title: Prediction of the association state of insulin using spectral parameters.
abstract: Human insulin exists in different association states, from monomer to hexamer, depending on the conditions. In the presence of zinc the "normal" state is a hexamer. The structural properties of 20 variants of human insulin were studied by near-UV circular dichroism, fluorescence spectroscopy, and small-angle X-ray scattering (SAXS). The mutants showed different degrees of association (monomer, dimers, tetramers, and hexamers) at neutral pH. A correlation was shown between the accessibility of tyrosines to acrylamide quenching and the degree of association of the insulin mutants. The near-UV CD spectra of the insulins were affected by protein association and by mutation-induced structural perturbations. However, the shape and intensity of difference CD spectra, obtained by subtraction of the spectra measured in 20% acetic acid (where all insulin species were monomeric) from the corresponding spectra measured at neutral pH, correlate well with the degree of insulin association. In fact, the near-UV CD difference spectra for monomeric, dimeric, tetrameric, and hexameric insulin are very distinctive, both in terms of intensity and shape. The results show that the spectral properties of the insulins reflect their state of association, and can be used to predict their oligomeric state.
authors.affiliation: Institute for Biological Instrumentation, Russian Academy of Sciences, 142292 Pushchino, Moscow Region, Russia. uversky@hydrogen.ucsc.edu
authors.forename: Vladimir N
authors.initials: VN
authors.lastname: Uversky
authors.forename: Liza Nielsen
authors.initials: LN
authors.lastname: Garriques
authors.forename: Ian S
authors.initials: IS
authors.lastname: Millett
authors.forename: Sven
authors.initials: S
authors.lastname: Frokjaer
authors.forename: Jens
authors.initials: J
authors.lastname: Brange
authors.forename: Sebastian
authors.initials: S
authors.lastname: Doniach
authors.forename: Anthony L
authors.initials: AL
authors.lastname: Fink
mesh_terms: D019943:Amino Acid Substitution; D002942:Circular Dichroism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000032:analysis* / Q000235:genetics; D009154:Mutation; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D014965:X-Rays
chemical_list: D007328:Insulin