Effects of tethering a multistate folding protein to a surface

Abstract

Protein/surface interactions are important in a variety of fields and devices, yet fundamental understanding of the relevant phenomena remains fragmented due to resolution limitations of experimental techniques. Molecular simulation has provided useful answers, but such studies have focused on proteins that fold through a two-state process. This study uses simulation to show how surfaces can affect proteins which fold through a multistate process by investigating the folding mechanism of lysozyme (PDB ID: 7LZM). The results demonstrate that in the bulk 7LZM folds through a process with four stable states: the folded state, the unfolded state, and two stable intermediates. The folding mechanism remains the same when the protein is tethered to a surface at most residues; however, in one case the folding mechanism changes in such a way as to eliminate one of the intermediates. An analysis of the molecular configurations shows that tethering at this site is advantageous for protein arrays because the active site is both presented to the bulk phase and stabilized. Taken as a whole, the results offer hope that rational design of protein arrays is possible once the behavior of the protein on the surface is ascertained.

Communication: Using multiple tethers to stabilize proteins on surfaces

“…Prior simulation work using the same models and simulation protocols as those used in the present work has also shown that when bound to a surface using only one tether, the four-state mechanism was maintained except when the tether was placed at residue 91 which resulted in a three-state mechanism that eliminated the most unfolded intermediate. 9 Subsequent experimental studies have confirmed that tethering to residue 91 does indeed result in improved activity compared to other residues which validates the simulation methodology. 19 The purpose of this research was to determine if attaching 7LZM to a surface using multiple tethers will stabilize the protein more than has been seen previously with a single tether.…”

“…9,18 The stable states are the folded state, the unfolded state, and two partially folded intermediates. Prior simulation work using the same models and simulation protocols as those used in the present work has also shown that when bound to a surface using only one tether, the four-state mechanism was maintained except when the tether was placed at residue 91 which resulted in a three-state mechanism that eliminated the most unfolded intermediate.…”

“…Wei and Knotts 8 showed that there are two basic criteria that must be fulfilled to keep proteins stable on surfaces and also showed that selecting appropriate tethers can remove stable but unfolded intermediate states from multistate folding proteins. 9 Other studies have also been done by various groups showing that protein stability on surfaces is dependent on the location of the tether site in the protein. [10][11][12] These have yielded valuable insights; however, a careful study of the literature reveals that all of the simulation work done to date has only used one tether.…”

The role of fluctuations in bistability and oscillations during the H2 + O2 reaction on nanosized rhodium crystals

“…For small proteins, the entire molecule is close to the surface and this close proximity impacts the rotational and vibrational freedom of the entire molecule. 12,13 In contrast, the size of the Ab and the flexible loops that connect the Fabs and Fc domains impart a high degree of rotational and vibrational freedom even when tethered to the surface. Rotational and vibrational freedom also correlate to the changes in stability among different types of surfaces.…”

“…10,11 As such, molecular simulation has emerged as the primary method used a) Electronic mail: thomas.knotts@byu.edu when studying protein/surface interactions. [12][13][14][15][16] This work uses molecular simulation to determine how tethering an Ab to different types of surfaces in different orientations affects the stability of the molecule. Specifically, two different orientations (flat and upright) and two types of surfaces (hydrophobic and hydrophilic) have been investigated using the IgG Ab 1IGT.…”