(191cn) Impact of Linker Attachment Site on Structure and Dynamics of Enzymes

Immobilized enzymes have several applications in a broad range of industries, such as, food and beverages, production of biofuels, biosensors, textiles, and pharmaceuticals. Enzymes are immobilized on surfaces by covalent linkers to enhance their stability and consequently their lifetime. These stabilizing effects of enzyme immobilization often come at the expense of specific enzyme activity. This is due to the complex interplay of enzyme-linker-surface interactions, mass transfer limitations, and immobilization related enzyme unfolding. Accordingly, current immobilization methods are limited to certain enzyme-surface systems. An understanding of the molecular interactions between the enzyme-linker-substrate for a wide range of systems is therefore necessary to generalize immobilization approach.

We use combined experimental and simulation techniques to develop such a widely applicable immobilization strategy. We first explore the role of linker molecules on the catalytic activity of enzymes. Our hypothesis is that given identical linker attachment chemistry, the linker effect on enzyme activity is primarily related to the structural changes and steric hindrance caused by enzyme-linker interactions. In this regard, we perform all-atom molecular dynamics simulations of modified T4 lysozyme (WT*) and its cysteine mutants in the presence and absence of succinimidyl polyethylene glycol ester (SM (PEG)2) linker molecules. This combination of linker chemistry and cysteine mutants provides an absolute certainty on the linker attachment site. The simulations are performed on selected cysteine mutants that are experimentally observed to be either influenced or not influenced by linker attachment. We compare the structure and dynamics of the WT* and its variants in the presence and absence of linker molecules. In our presentation, we will discuss these results and comment on the strategies to attach linker molecules with less impact on structure and dynamics of enzymes.