Folding Myoglobin within a Sol-Gel Glass: Protein Folding Constrained to a Small Volume

Abstract

The unfolding and refolding reaction of myoglobin was examined in solution and within a porous silica sol-gel glass. The sol-gel pores constrain the protein to a volume that is the same size and shape as the folded native state accompanied by a few layers of water solvation. Denaturants such as low pH buffers can be diffused through the gel pores to the protein to initiate unfolding and refolding. Acid-induced unfolding was hindered by the steric constraints imposed by the gel pores such that more denaturing conditions were required within the gel than in solution to create the unfolded state. No new folding intermediates were observed. Refolding of myoglobin was not complete in millimolar pH 7 buffer alone. Addition of 25% glycerol to the pH 7 buffer resulted in nearly complete refolding, and the use of 1 M phosphate buffer resulted in complete refolding. The role of this cosolvent and salt in disrupting the ordered water surrounding the protein within the gel is discussed in light of the Hofmeister series and entropic trapping via a diminished hydrophobic effect within the gel. These results are consistent with the premises of folding models in which secondary and tertiary structures are considered to form within a compact conformation of the protein backbone.

Models of macromolecular crowding effects and the need for quantitative comparisons with experiment

“…Since these and other experimental works (e.g. [58,59]) continue to advance our understanding of protein folding in sol-gel system s, and since significant computational and theoretical attention has already been focused on modeling solvent-driven effects that control behavior of biomolecules in confined, hydrophobic environments (e.g. [60–62]), it appears that there should be opportunities for using simulations to understand sol-gel behavior at a level beyond that of pure excluded volume effects.…”

Reverse micelles as a tool for probing solvent modulation of protein dynamics: Reverse micelle encapsulated hemoglobin

“…This conclusion is supported by our observation (not shown) that for Hb-Bd in solution and in a sol–gel, the initial peak position shifts from 470 nm to 527 nm and 506 nm, respectively when denaturants are added (either 6 M urea or 5 M GdHCl). The confinement condition of the sol–gel has been shown to limit the degree of denaturant- induced “unfolding” [47,76,77]. The implication is that added osmolytes can be used to reverse the seemingly minor loosening effect of the RM on at least some aspects of conformation.…”

Immobilization of Proteins in Silica Gel: Biochemical and Biophysical Properties

“…Hemoglobin and myoglobin were also used as test molecules to investigate the effects of encapsulation on protein stability and folding [71,[97][98][99]. However, the most striking result about Hb encapsulated in silica gels is the perfect conservation of equilibrium oxygen binding properties observed in solution under comparable conditions [54,57,58,82,100,101] (Fig.…”