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N‐terminal extension changes the folding mechanism of the FK506‐binding protein
Author(s) -
Korepanova Alla,
Douglas Chanel,
Leyngold Ilya,
Logan Timothy M.
Publication year - 2001
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.14801
Subject(s) - fkbp , protein folding , chemistry , denaturation (fissile materials) , amino acid , folding (dsp implementation) , biophysics , n terminus , phi value analysis , peptide sequence , equilibrium unfolding , crystallography , stereochemistry , biochemistry , biology , gene , electrical engineering , nuclear chemistry , engineering
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 α‐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.