In vitro association of fragments of a beta-sheet membrane protein

TY - JOUR

T1 - In vitro association of fragments of a beta-sheet membrane protein

AU - Debnath, D

AU - Nielsen, K L

AU - Otzen, D E

PY - 2010/5/1

Y1 - 2010/5/1

N2 - Although the beta-barrel membrane protein OmpA can be produced in a biologically active form in E. coli from co-expressed fragments, the fragments have not been demonstrated to associate in vitro. We have produced 3 complementary fragment pairs of OmpA which can associate to form a folded complex according to the SDS band-shift assay. We are able to convert 25-35% of the fragment populations to non-covalent but SDS-stable complexes. The periplasmic chaperone Skp effectively prevented this association. Two separately expressed and purified overlapping fragments of OmpA can form a protease-resistant complex that undergoes the characteristic band-shift upon heating. Our work demonstrates that although membrane insertion and folding of beta-barrel membrane proteins may be a cooperative process, the fragments can associate in vitro without any additional components. However, the low yield and slow folding rates indicate that partially unfolded or destabilized beta-sheet membrane proteins can potentially engage in many non-native interactions.

AB - Although the beta-barrel membrane protein OmpA can be produced in a biologically active form in E. coli from co-expressed fragments, the fragments have not been demonstrated to associate in vitro. We have produced 3 complementary fragment pairs of OmpA which can associate to form a folded complex according to the SDS band-shift assay. We are able to convert 25-35% of the fragment populations to non-covalent but SDS-stable complexes. The periplasmic chaperone Skp effectively prevented this association. Two separately expressed and purified overlapping fragments of OmpA can form a protease-resistant complex that undergoes the characteristic band-shift upon heating. Our work demonstrates that although membrane insertion and folding of beta-barrel membrane proteins may be a cooperative process, the fragments can associate in vitro without any additional components. However, the low yield and slow folding rates indicate that partially unfolded or destabilized beta-sheet membrane proteins can potentially engage in many non-native interactions.

KW - Amino Acid Sequence

KW - Bacterial Outer Membrane Proteins

KW - DNA-Binding Proteins

KW - Escherichia coli Proteins

KW - Hot Temperature

KW - Kinetics

KW - Models, Molecular

KW - Molecular Chaperones

KW - Peptide Hydrolases

KW - Protein Folding

KW - Protein Structure, Secondary

KW - Protein Structure, Tertiary

KW - Substrate Specificity

U2 - 10.1016/j.bpc.2010.03.004

DO - 10.1016/j.bpc.2010.03.004

M3 - Journal article

C2 - 20356666

SN - 1057-8943

VL - 148

SP - 112

EP - 120

JO - Advances in Biophysical Chemistry

JF - Advances in Biophysical Chemistry

IS - 1-3

ER -