Exploring Enzymatic Conformational Dynamics at Surfaces through μ-FTIR Spectromicroscopy

Abstract

Immobilization of proteins onto solid supports has critical industrial, technological, and medical applications, and is a daily task in chemical research. Significant conformational rearrangements often occur due to enzyme–surface interactions, and it is of broad interest to develop methods to probe and better understand these molecular-level changes that contribute to the enzyme’s catalytic activity and stability. While circular dichroism is a common method for solution-phase conformational study, the application to surface-supported proteins is not trivial and spatial mapping is not viable. On the other hand, a nonlinear laser spectroscopy technique used to analyze surfaces and interfaces is not often found in most laboratories, therefore requiring an alternative and reliable method. Here, we employed high-dimensional data spectromicroscopy analysis in the infrared region (μ-FTIR) to investigate the enzyme’s conformational change when adsorbed onto solid matrices, across a ca. 20 mm2 area. Alcohol dehydrogenase (ADH) enzyme was adopted as a model enzyme to interact with CaF2, Au, and Au-thiol model substrates, strategically chosen for mapping the enzyme dynamics on solid surfaces with different polarity/hydrophobicity properties and extendable to other materials. Two-dimensional chemical maps indicate that the enzyme adsorbs with different patterns in which secondary structures dynamically adjust to optimize interprotein and enzyme–surface interactions. The results suggest an experimental approach to identify and map enzyme conformational dynamics onto different solid surfaces across space and provide insights into immobilized protein structure investigations for areas such as biosensing and bioenergy.

Unraveling the Nano–Bio Interface Interactions of a Lipase Adsorbed on Gold Nanoparticles under Laser Excitation

“…It is well established that the surface of AuNPs is highly favorable for protein adsorption, mainly through electrostatic interactions or covalent bonding via thiol groups. , The amino groups present in the CALB structure are also favorable for interacting with the carboxyl groups on the surface of the AuNPs that remain from citrate-driven reduction and stabilization. Additionally, the structure of CALB contains 6 cysteine and 4 methionine residues, which possess thiolated groups that thermodynamically preferentially interact with the AuNP surface. , Ultimately, CALB and AuNPs are expected to interact through subsequent anchoring, crawling, and binding kinetic processes. , …”

Hydrogen bioelectrogeneration with pH-resilient and oxygen-tolerant cobalt apoenzyme-saccharide

“…To assess the impact of reconstitution and subsequent environmental conditions on the secondary structure of SacCoMyo, it underwent a test of structural stability in an undisturbed H 2 SO 4 0.5 mol L −1 solution with a pH of 1 for a month. Throughout this incubation, we continuously monitored the protein's secondary structure using high-resolution FTIR spectromicroscopy 11 (micro-FTIR) (Fig. S3, ESI†).…”