N-terminal extension changes the folding mechanism of the FK506-binding protein.

Journal Article

Many of the protein fusion systems used to enhance the yield of recombinant proteins result in the addition of a small number of amino acid residues onto the desired protein. Here, we investigate the effect of short (three amino acid) N-terminal extensions on the equilibrium denaturation and kinetic folding and unfolding reactions of the FK506-binding protein (FKBP) and compare the results obtained with data collected on an FKBP variant lacking this extension. Isothermal equilibrium denaturation experiments demonstrated that the N-terminal extension had a slight destabilizing effect. NMR investigations showed that the N-terminal extension slightly perturbed the protein structure near the site of the extension, with lesser effects being propagated into the single alpha-helix of FKBP. These structural perturbations probably account for the differential stability. In contrast to the relatively minor equilibrium effects, the N-terminal extension generated a kinetic-folding intermediate that is not observed in the shorter construct. Kinetic experiments performed on a construct with a different amino acid sequence in the extension showed that the length and the sequence of the extension both contribute to the observed equilibrium and kinetic effects. These results point to an important role for the N terminus in the folding of FKBP and suggest that a biological consequence of N-terminal methionine removal observed in many eukaryotic and prokaryotic proteins is to increase the folding efficiency of the polypeptide chain.

Full Text

Duke Authors

Cited Authors

  • Korepanova, A; Douglas, C; Leyngold, I; Logan, TM

Published Date

  • September 2001

Published In

Volume / Issue

  • 10 / 9

Start / End Page

  • 1905 - 1910

PubMed ID

  • 11514681

Electronic International Standard Serial Number (EISSN)

  • 1469-896X

International Standard Serial Number (ISSN)

  • 0961-8368

Digital Object Identifier (DOI)

  • 10.1110/ps.14801

Language

  • eng