Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 293, Issue 28, Pages 10895-10910Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA118.003650
Keywords
molecular pharmacology; protein design; insulin; protein self-assembly; molecular dynamics
Categories
Funding
- United States Department of Energy (DOE), Office of Science and Office of Basic Energy Sciences [DE-AC02-76SF00515]
- National Institutes of Health NIGMS [P41GM103393]
- DOE Office of Biological and Environmental Research
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Key contributions to protein structure and stability are provided by weakly polar interactions, which arise from asymmetric electronic distributions within amino acids and peptide bonds. Of particular interest are aromatic side chains whose directional -systems commonly stabilize protein interiors and interfaces. Here, we consider aromatic-aromatic interactions within a model protein assembly: the dimer interface of insulin. Semi-classical simulations of aromatic-aromatic interactions at this interface suggested that substitution of residue Tyr(B26) by Trp would preserve native structure while enhancing dimerization (and hence hexamer stability). The crystal structure of a [Trp(B26)]insulin analog (determined as a T3R3f zinc hexamer at a resolution of 2.25 ) was observed to be essentially identical to that of WT insulin. Remarkably and yet in general accordance with theoretical expectations, spectroscopic studies demonstrated a 150-fold increase in the in vitro lifetime of the variant hexamer, a critical pharmacokinetic parameter influencing design of long-acting formulations. Functional studies in diabetic rats indeed revealed prolonged action following subcutaneous injection. The potency of the Trp(B26)-modified analog was equal to or greater than an unmodified control. Thus, exploiting a general quantum-chemical feature of protein structure and stability, our results exemplify a mechanism-based approach to the optimization of a therapeutic protein assembly.
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