Biochemical and biophysical characterization of a monomeric porin, OmpG /
A recombinant form of the E. coli porin OmpG, has been over-expressed and purified under denaturing conditions. OmpG was folded in detergent solution, either by dialysis or by direct addition of detergent to a water-soluble form of the protein. Folding of the OmpG barrel in detergent is hierarchic...
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| Format: | Thesis Book |
| Language: | English |
| Published: |
[Place of publication not identified] :
[publisher not identified] ;
2003.
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| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=765866841&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | A recombinant form of the E. coli porin OmpG, has been over-expressed and purified under denaturing conditions. OmpG was folded in detergent solution, either by dialysis or by direct addition of detergent to a water-soluble form of the protein. Folding of the OmpG barrel in detergent is hierarchical. Secondary structure, monitored by CD, forms with a t₁/₂= 6 min at 25⁰C, while tertiary structure, monitored by heat modifiability and protease resistance, develops with a t₁/₂ = 60 min at 23⁰C. The formation of a β barrel was further demonstrated using intramolecular disulfide bond formation of a double cysteine mutant. Conditions are presented for folding OmpG in detergent with >90% efficiency. Two putative β strands were probed using cysteine scanning mutagenesis in conjunction with a modified cell-free transcription-translation system and validated a region of the predicted topology. Previous biochemical studies indicated that OmpG functions as a monomer. This is in contrast to most porins which function as trimers. The monomeric state of OmpG was confirmed using the functional protein in planar bilayers. The voltage-induced and pH-induced gating of OmpG was consistent with a monomeric porin. In addition, non-covalent blockade by gadolinium and covalent blockade of a cysteine mutant, demonstrated that OmpG is a monomer. |
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| Item Description: | Vita. "Major Subject: Medical Sciences". |
| Physical Description: | xi, 180 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 163-179). |