that screening for a derivatizing agent of suitable polarity and size be carefully performed. MALDI-MS allowed a rapid screening of derivatized-KcsA. Methyl mercury chloride was deemed to yield the highest incorporation of mercury. KcsA crystals were also analyzed directly by MALDI-MS for incorporation of methyl mercury, hastening the selection of optimum crystallization and mercuration conditions. The work has resulted in three papers: D.A. Doyle, J.M. Cabral, R.A. Pfuetzner, A. Kuo, J.M. Gulbis, S.L. Cohen, B.T. Chait, R. Mackinnon, """"""""The Structure of the Potassium Channel Molecular Basis of K+ Conduction and Selectivity"""""""" Science 280, (1998) 69-77. R. Mackinnon, S.L. Cohen, A. Kuo, A. Lee, B.T. Chait """"""""Structural Conservation in Prokaryotic and Eukaryotic Potassium Channels"""""""" Science 280 (1998) 106-109. J.H. Morais Cabral, A. Lee, S. Cohen, B.T. Chait, M. Li, R. Mackinnon """"""""Structure of the HERG potassium channel amino-terminal domain: definition of a structural family of PAS domains"""""""" Cell in press.
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