Conformational analysis by nuclear magnetic resonance: insulin.

Area of Science:

• Biochemistry
• Structural Biology
• Spectroscopy

Background:

• Insulin hexamers are crucial for insulin storage and function.
• Understanding insulin's conformational dynamics is key to its biological activity.
• Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for studying protein structure and dynamics.

Purpose of the Study:

• To investigate the conformational changes of the two-zinc insulin hexamer using high-resolution proton NMR.
• To determine the effect of zinc ion and anion binding on insulin hexamer structure.
• To characterize the binding of thiocyanate (SCN-) to the insulin hexamer.

Main Methods:

• Acquisition of high-resolution 270-MHz proton NMR spectra of the native two-zinc insulin hexamer at pH 9.
• Titration experiments involving zinc ions (Zn2+) and thiocyanate (SCN-) anions.
• Analysis of NMR spectral changes to infer conformational alterations and binding characteristics.

Main Results:

• The conformation of the insulin hexamer is established with the addition of one equivalent of zinc per hexamer.
• Titration with thiocyanate (SCN-) induces significant conformational changes, forming a new hexameric structure with altered symmetry.
• The new thiocyanate-bound hexamer binds two equivalents of SCN- at independent sites with dissociation constants K1 = 10(3) M-1 and K2 = 2.5 x 10(2) M-1.

Conclusions:

• The two-zinc insulin hexamer exhibits a stable conformation that is largely unaffected by the second zinc ion.
• Anions high on the Hofmeister series, like SCN-, can induce substantial structural rearrangements in the insulin hexamer.
• The binding of SCN- suggests a potential mechanism for regulating insulin structure and function.