Interfacial Protein Research Articles

Freeze-thaw stability of Pickering emulsions stabilized by soy protein nanoparticles. Influence of ionic strength before or after emulsification

There is still a debate about the influence of salts on the freeze-thaw stability of emulsions. In this research, the influence of the NaCl addition (before or after the emulsification; 100–500 mM) on the freeze-thaw stability of the Pickering emulsions stabilized by heat-induced soy protein isolate (SPI) nanoparticles, at a given protein concentration of 1.0% (w/v) and oil fraction of 0.4, was investigated. The addition of NaCl resulted in considerable changes in particle characteristics (including particle size, surface hydrophobicity and ζ-potential) of the SPI nanoparticles, with the extent of the changes varying with the applied ionic strength (μ). Despite the NaCl addition before or after the emulsification, the presence of NaCl remarkably improved the freeze-thaw stability against droplet coalescence and creaming of the emulsions, though the improvement was related to the applied μ. The improvement of the freeze-thaw stability would be largely attributed to the strengthening of the interfacial protein or protein nanoparticle films coating droplets, rather than the inhibition of ice crystal formation by the presence of NaCl. If the salt was introduced before the emulsification, a gel-like network might be formed in the corresponding emulsions, which would further provide an additional stabilization against the freeze-thaw treatment. Optimal freeze-thaw stability was observed in the presence of appropriate concentrations of NaCl (e.g., 100–200 mM). This is the first report to indicate the importance of the electrostatic screening to the improvement of the Pickering emulsions stabilized by charged particles. The findings would be of great importance for the formulation of food grade emulsions with excellent freeze-thaw stability.

Modulating in vitro gastric digestion of emulsions using composite whey protein-cellulose nanocrystal interfaces

In this study, we designed emulsions with an oil-water interface consisting of a composite layer of whey protein isolate (WPI, 1wt%) and cellulose nanocrystals (CNCs) (1–3wt%). The hypothesis was that a secondary layer of CNCs at the WPI-stabilized oil-water interface could protect the interfacial protein layer against in vitro gastric digestion by pepsin at 37°C. A combination of transmission electron microscopy, ζ-potential measurements, interfacial shear viscosity measurements and theoretical surface coverage considerations suggested the presence of CNCs and WPI together at the O/W interface, owing to the electrostatic attraction between complementarily charged WPI and CNCs at pH 3. Microstructural analysis and droplet sizing revealed that the presence of CNCs increased the resistance of the interfacial protein film to rupture by pepsin, thus inhibiting droplet coalescence in the gastric phase, which occurs rapidly in an emulsion stabilized by WPI alone. It appeared that there was an optimum concentration of CNCs at the interface for such barrier effects. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) results further confirmed that the presence of 3wt% of CNCs reduced the rate and extent of proteolysis of protein at the interface. Besides, evidence of adsorption of CNCs to the protein-coated droplets to form more rigid layers, there is also the possibility that network formation by the CNCs in the bulk (continuous) phase reduced the kinetics of proteolysis. Nevertheless, structuring emulsions with mixed protein-particle layers could be an effective strategy to tune and control interfacial barrier properties during gastric passage of emulsions.

Determination of Absolute Orientation of Protein α-Helices at Interfaces Using Phase-Resolved Sum Frequency Generation Spectroscopy.

Understanding the structure of proteins at surfaces is key in fields such as biomaterials research, biosensor design, membrane biophysics, and drug design. A particularly important factor is the orientation of proteins when bound to a particular surface. The orientation of the active site of enzymes or protein sensors and the availability of binding pockets within membrane proteins are important design parameters for engineers developing new sensors, surfaces, and drugs. Recently developed methods to probe protein orientation, including immunoessays and mass spectrometry, either lack structural resolution or require harsh experimental conditions. We here report a new method to track the absolute orientation of interfacial proteins using phase-resolved sum frequency generation spectroscopy in combination with molecular dynamics simulations and theoretical spectral calculations. As a model system we have determined the orientation of a helical lysine-leucine peptide at the air-water interface. The data show that the absolute orientation of the helix can be reliably determined even for orientations almost parallel to the surface.

BslA-stabilized emulsion droplets with designed microstructure.

