The alpha helix is the most common secondary structure in the native state of proteins, involving nearly one third of the amino acids present. This project seeks to identify the interactions responsible for helix stabilization, and to determine at what stage(s) in folding a helical structure is important. The free energies of individual interactions involved in stabilizing helical structure will be determined, using synthetic model peptides and measurement of hydrogen exchange rates by 1/H NMR. The interactions to be monitored include the helical propensity of a given side chain, in various contexts; side chain-side chain interactions, including charged and hydrophobic side chains, and helix capping interactions. The role of helix stabilizing interactions in the native state and in folding intermediates of a helical protein will be investigated, using a series of mutant myoglobins and apomyoglobins. Methods include spectroscopic definition of mutant proteins in the presence of heme, using absorbance and CD. The stability of the mutant myoglobins will be determined by thermal and solvent induced unfolding. Structures of interesting proteins will be further investigated using a new surface fingerprinting technique as well as standard techniques. The structure and stability of compact, molten globular intermediate states in apomyoglobin and the protein with heme and dyes such as ANS which bind at the heme pocket will be investigated. Specific questions to be answered include: (i) What interactions control the stability of isolated alpha helical structure? (ii) Do these play a role in early intermediates in folding a protein? (iii) How is the helix content of intermediates in myoglobin folding established? (iv) Can the globin fold be redesigned using mutants at helix-helix cross-overs and interfaces? The results should provide a detailed picture of alpical structure in models and in folding of helical proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM040746-06A1
Application #
2180567
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1989-04-01
Project End
1997-11-30
Budget Start
1994-12-01
Budget End
1995-11-30
Support Year
6
Fiscal Year
1995
Total Cost
Indirect Cost
Name
New York University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
004514360
City
New York
State
NY
Country
United States
Zip Code
10012
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Wang, L; Kallenbach, N R (1998) Proteolysis as a measure of the free energy difference between cytochrome c and its derivatives. Protein Sci 7:2460-4
Wang, L; Chen, R X; Kallenbach, N R (1998) Proteolysis as a probe of thermal unfolding of cytochrome c. Proteins 30:435-41
Yang, J; Spek, E J; Gong, Y et al. (1997) The role of context on alpha-helix stabilization: host-guest analysis in a mixed background peptide model. Protein Sci 6:1264-72
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Gong, Y; Zhou, H X; Guo, M et al. (1995) Structural analysis of the N- and C-termini in a peptide with consensus sequence. Protein Sci 4:1446-56
Zhong, M; Lin, L; Kallenbach, N R (1995) A method for probing the topography and interactions of proteins: footprinting of myoglobin. Proc Natl Acad Sci U S A 92:2111-5
Zhou, H X; Lyu, P; Wemmer, D E et al. (1994) Alpha helix capping in synthetic model peptides by reciprocal side chain-main chain interactions: evidence for an N terminal ""capping box"". Proteins 18:1-7
Lin, L; Pinker, R J; Phillips, G N et al. (1994) Stabilization of myoglobin by multiple alanine substitutions in helical positions. Protein Sci 3:1430-5
Lin, L; Pinker, R J; Kallenbach, N R (1993) Alpha-helix stability and the native state of myoglobin. Biochemistry 32:12638-43

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