Probing protein aggregation at buried interfaces: distinguishing between adsorbed protein monomers, dimers, and a monomer-dimer mixture in situ.

Tieyi Lu,Yue Xin,E Neil G Marsh,Prathamesh M Datar,Zhan Chen,Wen Guo

doi:10.1039/d1sc04300e

Abstract

Protein adsorption on surfaces greatly impacts many applications such as biomedical materials, anti-biofouling coatings, bio-separation membranes, biosensors, antibody protein drugs etc. For example, protein drug adsorption on the widely used lubricant silicone oil surface may induce protein aggregation and thus affect the protein drug efficacy. It is therefore important to investigate the molecular behavior of proteins at the silicone oil/solution interface. Such an interfacial study is challenging because the targeted interface is buried. By using sum frequency generation vibrational spectroscopy (SFG) with Hamiltonian local mode approximation method analysis, we studied protein adsorption at the silicone oil/protein solution interface in situ in real time, using bovine serum albumin (BSA) as a model. The results showed that the interface was mainly covered by BSA dimers. The deduced BSA dimer orientation on the silicone oil surface from the SFG study can be explained by the surface distribution of certain amino acids. To confirm the BSA dimer adsorption, we treated adsorbed BSA dimer molecules with dithiothreitol (DTT) to dissociate these dimers. SFG studies on adsorbed BSA after the DTT treatment indicated that the silicone oil surface is covered by BSA dimers and BSA monomers in an approximate 6 : 4 ratio. That is to say, about 25% of the adsorbed BSA dimers were converted to monomers after the DTT treatment. Extensive research has been reported in the literature to determine adsorbed protein dimer formation using ex situ experiments, e.g., by washing off the adsorbed proteins from the surface then analyzing the washed-off proteins, which may induce substantial errors in the washing process. Dimerization is a crucial initial step for protein aggregation. This research developed a new methodology to investigate protein aggregation at a solid/liquid (or liquid/liquid) interface in situ in real time using BSA dimer as an example, which will greatly impact many research fields and applications involving interfacial biological molecules.

Highlights

By using sum frequency generation vibrational spectroscopy (SFG) with Hamiltonian local mode approximation method analysis, we studied protein adsorption at the silicone oil/protein solution interface in situ in real time, using bovine serum albumin (BSA) as a model
The sample geometry used in this study to collect SFG spectra from the silicone oil/solution interface is shown in the Electronic supplementary information (ESI) (Fig. S1).† Time-dependent SFG signals were rst collected by tracking the signal intensity of the characteristic peak from a-helices in BSA a er the silicone oil surface was placed in contact with the BSA solution
The use of DTT in this study is aimed to decrease the BSA dimer/ monomer ratio, which is successful for interfacial BSA according to our SFG results presented above and for bulk BSA according to the PAGE data shown in the ESI.†

Summary

Introduction

Protein molecular behavior on surfaces or at interfaces determines protein properties and function, and greatly impacts many applications and research elds, especially those involving interfacial proteins, such as biosensors, anti-fouling coatings, biomedical implants, and membranes used for bioseparation, and protein antibody drugs.[1,2,3,4,5,6,7,8,9,10,11] In order to rationally design surfaces/interfaces to optimize interfacial protein properties and functions through controlling protein interfacial behavior, it is important to develop powerful tools to investigateSum frequency generation vibrational spectroscopy (SFG) is a unique and powerful second-order nonlinear vibrational spectroscopic tool,[15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36] which can detect amide I signals from interfacial protein molecules, providing molecular levelEdge Article information regarding the protein structure.[18,19,31,37,38] According to the selection rule of a second-order nonlinear optical process, SFG is a highly surface/interface-sensitive method with a sub-monolayer speci city.[15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39] It allows probing protein behavior at buried solid/liquid and liquid/liquid interfaces in situ directly.[18,19,31,39,40] SFG can monitor interfacial adsorption of proteins in real time and probe detailed structural information of interfacial proteins (such as protein orientation).[18,19,31,37,38,39,40,41] In previous studies by our group, we have reported the successful detection and orientation analysis of many proteins at different interfaces, including cell membrane associated proteins,[42] surface chemically immobilized enzymes,[43] and physically adsorbed proteins.[37,40]. Sum frequency generation vibrational spectroscopy (SFG) is a unique and powerful second-order nonlinear vibrational spectroscopic tool,[15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36] which can detect amide I signals from interfacial protein molecules, providing molecular level.

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Conclusion