Emulsions are a central component of many modern formulations in food, pharmaceuticals, agrichemicals and personal care products. The droplets in these formulations are limited to being spherical as a consequence of the interfacial tension between the dispersed phase and continuous phase. The ability to control emulsion droplet morphology and stabilize non-spherical droplets would enable the modification of emulsion properties such as stability, substrate binding, delivery rate and rheology. One way of controlling droplet microstructure is to apply an elastic film around the droplet to prevent it from relaxing into a sphere. We have previously shown that BslA, an interfacial protein produced by the bacterial genus Bacillus, forms an elastic film when exposed to an oil- or air–water interface. Here, we highlight BslA's ability to stabilize anisotropic emulsion droplets. First, we show that BslA is capable of arresting dynamic emulsification processes leading to emulsions with variable morphologies depending on the conditions and emulsification technique applied. We then show that frozen emulsion droplets can be manipulated to induce partial coalescence. The structure of the partially coalesced droplets is retained after melting, but only when there is sufficient free BslA in the continuous phase. That the fidelity of replication can be tuned by adjusting the amount of free BslA in solution suggests that freezing BslA-stabilized droplets disrupts the BslA film. Finally, we use BslA's ability to preserve emulsion droplet structural integrity throughout the melting process to design emulsion droplets with a chosen shape and size.

Characterization of Sodium Mobility and Binding by 23 Na NMR Spectroscopy in a Model Lipoproteic Emulsion Gel for Sodium Reduction.

The effects of formulation and processing parameters on sodium availability in a model lipid/protein-based emulsion gel were studied for purposes of sodium reduction. Heat-set model gels were prepared with varying levels of protein, lipid, and NaCl contents and high pressure homogenization treatments. Single quantum and double quantum-filtered 23 Na NMR spectroscopy experiments were used to characterize sodium mobility, structural order around "bound" (restricted mobility) sodium, and sodium binding, which have been correlated to saltiness perception in food systems previously. Total sodium mobility was lower in gels with higher protein or fat content, and was not affected by changes in homogenization pressure. The gels with increased protein, fat, or homogenization pressure had increased structure surrounding "bound" sodium and more relative "bound" sodium due to increased interfacial protein interactions. The data obtained in this study provide information on factors affecting sodium availability, which can be applied towards sodium reduction in lipid/protein-based foods.

State of the art in rumen lipid protection technologies and emerging interfacial protein cross‐linking methods

Polyunsaturated fatty acids given to ruminants are to a large extent hydrogenated to more saturated forms by microbial metabolism. Numerous protection technologies have been developed to overcome this hydrogenation process in order to increase the amount of unsaturated fatty acids bypassing the rumen and resulting in an effective transfer to the peripheral tissues. This review gives an overview of the current state of the art in rumen lipid bypass technologies, with the focus on both patent‐described protection mechanisms, possible advantages or drawbacks of the technologies, and protection results being described in recent scientific literature. Lipid bypass techniques which are dealt with include calcium salts, fatty acyl amides, aldehyde treatment, non‐enzymatic browning, lipid composite gels, and encapsulation within lipids. Further, the potential of a novel rumen lipid protection technology, based on interfacial cross‐linking of emulsions, is explored. Therefore, an overview is given on current knowledge of different types of enzymatically induced cross‐linking of protein at emulsion interfaces, both for existing food and possible ruminal bypass applications.Practical applications: Both from a human and animal health care as well as from a resource‐saving and economic perspective, protection of PUFA from ruminal BH is of interest.Protection, release, and absorption principle of a lipophilic rumen bypass product: As polyunsaturated fatty acids (P) are hydrogenated to more saturated fatty acids (S) upon ruminal passage (panel A), P sources administered to ruminants have to be protected against microbial turnover to transfer them to the peripheral tissues (panel B).

Foaming and adsorption behavior of bovine and camel proteins mixed layers at the air/water interface

The aim of this work was to examine foaming and interfacial behavior of three milk protein mixtures, bovine α-lactalbumin-β-casein (M1), camel α-lactalbumin-β-casein (M2) and β-lactoglobulin-β-casein (M3), alone and in binary mixtures, at the air/water interface in order to better understand the foaming properties of bovine and camel milks. Different mixture ratios (100:0; 75:25; 50:50; 25:75; 0:100) were used during foaming tests and interfacial protein interactions were studied with a pendant drop tensiometer. Experimental results evidenced that the greatest foam was obtained with a higher β-casein amount in all camel and bovine mixtures. Good correlation was observed with the adsorption and the interfacial rheological properties of camel and bovine protein mixtures. The proteins adsorbed layers are mainly affected by the presence of β-casein molecules, which are probably the most abundant protein at interface and the most efficient in reducing the interfacial properties. In contrast of, the globular proteins, α-lactalbumin and β-lactoglobulin that are involved in the protein layer composition, but could not compact well at the interface to ensure foams creation and stabilization because of their rigid molecular structure.

