Application of SIMSTEX to oligomerization of insulin analogs and mutants.
The propensity of various insulins and their analogs to oligomerize was investigated by mass spectrometric methods including measurement of the relative abundances of oligomers in the gas phase and the kinetics of H/D amide exchange. The kinetics of deuterium uptake show a good fit when the exchanging amides are placed in three kinetic groups: fast, intermediate, and slow. r-Human insulin, of the insulins investigated, has fewer amides that exchange at intermediate rates and more that exchange at slow rates, in accord with its higher extent of association in solution. We adapted PLIMSTEX (protein ligand interactions by mass spectrometry, titration, and H/D exchange) to determine protein/ligand affinities in solution, to determine self-association equilibrium constants for proteins, and to apply them to various insulin analogs. We term this adaptation SIMSTEX (self-association interactions using mass spectrometry, self-titration and H/D exchange); it gives affinity constants that compare well with the literature results. The results from SIMSTEX show that some mutants (e.g., GlnB13) have an increased tendency to self-associate, possibly slowing down their action in vivo. Other mutants (e.g., lispro and AspB9) have lower propensities for self-association, thus providing potentially faster-acting analogs for use in controlling diabetes.
Chitta
Raghu K
RK
Department of Chemistry, Washington University, One Brookings Drive, Box 1134, 63130, St. Louis, MO, USA.
Rempel
Don L
DL
Department of Chemistry, Washington University, One Brookings Drive, Box 1134, 63130, St. Louis, MO, USA.
Grayson
Michael A
MA
Department of Chemistry, Washington University, One Brookings Drive, Box 1134, 63130, St. Louis, MO, USA.
Remsen
Edward E
EE
Department of Chemistry, Washington University, One Brookings Drive, Box 1134, 63130, St. Louis, MO, USA.
Gross
Michael L
ML
Department of Chemistry, Washington University, One Brookings Drive, Box 1134, 63130, St. Louis, MO, USA. mgross@wustl.edu.
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D041961:Deuterium Exchange Measurement / Q000379:methods*; D006801:Humans; D006859:Hydrogen / Q000737:chemistry*; D007328:Insulin / Q000031:analogs & derivatives* / Q000737:chemistry*; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins; D013552:Swine
D007328:Insulin; D011994:Recombinant Proteins; D006859:Hydrogen