Experimental characterization of adsorbed protein orientation, conformation, and bioactivity

Abstract

Protein adsorption on material surfaces is a common phenomenon that is of critical importance in many biotechnological applications. The structure and function of adsorbed proteins are tightly interrelated and play a key role in the communication and interaction of the adsorbed proteins with the surrounding environment. Because the bioactive state of a protein on a surface is a function of the orientation, conformation, and accessibility of its bioactive site(s), the isolated determination of just one or two of these factors will typically not be sufficient to understand the structure-function relationships of the adsorbed layer. Rather a combination of methods is needed to address each of these factors in a synergistic manner to provide a complementary dataset to characterize and understand the bioactive state of adsorbed protein. Over the past several years, the authors have focused on the development of such a set of complementary methods to address this need. These methods include adsorbed-state circular dichroism spectropolarimetry to determine adsorptioninduced changes in protein secondary structure, amino-acid labeling/mass spectrometry to assess adsorbed protein orientation and tertiary structure by monitoring adsorption-induced changes in residue solvent accessibility, and bioactivity assays to assess adsorption-induced changes in protein bioactivity. In this paper, the authors describe the methods that they have developed and/or adapted for each of these assays. The authors then provide an example of their application to characterize how adsorption-induced changes in protein structure influence the enzymatic activity of hen eggwhite lysozyme on fused silica glass, high density polyethylene, and poly(methyl-methacrylate) as a set of model systems.

How enzymes are adsorbed on soil solid phase and factors limiting its activity: A Review

“…Many immobilized enzymes obtained with different methods have been analysed using CD. Modification in the enzyme secondary structure after immobilization has also been observed by CD spectroscopy (Barth, 2007;Thyparambil et al, 2015;Verma et al, 2013).…”

“…Raman spectroscopy is also potentially a strong technique to analyse enzyme structure in dried or frozen solution samples (Thyparambil et al, 2015;Xiao et al, 2012). There is increasing use of Raman spectroscopy to characterize immobilized enzyme.…”

“…Latest technique such as Foster resonance energy transfer (FRET) measurements (Mohamad et al, 2015), solid-state nuclear magnetic resonance spectroscopy (NMR) (Noureddini and Gao, 2007), Trp/Tyr fluorescence measurements (da Silva et al, 2015), and TOF-secondary ion mass spectrometry (Thyparambil et al, 2015) are gathering strength and more advancement are expected in near future.…”

“…CD elucidates the structure information of enzyme solid surface interaction (Carlsson et al, 2014;Thyparambil et al, 2015). CD spectroscopy is an alternative to FTIR spectroscopy in the characterization of an immobilized enzyme.…”

Absence of conformational change in complement factor 3 and factor XII adsorbed to acrylate polymers is related to a high degree of polymer backbone flexibility

“…Recently, Latour and coworkers addressed conformational change upon adsorption by implementing an improved experimental circular dichroism set-up as well as mass spectroscopy. 41,42 By the use of these methods, eventual conformational change can be directly assessed and the degree of unfolding, and accessibility of the antibody binding site can be investigated.…”