Protein-crystal interface mediates cell adhesion and proangiogenic secretion.

The nanoscale materials properties of bone apatite crystals have been implicated in breast cancer bone metastasis and their interactions with extracellular matrix proteins are likely involved. In this study, we used geologic hydroxyapatite (HAP, Ca10(PO4)6(OH)2), closely related to bone apatite, to investigate how HAP surface chemistry and nano/microscale topography individually influence the crystal-protein interface, and how the altered protein deposition impacts subsequent breast cancer cell activities. We first utilized Förster resonance energy transfer (FRET) to assess the molecular conformation of fibronectin (Fn), a major extracellular matrix protein upregulated in cancer, when it adsorbed onto HAP facets. Our analysis reveals that both low surface charge density and nanoscale roughness of HAP facets individually contributed to molecular unfolding of Fn. We next quantified cell adhesion and secretion on Fn-coated HAP facets using MDA-MB-231 breast cancer cells. Our data show elevated proangiogenic and proinflammatory secretions associated with more unfolded Fn adsorbed onto nano-rough HAP facets with low surface charge density. These findings not only deconvolute the roles of crystal surface chemistry and topography in interfacial protein deposition but also enhance our knowledge of protein-mediated breast cancer cell interactions with apatite, which may be implicated in tumor growth and bone metastasis.

Switch of Surface Adhesion to Cohesion by Dopa-Fe3+ Complexation, in Response to Microenvironment at the Mussel Plaque/Substrate Interface

Although Dopa-Fe3+ complexation is known to play an important role in mussel adhesion for providing mechanical properties, its function at the plaque/substrate interface, where actual surface adhesion occurs, remains unknown, with regard to interfacial mussel adhesive proteins (MAPs) type 3 fast variant (fp-3F) and type 5 (fp-5). Here, we confirmed Dopa-Fe3+ complexation of interfacial MAPs and investigated the effects of Dopa-Fe3+ complexation regarding both surface adhesion and cohesion. The force measurements using surface forces apparatus (SFA) analysis showed that intrinsic strong surface adhesion at low pH, which is similar to the local acidified environment present during the secretion of adhesive proteins, vanishes by Dopa-Fe3+ complexation and alternatively, strong cohesion is generated in higher pH conditions similar to seawater. A high Dopa content increased the capacity for both surface adhesion and cohesion, but not at the same time. In contrast, a lack of Dopa resulted in both weak surface a...

Improved stabilization of nanoemulsions by partial replacement of sodium caseinate with pea protein isolate

In this research, pea protein isolate (PPI) was used to partially replace sodium caseinate (SC) in the formation and long-term stabilization of 5 wt% oil-in-water nanoemulsions. Both SC and PPI alone and their 1:1 mixture, at various total protein concentrations, were used to prepare the nanoemulsions using a high-pressure homogenizer. The presence of both the proteins at the oil droplet interface was confirmed by SDS-PAGE analysis of interfacial protein from the mixed-protein-stabilized nanoemulsions. All SC-stabilized nanoemulsions (d32 < 200 nm) displayed creaming due to depletion flocculation by excess proteins. PPI alone also failed to produce stable flowable nanoemulsions (d32 > 200 nm) due to excessive droplet and protein aggregation. Interestingly, nanoemulsions stabilized by 1:1 mixture of SC and PPI did not display any creaming or aggregation and remained stable for more than 6 months (d32 < 200 nm). It was proposed that under high-pressure homogenization disrupted pea proteins aggregate interacted with SC in the continuous phase of the nanoemulsions, which prevented depletion flocculation effect of SC by increasing the size and decreasing the number-density of the depletants and interfered with PPI aggregation thereby blocking both the destabilization mechanisms seen in individual protein-stabilized nanoemulsions. The interaction between the SC and PPI in the aqueous phase were confirmed by the change in surface hydrophobicity and intrinsic fluorescence of the homogenized mixed-protein solutions. The proposed method could be a novel way of utilizing plant proteins in the long-term stabilization of nanoemulsions in the food and beverage industry.

DMSO enhanced conformational switch of an interfacial enzyme.

