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.
more »
« less
Early sum frequency generation vibrational spectroscopic studies on peptides and proteins at interfaces
This paper summarizes the early research results on studying proteins and peptides at interfaces using sum frequency generation (SFG) vibrational spectroscopy. SFG studies in the C—H stretching frequency region to examine the protein side-chain behavior and in the amide I frequency region to investigate the orientation and conformation of interfacial peptides/proteins are presented. The early chiral SFG research and SFG isotope labeling studies on interfacial peptides/proteins are also discussed. These early SFG studies demonstrate the feasibility of using SFG to elucidate interfacial molecular structures of peptides and proteins in situ, which built a foundation for later SFG investigations on peptides and proteins at interfaces.
more »
« less
- Award ID(s):
- 1904380
- NSF-PAR ID:
- 10332742
- Date Published:
- Journal Name:
- Biointerphases
- Volume:
- 17
- Issue:
- 3
- ISSN:
- 1934-8630
- Page Range / eLocation ID:
- 031202
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Protein structures at solid/liquid interfaces mediate interfacial protein functions, which are important for many applications. It is difficult to probe interfacial protein structures at buried solid/liquid interfaces in situ at the molecular level. Here, a systematic methodology to determine protein molecular structures (orientation and conformation) at buried solid/liquid interfaces in situ was successfully developed with a combined approach using a nonlinear optical spectroscopic technique – sum frequency generation (SFG) vibrational spectroscopy, isotope labeling, spectra calculation, and computer simulation. With this approach, molecular structures of protein GB1 and its mutant (with two amino acids mutated) were investigated at the polymer/solution interface. Markedly different orientations and similar (but not identical) conformations of the wild-type protein GB1 and its mutant at the interface were detected, due to the varied molecular interfacial interactions. This systematic strategy is general and can be widely used to elucidate protein structures at buried interfaces in situ .more » « less
-
Choose your own adventure: Picosecond or broadband vibrational sum-frequency generation spectroscopyVibrational sum-frequency generation (VSFG) spectroscopy is a method capable of measuring chemical structure and dynamics within the interfacial region between two bulk phases. At the core of every experimental system is a laser source that influences the experimental capabilities of the VSFG spectrometer. In this article, we discuss the differences between VSFG spectrometers built with picosecond and broadband laser sources as it will impact everything from material costs, experimental build time, experimental capabilities, and more. A focus is placed on the accessibility of the two different SFG systems to newcomers in the SFG field and provides a resource for laboratories considering incorporating VSFG spectroscopy into their research programs. This Tutorial provides a model decision tree to aid newcomers when determining whether the picosecond or femtosecond laser system is sufficient for their research program and navigates through it for a few specific scenarios.more » « less
-
Sum frequency generation (SFG) * Equal contributors. spectroscopy was used to deduce the orientation of the terminal methyl (CH 3 ) group of self-assembled monolayers (SAMs) at the air–solid and air–liquid interfaces at surface concentrations as low as 1% protonated molecules in the presence of 99% deuterated molecules. The SFG spectra of octadecanethiol (ODT) and deuterated octadecanethiol (d 37 ODT) SAMs on gold were used for analysis at the air–solid interface. However, the eicosanoic acid (EA) and deuterated EA (d 39 EA) SAMs on the water were analyzed at the air–liquid interface. The tilt angle of the terminal CH 3 group was estimated to be ∼39 ° for a SAM of 1% ODT : 99% d 37 ODT, whereas the tilt angle of the terminal CH 3 group of the 1% EA : 99% d 39 EA monolayer was estimated to be ∼32 °. The reliability of the orientational analysis at low concentrations was validated by testing the sensitivity of the SFG spectroscopy. A signal-to-noise (S/N) ratio of ∼60 and ∼45 was obtained for the CH 3 symmetric stretch (SS) of 1% ODT : 99% d 37 ODT and 1% EA : 99% d 39 EA, respectively. The estimated increase in S/N ratio values, as a measure of the sensitivity of the SFG spectroscopy, verified the capacity to acquire the SFG spectra at low concentrations of interfacial molecules under ambient conditions. Overall, the orientational analysis of CH 3 SS vibrational mode was feasible at low concentrations of protonated molecules due to increased S/N ratio. In support, the improved S/N ratio on varying incident power density of the visible beam was also experimentally demonstrated.more » « less
-
Eddy, Charles R. (Ed.)more » « less
An often-quoted statement attributed to Wolfgang Pauli is that God made the bulk, but the surface was invented by the devil. Although humorous, the statement really reflects frustration in developing a detailed picture of a surface. In the last several decades, that frustration has begun to abate with numerous techniques providing clues to interactions and reactions at surfaces. Often these techniques require considerable prior knowledge. Complex mixtures on irregular or soft surfaces—complex interfaces—thus represent the last frontier. Two optical techniques: sum frequency generation (SFG) and second harmonic generation (SHG) are beginning to lift the veil on complex interfaces. Of these techniques, SFG with one excitation in the infrared has the potential to provide exquisite molecular- and moiety-specific vibrational data. This Perspective is intended both to aid newcomers in gaining traction in this field and to demonstrate the impact of high-phase resolution. It starts with a basic description of light-induced surface polarization that is at the heart of SFG. The sum frequency is generated when the input fields are sufficiently intense that the interaction is nonlinear. This nonlinearity represents a challenge for disentangling data to reveal the molecular-level picture. Three, high-phase-resolution methods that reveal interactions at the surface are described.
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1116/6.0001859
