1H NMR investigations of the nature of fluctuations in structure of native protein molecules responsible for the chemical exchange of N-H protons with solvent are proposed. Two model systems will be used. First I have prepared a set of closely related short peptides (18-24 amino acids) from lyophilized venom of the honey bee, A. mellifera. These include the central nervous neurotoxin apamin, the mast cell degranulating peptide (P401), and two molecules of unknown function, secapin and tertiapin (1-3). Each represents a minor component (less than 3 percent) of the total venom compared to mellitin (greater than 50 percent) and phospholipase A. Their structure, stability and spectroscopic properties (4-6) make them ideal models for Alpha-helices in solution; in each case the stability is very high due to the presence of disulfide bridges. Measurements of 1H-2H exchange rates as functions of concentration, pH and temperature using the fully assigned amide IH NMR spectrum of apamin (7) should provide direct tests of the magnitude, cooperativity, and possibly kinetics of the """"""""opening"""""""" reactions mediating hydrogen exchange in these molecules. Studies of the folding of these chains can also be carried out, since apamin, and possibly the others, refold following reduction and reoxidation of the disulfides (4,7). The second experimental system involves the use of general base catalysts to catalyse the exchange of certain side chain residues in myoglobin and other heme proteins. In a number of heme proteins, the 1H resonances corresponding to exchangeable side chains and peptides in the vicinity of the heme have been assigned (10). These include several histidine ring N-H protons, which I have shown to respond to catalysis by organic bases (e.g., piperidine) as well as hydroxide. Preliminary results indicate that the simple local unfolding model fails to explain the differences in catalysis observed for piperidine and OH-. Further work using other basis and experimental conditions, together with a model for the """"""""open"""""""" state of the ligand -- the aquo cobalamin imidazole complex -- should provide the first detailed picture of the exchange reaction in which access to the extremely hydrophobic heme pocket is involved.
| Kallenbach, N R; Lu, M; Vasant Kumar, N et al. (1990) Distribution of charged residues stabilizes individual helices in myoglobins. J Biomol Struct Dyn 7:973-83 |
| Pease, J H; Kumar, N V; Schweitz, H et al. (1989) NMR studies of toxin III from the sea anemone Radianthus paumotensis and comparison of its secondary structure with related toxins. Biochemistry 28:2199-204 |
| Nelson, J W; Kallenbach, N R (1989) Persistence of the alpha-helix stop signal in the S-peptide in trifluoroethanol solutions. Biochemistry 28:5256-61 |
| Kumar, N V; Wemmer, D E; Kallenbach, N R (1988) Structure of P401 (mast cell degranulating peptide) in solution. Biophys Chem 31:113-9 |
| Kumar, N V; Kallenbach, N R (1985) Hydrogen exchange of individual amide protons in the F helix of cyanometmyoglobin. Biochemistry 24:7658-62 |
