The proposal lists five specific aims: 1. To prepare sequential mutants spanning much of the C-terminal domain of aA-crystallin, replacing native amino acids with cysteines. 2. To evaluate the effects of these mutations on the stability and function of aA-crystallin using thermal denaturation circular dichroism spectroscopy, chaperone assays and gel filtration chromatography. 3. To characterize the local environment of each mutated site, assign its structural class and determine the sequence-dependent secondary structure of the C-terminal domain by nitroxide scanning experiments. 4. To explore the folding pattern of the C-terminal domain and identify the surfaces of subunit contacts in the domain using internitroxide distance measurements. 5. To develop a molecular model of the C-terminal domain backbone fold. By accomplishing the proposed specific aims, the following questions will be addressed: a. What is the sequence-dependent secondary and tertiary structure of the C-terminal domain? b. What residues are involved in subunit-subunit interactions in the quaternary structure? c. What are the key regions involved in the binding to unfolded proteins?
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