Studies of the association and conformational properties of metal-free insulin in alkaline sodium chloride solutions by one- and two-dimensional 1H NMR

• Published in: The Journal of biological chemistry Vol. 267; no. 13; pp. 8963 - 8970
• Main Authors: KADIMA, W, ROY, M, LEE, R. W.-K, KAARSHOLM, N. C, DUNN, M. F
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 05-05-1992
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Humans; Hydrogen-Ion Concentration; Insulin - chemistry; Magnetic Resonance Spectroscopy - methods; Metals - chemistry; Protein Conformation; Protein hormones. Growth factors. Cytokines; Proteins; Sodium Chloride - chemistry; Solutions; Swine; Protein hormone; Sodium; Chlorides; Molecular association; NMR spectrometry; Alkalinity; Insulin; Conformation
• DOI: 10.1016/S0021-9258(19)50374-X
• EISSN: 1083-351X
• ISSN: 0021-9258

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.