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oai:www.ideals.illinois.edu:2142/87260 2023-07-11 col_2142_5131 col_2142_16508 com_2142_5130 com_2142_16507 com_2142_14793 com_2142_8903

Byron Kemper Chen, Ci-Di 2015-09-28T15:50:16Z 2015-09-28T15:50:16Z 10000-01-01 1998 1998 The linker region connecting the N-terminal signal anchor and the cytoplasmic catalytic domain of P450 2C2 contains two segments with distinct sequence properties: a Gly-rich and a Pro-rich region. The activities and spectral properties were determined for both Pro- and Gly-rich region mutants expressed in COS-1 cells, bacteria, and insect cells. In COS-1 cells, mutations at Pro 30 and Pro33 in the Pro-rich region dramatically reduced activity, suggesting that a PXXP motif may be important for the formation or activity of a functional P450, and further that this sequence might have a helical structure with a repeat of three, as in the left-handed polyproline II helix. Substitution of Pro30 and Pro33 with Ala resulted in a reduced P450 and increased P420 for the mutants expressed in bacteria or insect cells which correlated with the decreased activity in COS-1 cells. These data suggest the Pro-rich region is critical for the folding of P450. Substitution of the Gly residues in the Gly-rich region with Ala or Pro or the entire sequence from 22 to 28 with Ala did not reduce laurate hydroxylase activity of the proteins expressed in COS-1 cells. Deletion of residues 22-28 or substitution with valine inactivated the protein. Substitution of two Ala resulted in loss of activity in COS-1 cells, but activity increased progressively with substitution of 3 or 4 Ala to activities similar to wild-type. Lengthening the linker from 2 to 7 Ala resulted in progressively increased P450 which corresponded with decreased inactive P420 for the mutants expressed in bacteria or insect cells. Substitution of 7 Val resulted in only the P420 form of the protein and deletion of 22-28 resulted in neither P450 nor P420 forms. The activities per nmole P450 were similar for wild type and the mutants with 2 to 7 Ala substituted. These data are consistent with a role for the Gly-rich region as a linker which facilitates the folding of P450 into a functional protein, but is not required for the activity of the folded protein. Made available in DSpace on 2015-09-28T15:50:16Z (GMT). No. of bitstreams: 2 license.txt: 4848 bytes, checksum: 96035ab3f5e1c23cc7138a224ce498bd (MD5) 9834662.pdf: 3517201 bytes, checksum: aa0ca4c0afe50962dde521dd630040c9 (MD5) Previous issue date: 1998 Embargo set by: Seth Robbins for item 88541 Lift date: Forever Reason: Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs U of I Only 89 p. Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1998. http://hdl.handle.net/2142/87260 (MiAaPQ)AAI9834662 eng Biology, Molecular Functional Analysis of the Region Linking the N-Terminal Transmembrane Anchor and the Catalytic Domain of Cytochrome P450 2C2 text Molecular and Integrative Physiology Molecular and Integrative Physiology University of Illinois at Urbana-Champaign Dissertation Ph.D.