Interfacial proteins function in unique heterogeneous solvent environments, such as water-oil interfaces. One important example is microbial lipase, which is activated in an oil-water emulsion phase and has many important enzymatic functions. A unique aprotic dipolar organic solvent, dimethyl sulfoxide (DMSO), has been shown to increase the activity of lipases, but the mechanism behind this enhancement is still unknown. Here, all-atom molecular dynamics simulations of lipase in a binary solution were performed to examine the effects of DMSO on the dynamics of the gating mechanism. The amphiphilic α5 region of the lipase was a focal point for the analysis, since the structural ordering of α5 has been shown to be important for gating under other perturbations. Compared to the closed-gorge ensemble in an aqueous environment, the conformational ensemble shifts towards open-gorge structures in the presence of DMSO solvents. Increased width of the access channel is particularly prevalent in 45% and 60% DMSO concentrations (w/w). As the amount of DMSO increases, the α5 region of the lipase becomes more α-helical, as we previously observed in studies that address water-oil interfacial and high pressure activation. We believe that the structural ordering of α5 plays an essential role on gating and lipase activity.

Accessibility of transglutaminase to induce protein crosslinking in gelled food matrices - Influence of network structure

Microbial transglutaminase (mTG) catalyzes the crosslinking of bulk and interfacial proteins, thereby influencing the physical properties of food dispersions. However, little is known about the impact of the spatial distribution of proteins in a gelled matrix on the enzymatic crosslinking. In this study, free and incorporated emulsions were subjected to mTG to induce crosslinking of emulsion interfaces. First, simple caseinate-stabilized emulsions were fabricated by high shear blending (20,000 rpm, 6 min). Second, emulsion samples were embedded into hydrogel beads using an injection technique. Different alginate (0.5–1.5%) and CaCl2 levels (50–500 mM) were used to modulate the hydrogel pore size and number of junction zones. Third, free and incorporated emulsions were mixed with mTG to investigate the diffusion behavior of the enzyme. Results showed that mTG diffused into the beads to an extent of more than 50%. A delay in ammonia release was observed when emulsions were incorporated into hydrogel beads, whereas protein in free emulsions was instantly crosslinked after mTG was added. These results illustrate that the spatial make-up of biopolymers in gelled matrices play a key role on the enzyme accessibility.

New insights about flocculation process in sodium caseinate-stabilized emulsions

Flocculation process was studied in emulsions formulated with 10wt.% sunflower oil, 2, 5 or 7.5wt.% NaCas, and with or without addition of sucrose (0, 5, 10, 15, 20 or 30wt.%). Two different processing conditions were used to prepare emulsions: ultraturrax homogenization or further homogenization by ultrasound. Emulsions with droplets with diameters above (coarse) or below (fine) 1μm were obtained. Emulsions were analyzed for droplet size distribution by static light scattering (SLS), stability by Turbiscan, and structure by confocal laser scanning microscopy (CLSM) and small angle X-ray scattering (SAXS). SAXS data were fitted by a theoretical model that considered a system composed of poly dispersed spheres with repulsive interaction and presence of aggregates. Flocculation behavior was caused by the self-assembly properties of NaCas, but the process was more closely related to interfacial protein content than micelles concentration in the aqueous phase. The results indicated that casein aggregation was strongly affected by disaccharide addition, hydrophobic interaction of the emulsion droplets, and interactions among interfacial protein molecules. The structural changes detected in the protein micelles in different environments allowed understanding the macroscopic physical behavior observed in concentrated NaCas emulsions.

Subcritical Water Induced Complexation of Soy Protein and Rutin: Improved Interfacial Properties and Emulsion Stability.

Rutin is a common dietary flavonoid with important antioxidant and pharmacological activities. However, its application in the food industry is limited mainly because of its poor water solubility. The subcritical water (SW) treatment provides an efficient technique to solubilize and achieve the enrichment of rutin in soy protein isolate (SPI) by inducing their complexation. The physicochemical, interfacial, and emulsifying properties of the complex were investigated and compared to the mixtures. SW treatment had much enhanced rutin-combined capacity of SPI than that of conventional method, ascribing to the well-contacted for higher water solubility of rutin with stronger collision-induced hydrophobic interactions. Compared to the mixtures of rutin with proteins, the complex exhibited an excellent surface activity and improved the physical and oxidative stability of its stabilized emulsions. This improving effect could be attributed to the targeted accumulation of rutin at the oil-water interface accompanied by the adsorption of SPI resulting in the thicker interfacial layer, as evidenced by higher interfacial protein and rutin concentrations. This study provides a novel strategy for the design and enrichment of nanovehicle providing water-insoluble hydrophobic polyphenols for interfacial delivery in food emulsified systems.

