Novel procedures for genetic engineering [i.e. Target Set Mutagenesis (TSM) and Recursive Ensemble Mutagenesis (REM)] will be used to construct populations of phenotypically related proteins whose members differ in amino acid sequence. TSM and REM enable a genetic engineer to mutagenize (in a combinatorial fashion) more positions in a protein than would otherwise be possible by taking advantage of the """"""""structure"""""""" of the genetic code and by reducing the sequence complexity of DNA cassettes used in mutagenesis. REM employs a feedback mechanism to further restrict sequence complexity: Each iteration of REM uses information gained from previous iterations to search """"""""sequence space"""""""" more efficiently for mutants fitting the experimenter's screening or selection criteria. Computer simulations and preliminary experiments show that TSM and REM produce diverse populations of proteins fitting specific phenotypic criteria. An efficient means of screening very large numbers of mutants uses Digital Imaging Spectroscopy (DIS), i.e. instrumentation that simultaneously records the spectra of hundreds of colonies on a petri dish. Initial experiments will utilize a pigment binding protein (LHII), because simple absorption spectroscopy can be used to assess protein expression, structure, and function. These combinatorial optimization methods have been designed to be independent of the type of protein, selection, or screen actually used. TSM and REM technology will be transferred to other areas of protein engineering, such as the expression of health-related peptides and proteins on phage display libraries to obtain altered proteins with desirable properties. This constitutes a """"""""reverse engineering"""""""" of the protein design problem.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM042645-04
Application #
3301392
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1989-07-01
Project End
1993-12-31
Budget Start
1993-04-01
Budget End
1993-12-31
Support Year
4
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Type
Schools of Arts and Sciences
DUNS #
City
Cambridge
State
MA
Country
United States
Zip Code
02139
Coleman, W J; Mattioli, T A; Youvan, D C et al. (1997) Site-directed mutations near the L-subunit D-helix of the purple bacterial reaction center: a partial model for the primary donor of photosystem II. Biochemistry 36:2178-87
Yang, T T; Kain, S R; Kitts, P et al. (1996) Dual color microscopic imagery of cells expressing the green fluorescent protein and a red-shifted variant. Gene 173:19-23
Mitra, R D; Silva, C M; Youvan, D C (1996) Fluorescence resonance energy transfer between blue-emitting and red-shifted excitation derivatives of the green fluorescent protein. Gene 173:13-7
Delagrave, S; Goldman, E R; Youvan, D C (1995) Context dependence of phenotype prediction and diversity in combinatorial mutagenesis. Protein Eng 8:237-42
Delagrave, S; Hawtin, R E; Silva, C M et al. (1995) Red-shifted excitation mutants of the green fluorescent protein. Biotechnology (N Y) 13:151-4
Delagrave, S; Goldman, E R; Youvan, D C (1993) Recursive ensemble mutagenesis. Protein Eng 6:327-31
Delagrave, S; Youvan, D C (1993) Searching sequence space to engineer proteins: exponential ensemble mutagenesis. Biotechnology (N Y) 11:1548-52
Robles, S J; Youvan, D C (1993) Hydropathy and molar volume constraints on combinatorial mutants of the photosynthetic reaction center. J Mol Biol 232:242-52
Nabedryk, E; Robles, S J; Goldman, E et al. (1992) Probing the primary donor environment in the histidineM200-->leucine and histidineL173-->leucine heterodimer mutants of Rhodobacter capsulatus by light-induced Fourier transform infrared difference spectroscopy. Biochemistry 31:10852-8
Arkin, A P; Youvan, D C (1992) An algorithm for protein engineering: simulations of recursive ensemble mutagenesis. Proc Natl Acad Sci U S A 89:7811-5

Showing the most recent 10 out of 20 publications