Unfolding and refolding of juvenile hormone binding protein.
Juvenile hormone (JH) regulates insect development. JH present in the hemolymph is bound to a specific glycoprotein, juvenile hormone binding protein (JHBP), which serves as a carrier to deploy the hormone to target tissues. In this report structural changes of JHBP from Galleria mellonella induced by guanidine hydrochloride have been investigated by a combination of size-exclusion chromatography, protein activity measurements, and spectroscopic methods. Molecules of JHBP change their conformation from a native state via two unstable intermediates to a denatured state. The first intermediate appears in a compact state, because it slightly changes its molecular size and preserves most of the JHBP secondary structure of the native state. Although the second intermediate also preserves a substantial part of the secondary structure, it undergoes a change into a noncompact state changing its Stokes radius from approximately 30 to 39 A. Refolding experiments showed that JHBP molecules recover their full protein structure, as judged from the CD spectrum, fluorescence experiments, and JH binding activity measurements. The free energy of unfolding in the absence of the denaturant, DeltaG(D-N), is calculated to be 4.1 kcal mol(-1).
Dobryszycki
Piotr
P
Division of Biochemistry, Institute of Organic Chemistry, Biochemistry and Biotechnology, Wrocław University of Technology, Wrocław, Poland.
Kołodziejczyk
Robert
R
Krowarsch
Daniel
D
Gapiński
Jacek
J
Ozyhar
Andrzej
A
Kochman
Marian
M
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry*; D019791:Guanidine / Q000737:chemistry*; D019476:Insect Proteins; D007605:Juvenile Hormones / Q000737:chemistry*; D009036:Moths; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D014443:Tyrosine / Q000737:chemistry*
D002352:Carrier Proteins; D019476:Insect Proteins; D007605:Juvenile Hormones; C035606:juvenile hormone-binding protein, insect; D014443:Tyrosine; D019791:Guanidine