Understanding the stability mechanisms of lentil legumin-like protein and polysaccharide foams

Foaming properties of lentil legumin-like protein were investigated in the presence of guar gum, xanthan gum and pectin at different environmental pH conditions (3.0, 5.0, and 7.0). The protein foaming capacity was not significantly impacted by adding polysaccharides, whereas foam stability was greatly enhanced at pH 3.0 and 5.0, leading to formation of long-life foams in most samples with the highest mean life value of 275 min at pH 5.0 in the presence of pectin. Investigation of the stability mechanisms revealed that at pH 3.0, the presence of the coacervates stabilized the foams against collapse due to the formation of an electrostatically cross-linked gel-like interfacial network. At pH 5.0, aggregates were formed that adsorbed to the interface to form stiff and thick interfacial network, avoiding foam coarsening. Aggregates also plugged the junctions of the Plateau borders, slowing down the drainage by a jamming effect, and dramatically increased apparent viscosity of the foams, thus favoring the immobilization of the lamellar water surrounding the gas bubbles. The thermodynamic incompatibility at pH 7.0 resulted in a phase separation of protein and polysaccharide in the interfacial protein membrane. This induced a disruption of the protein layer around the bubbles making it weaker and easier to break, leading to reduced foaming stability. The findings revealed that guar, xanthan, and pectin can improve the stability of lentil legumin-like protein foams at mild acidic pH, creating long-life foams, which would be particularly useful in the food industry where aerated structures must be preserved for a long period of time before solidifying or gelling.

Adsorption and rheological behavior of an amphiphilic protein at oil/water interfaces

Hydrophobins are highly surface active proteins which self-assemble at hydrophilic-hydrophobic interfaces into amphipathic membranes. We investigate hydrophobin self-assembly at oil/water interfaces to deepen the understanding of protein behavior in order to improve our biomimetic synthesis. Therefore, we carried out pendant drop measurements of hydrophobin stabilized oil/water systems determining the time-dependent IFT and the dilatational rheology with additional adaptation to the Serrien protein model. We show that the class I hydrophobin H∗Protein B adsorbs at an oil/water interface where it forms a densely-packed interfacial protein layer, which dissipates energy during droplet oscillation. Furthermore, the interfacial protein layer exhibits shear thinning behavior.

Sugary interfaces mitigate contact damage where stiff meets soft.

The byssal threads of the fan shell Atrina pectinata are non-living functional materials intimately associated with living tissue, which provide an intriguing paradigm of bionic interface for robust load-bearing device. An interfacial load-bearing protein (A. pectinata foot protein-1, apfp-1) with L-3,4-dihydroxyphenylalanine (DOPA)-containing and mannose-binding domains has been characterized from Atrina's foot. apfp-1 was localized at the interface between stiff byssus and the soft tissue by immunochemical staining and confocal Raman imaging, implying that apfp-1 is an interfacial linker between the byssus and soft tissue, that is, the DOPA-containing domain interacts with itself and other byssal proteins via Fe3+–DOPA complexes, and the mannose-binding domain interacts with the soft tissue and cell membranes. Both DOPA- and sugar-mediated bindings are reversible and robust under wet conditions. This work shows the combination of DOPA and sugar chemistry at asymmetric interfaces is unprecedented and highly relevant to bionic interface design for tissue engineering and bionic devices.

Effects of the conjugation of whey proteins with gellan polysaccharides on surfactant-induced competitive displacement from the air-water interface

Whey proteins can be used to stabilize foams and emulsions against coalescence because of their ability to form viscoelastic films at the interface that resist film rupture on collision between colloidal particles. However, whey proteins are competitively displaced from the interface if small-molecule surfactants are added, leading to destabilization of the entire system. This is because surfactants are more effective in molecular packing at the interface, and they lower interfacial tension to a greater degree than whey proteins do, but their interfacial films are poor in viscoelasticity. We hypothesized that whey proteins would become more resistant to surfactant-induced competitive displacement if they were conjugated with network-forming polysaccharides. The protein moiety of the conjugate would be expected to enable its adsorption to the interface, and the polysaccharide moiety would be expected to form self-assembled networks, strengthening the interfacial film as a whole. In this study, whey proteins were conjugated with gellan polysaccharides using the Maillard reaction. Atomic force microscopy images of interfacial films formed by the whey protein–gellan conjugate at the air-water interface and transferred onto mica sheets using the Langmuir-Blodgett method revealed that gellan did form self-assembled networks at the interface and that interfacial films also contained a large number of unconjugated whey protein molecules. Following the addition of a small-molecule surfactant (Tween 20) to the sub-phase, surface pressure increased, indicating spontaneous adsorption of surfactants to the interface. Atomic force microscopy images showed decreases in interfacial area coverage by whey proteins as surface pressure increased. At a given surface pressure, the interfacial area coverage by whey protein–gellan conjugates was greater than coverage by unconjugated whey proteins, confirming that whey proteins became more resistant to surfactant-induced displacement after conjugation with gellan. Furthermore, gellan molecules added to the sub-phase after the formation of a monolayer of whey proteins at the air-water interface did not adsorb to the interfacial protein film. These results provide a molecular basis for designing interfacial structures to enhance the stability of colloidal systems.

