Studies of the association and conformational properties of metal-free insulin in alkaline sodium chloride solutions by one- and two-dimensional 1H NMR.
One- and two-dimensional 1H NMR spectroscopy have been employed to probe the association and subsequent conformational changes of metal-free insulin in sodium chloride solution at pH 9 and 9.4. These studies establish that the proton resonances of His(B5) and His(B10) are useful signatures of aggregation and conformation. Changes in chemical shifts and areas of resonances due to the C2 protons of His(B10) and His(B5) and transfer of magnetization experiments served to identify the association as the assembly of tetramer from dimers under our experimental conditions (pH 9.4, [insulin] greater than 1 mM, [NaCl] = 0.1 M). Sodium chloride also alters the equilibrium distribution of species in favor of a tetrameric species. The association equilibrium constant was estimated from area measurements to be approximately 5 x 10(3) M-1 at pH 9.4, 26 +/- 0.1 degrees C, and 0.1 M sodium chloride. Under conditions of 0.1 M sodium chloride concentration, nuclear Overhauser effect experiments in the one- and two-dimensional modes revealed an operative nuclear Overhauser effect between the His(B5) C2 protons and the 2,6 ring protons of a Tyr residue provisionally assigned as Tyr(B16). We conclude that this interaction is a diagnostic signature of a conformational transition whereupon an extended chain from residues B1 to B9 (T-state) is transformed into an alpha-helix (R-state) thus bringing the rings of His(B5) and Tyr(B16) from adjacent subunits across the monomer-monomer interface into van der Waals contact. This conformational flexibility is an added consideration to the discussion of the relevant structure of insulin for receptor binding.
Kadima
W
W
Department of Biochemistry, University of California, Riverside 92521-0129.
Roy
M
M
Lee
R W
RW
Kaarsholm
N C
NC
Dunn
M F
MF
D000818:Animals; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008670:Metals / Q000737:chemistry*; D011487:Protein Conformation; D012965:Sodium Chloride / Q000737:chemistry; D012996:Solutions; D013552:Swine
D007328:Insulin; D008670:Metals; D012996:Solutions; D012965:Sodium Chloride