Conformational dynamics of insulin in solution. Circular dichroic studies

Abstract

Conformational changes of bovine insulin in solution with concentration and pH detected by circular dichroic (CD) studies are reported. The change in the CD spectrum of insulin in the higher concentration range (from 100 microM down to 2 microM) is relatively small, but in the lower concentration range (from 2 microM down to 60 nM) the CD spectrum changes substantially with concentration. A detailed analysis of the data indicates that the hormone has two major conformational states: conformation I, a form which predominates in extremely dilute solutions and corresponds to the monomeric state, and conformation II, a form present in the crystalline state and also, with but minor changes, in all associated states in solution. The apparent conformation of insulin at various concentrations is computed by using a nonlinear least-squares iterative computer program. The mean residue ellipticities at 223 and 208 nm are extrapolated by using [theta] lambda VS. fmonomer plots to calculate the conformations of monomeric and dimeric insulin. These calculations indicate that conformation I of insulin has 21% less helix content than conformation II, the latter conformation being very similar to that found in the crystalline state. It is also evident from these calculations that the conformational transition is of the helix--coil type. Studies pertaining to the dependence of the CD spectrum of insulin on pH are also reported, and a comparison is made with earlier sedimentation coefficient studies. An analysis of the data indicates that changes in the sedimentation coefficient correspond quite closely to changes of CD spectra with pH. This paper is the first report known to us pertaining to conformational studies of insulin in the monomeric state; it presents evidence for conformational transitions of the protein hormone induced by concentration and pH. Since insulin is biologically active mainly in the monomeric state, a knowledge of its conformation in this state should be an important tool in deciphering the molecular basis of insulin action.

Liposomes containing glycocholate as potential oral insulin delivery systems: preparation, in vitro characterization, and improved protection against enzymatic degradation

“…35 Therefore, it was employed as an easy tool to study the conformational stability of the rhINS. Preparative variables, such as homogenization pressure and number of cycles, hydration time, amount of soybean phosphotidylcholine and insulin, and volume of organic solvents volume, at their extreme levels, may have an impact on the conformational stability of rhINS.…”

“…35,36 The ratio of θ 208 /θ 223 can be used as a qualitative measure of rhINS association. 34 This is due to the antiparallel β structure which forms when two monomers dimerize.…”

Molecular-replacement studies on crystal forms of despentapeptide insulin

“…It has been assumed that the hormone's structure does not change significantly when it dimerizes but it is difficult to prove this unequivocally. Indeed, spectroscopic studies have suggested that the insulin monomer partially unwinds in solution (Pocker & Biswas, 1980), which would invalidate the structure-function arguments (Blundell, Dodson, Hodgkin & Mercola, 1972;Peking Insulin Structure Research Group, 1974). However, the crystal structure of DPI shows that this modified monomer is closely similar to the insulin structure present in 2-Zn insulin and in the dimer forms of hagfish insulin and pig insulin (Cutfield, Cutfield, Dodson, Dodson, Reynolds & Vallely, 1981;Dodson, Dodson, Hodgkin & Reynolds, 1979).…”

Nanoemulsions coated with alginate/chitosan as oral insulin delivery systems: preparation, characterization, and hypoglycemic effect in rats

“…Generally, the secondary structures (α-helix and β-fold) can illustrate the efficacy of insulin, and CD is regarded as one of the most effective methods to evaluate the secondary structures of proteins and peptides. 27 Figure 4 shows the CD spectra of the insulin secondary structures. The CD spectra of insulin in mixture with emulsion and the coated nanoemulsion were totally identical to the insulin solution, which showed a peak valley at 209 nm and a shoulder at around 225 nm.…”