Diffusion Behavior of Microbial Transglutaminase to Induce Protein Crosslinking in Oil-in-Water Emulsions

Food-grade hydrogel particles composed of sodium alginate were used to investigate the diffusion behavior of microbial transglutaminase (mTG) to induce crosslinking of interfacially adsorbed protein. For this purpose, mTG-loaded hydrogel beads were mixed with caseinate-stabilized oil-in-water emulsions, whereas Bradford assay and ammonia measurements were utilized to monitor the enzyme-induced interfacial protein crosslinking. Different alginate (0.5–1.5%) and gelling concentrations (50–500 mM CaCl2) were used to modulate the hydrogel mesh size and number of junction zones. The results indicated that mTG was able to diffuse out of alginate beads. However, a decrease in NH3 concentration with increasing alginate and CaCl2 levels was observed due to the formation of tight and dense bead structures. These results illustrate that the spatial distribution of molecules in complex matrices plays a key role on the enzyme accessibility

Engineering and Characterization of Peptides and Proteins at Surfaces and Interfaces: A Case Study in Surface-Sensitive Vibrational Spectroscopy.

Understanding molecular structures of interfacial peptides and proteins impacts many research fields by guiding the advancement of biocompatible materials, new and improved marine antifouling coatings, ultrasensitive and highly specific biosensors and biochips, therapies for diseases related to protein amyloid formation, and knowledge on mechanisms for various membrane proteins and their interactions with ligands. Developing methods for measuring such unique systems, as well as elucidating the structure and function relationship of such biomolecules, has been the goal of our lab at the University of Michigan. We have made substantial progress to develop sum frequency generation (SFG) vibrational spectroscopy into a powerful technique to study interfacial peptides and proteins, which lays a foundation to obtain unique and valuable insights when using SFG to probe various biologically relevant systems at the solid/liquid interface in situ in real time. One highlighting feature of this Account is the demonstration of the power of combining SFG with other techniques and methods such as ATR-FTIR, surface engineering, MD simulation, liquid crystal sensing, and isotope labeling in order to study peptides and proteins at interfaces. It is necessary to emphasize that SFG plays a major role in these studies, while other techniques and methods are supplemental. The central role of SFG is to provide critical information on interfacial peptide and protein structure (e.g., conformation and orientation) in order to elucidate how surface engineering (e.g., to vary the structure) can ultimately affect surface function (e.g., to optimize the activity). This Account focuses on the most significant recent progress in research on interfacial peptides and proteins carried out by our group including (1) the development of SFG analysis methods to determine orientations of regular as well as disrupted secondary structures, and the successful demonstration and application of an isotope labeling method with SFG to probe the detailed local structure and microenvironment of peptides at buried interfaces, (2) systematic research on cell membrane associated peptides and proteins including antimicrobial peptides, cell penetrating peptides, G proteins, and other membrane proteins, discussing the factors that influence interfacial peptide and protein structures such as lipid charge, membrane fluidity, and biomolecule solution concentration, and (3) in-depth discussion on solid surface immobilized antimicrobial peptides and enzymes. The effects of immobilization method, substrate surface, immobilization site on the peptide or protein, and surrounding environment are presented. Several examples leading to high impact new research are also briefly introduced: The orientation change of alamethicin detected while varying the model cell membrane potential demonstrates the feasibility to apply SFG to study ion channel protein gating mechanisms. The elucidation of peptide secondary structures at liquid crystal interfaces shows promising results that liquid crystal can detect and recognize different peptides and proteins. The method of retaining the native structure of surface immobilized peptides or proteins in air demonstrates the feasibility to protect and preserve such structures via the use of hydromimetic functionalities when there is no bulk water. We hope that readers in many different disciplines will benefit from the research progress reported in this Account on SFG studies of interfacial structure-function relationships of peptides and proteins and apply this powerful technique to study interfacial biomolecules in the future.