St. Norbert College1, Chemistry, De Pere, WI 54115 University of California-Santa Barbara2, Physics, Santa Barbara, CA 93106 University of Wisconsin-Madison3, Chemistry, Madison, WI 53706 University of California-Los Angeles4, Chemistry & Biochemistry, Los Angeles, CA 90095 Universtiy of Wisconsin-Madison5, Chemistry, Madison, WI 53706
Exclusion of water from the hydrophobic core is a prerequisite for the folding of most proteins. This “hydrophobic collapse” is thought to be one of the main driving forces for correct protein folding. In the all-helical, single domain protein apomyoglobin, a molten globular equilibrium folding intermediate can be populated at pH=4.1. This intermediate contains a highly dynamic compact core with some degree of secondary structure. However, nothing is known about the water content (i.e., the degree of hydration) of the core. If the entropically favored exclusion of ordered water has already occurred in the molten globular state, other forces must be responsible for completing the folding of the protein to its native conformation. Cysteine point mutations were carried out at positions 41 (a solvent exposed site) and 131 (a deeply buried site) of apomyoglobin. Nucleophilic attack by the cysteine thiol was exploited to covalently attach a stable nitroxide radical spin label to the protein. The unpaired electron of this spin label is currently being employed as a sensitive probe through the use of a new technique known as dynamic nuclear polarization- (DNP-) enhanced nuclear magnetic resonance (NMR). DNP performs polarization transfer from the highly polarized electron spin to the surrounding nuclear spin, here the protons of water, which leads to amplification of the NMR signal of water. The key is that >80% of the signal amplification effect originates from water within 5Å of the spin label and that the signal amplification critically and quantitatively depends on the translational dynamics of the local water interacting with the spin label. Thus, DNP-enhanced NMR provides a tool to monitor the interaction between proteins and the local water with amino acid site-specific resolution. We expect to obtain direct evidence in favor or against the presence of water and its dynamics inside the molten globular core by comparing the DNP of molten globule, unfolded and native states.
[Abstract (DOC)]