Cell membranes are crucial to many biological processes. Because of their complexity, however, lipid bilayers are often used as model systems. Lipid structures influence the physical properties of bilayers, but their interplay, especially in multiple-component lipid bilayers, has not been fully explored. Here, we used the Langmuir–Blodgett method to make mono- and bilayers of 1,2-dihexadecanoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), and 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phospho-l-serine (POPS) as well as their 1:1 binary mixtures. We studied the fluidity, stability, and rigidity of these structures using sum frequency generation (SFG) spectroscopy combined with analyses of surface pressure–area isotherms, compression modulus, and stability. Our results show that single-component bilayers, both saturated and unsaturated, may not be ideal membrane mimics because of their low fluidity and/or stability. However, the binary saturated and unsaturated DPPG/POPG and DPPG/POPS systems show not only high stability and fluidity but also high resistance to changes in surface pressure, especially in the range of 25–35 mN/m, the range typical of cell membranes. Because the ratio of saturated to unsaturated lipids is highly regulated in cells, our results underline the possibility of modulating biological properties using lipid compositions. Also, our use of flat optical windows as solid substrates in SFG experiments should make the SFG method more compatible with other techniques, enabling more comprehensive future surface characterizations of bilayers.

We characterized BslA, a bacterial biofilm protein, at the air/water interface using vibrational sum frequency generation spectroscopy and observed one of the sharpest amide I bands ever reported. Combining methods of surface pressure measurements, thin film X-ray reflectivity, and atomic force microscopy, we showed extremely ordered BslA at the interface.

Correction for ‘A narrow amide I vibrational band observed by sum frequency generation spectroscopy reveals highly ordered structures of a biofilm protein at the air/water interface’ by Zhuguang Wang et al., Chem. Commun., 2016, 52, 2956–2959.

Proteins are desirable building blocks to create self-assembled, spatially defined structures and interfaces on length-scales that are inaccessible by traditional methods. Here, we describe a novel approach to create functionalized monolayers using the proteins BslA and SpyCatcher/SpyTag. BslA is a bacterial hydrophobin whose amphiphilic character underlies its ability to assemble into a monolayer at both air/water and oil/water interfaces. We demonstrate that Bsa1A having the SpyTag peptide fused at the N- or C-terminus does not affect the formation of such monolayers. We establish the creation of stable oil-in-water microcapsules using BslA, and also show the fabrication of capsules outwardly displaying the reactive SpyTag peptide by fusing it to the C-terminus of BslA. Such capsules can be covalently labeled by reacting the surface-displayed SpyTag with SpyCatcher fused to any desired protein. We demonstrate this principle by labeling microcapsules using green fluorescent protein (GFP). All components are genetically encodable, the reagents can be readily prepared in large quantities, and all reactions occur at ambient temperature in aqueous solution. Thus, this straightforward, modular, scalable strategy has myriad potential applications in the creation of novel, functional materials, and interfaces.Keywords: hydrophobin; microcapsules; modular; protein design; self-assembly; surface display

Two peptide–linker–lipid constructs were designed and prepared which target the parathyroid hormone 1 receptor, a family B G protein-coupled receptor. Both show increased agonist activity in a cell-based assay. The lipid moiety enables the formation of micelle-like nanostructures, which is shown to hinder proteolytic digestion and is expected to reduce renal clearance.

Characterization of protein secondary structures at interfaces is still challenging due to the limitations of surface-selective optical techniques. Here, we address the challenge of characterizing parallel β-sheets by combining chiral sum frequency generation (SFG) spectroscopy and computational modeling. We focus on human islet amyloid polypeptide aggregates and a de novo designed short polypeptide at lipid/water and air/glass interfaces. We find that parallel β-sheets adopt distinct orientations at various interfaces and exhibit characteristic chiroptical responses in the amide I and N–H stretch regions. Theoretical analysis indicates that the characteristic chiroptical responses provide valuable information on the symmetry, orientation, and vibrational couplings of parallel β-sheet at interfaces.

Characterizations of protein structures at interfaces are important in solving an array of fundamental and engineering problems, including understanding transmembrane signal transduction and molecular transport processes and development of biomaterials to meet the needs of biomedical and energy research. However, in situ and real-time characterization of protein secondary structures is challenging because it requires physical methods that are selective to both interface and secondary structures. Here, we summarize recent experimental developments in our laboratory of chiral vibrational sum frequency generation spectroscopy (SFG) for analyzing protein structures at interfaces. We showed that chiral SFG provides vibrational optical signatures of the peptide N–H stretch and amide I modes that can distinguish various protein secondary structures. Using these signatures, we further applied chiral SFG to probe orientations and folding kinetics of proteins at interfaces. Our results show that chiral SFG is a background-free, label-free, in situ, and real-time vibrational method for studying proteins at interfaces. This recent progress demonstrates the potential of chiral SFG in solving problems related to proteins and other chiral biopolymers at interfaces.

Visual pigments can be thermally activated via isomerization of the retinyl chromophore and hydrolysis of the Schiff base (SB) through which the retinyl chromophore is bound to the opsin protein. Here, we present the first combined experimental and theoretical study of the thermal activation of a Siberian hamster ultraviolet (SHUV) pigment. We measured the rates of thermal isomerization and hydrolysis in the SHUV pigment and bovine rhodopsin. We found that these rates were significantly faster in the UV pigment than in rhodopsin due to the difference in the structural and electrostatic effects surrounding the unprotonated Schiff base (USB) retinyl chromophore in the UV pigment. Theoretical (DFT-QM/MM) calculations of the cis–trans thermal isomerization revealed a barrier of ∼23 kcal/mol for the USB retinyl chromophore in SHUV compared to ∼40 kcal/mol for protonated Schiff base (PSB) chromophore in rhodopsin. The lower barrier for thermal isomerization in the SHUV pigment is attributed to the (i) lessening of the steric restraints near the β-ionone ring and SB ends of the chromophore, (ii) displacement of the transmembrane helix 6 (TM6) away from the binding pocket toward TM5 due to absence of the salt bridge between the USB and the protonated E113 residue, and (iii) change in orientation of the hydrogen-bonding networks (HBNs) in the extracellular loop 2 (EII). The results in comparing thermal stability of UV cone pigment and rhodopsin provide insight into molecular evolution of vertebrate visual pigments in achieving low discrete dark noise and high photosensitivity in rod pigments for dim-light vision.

We present measurements of rate constants for thermal-induced reactions of the 11-cis retinyl chromophore in vertebrate visual pigment rhodopsin, a process that produces noise and limits the sensitivity of vision in dim light. At temperatures of 52.0–64.6 °C, the rate constants fit well to an Arrhenius straight line with, however, an unexpectedly large activation energy of 114 ± 8 kcal/mol, which is much larger than the 60-kcal/mol photoactivation energy at 500 nm. Moreover, we obtain an unprecedentedly large prefactor of 1072±5 s−1, which is roughly 60 orders of magnitude larger than typical frequencies of molecular motions! At lower temperatures, the measured Arrhenius parameters become more normal: Ea = 22 ± 2 kcal/mol and Apref = 109±1 s−1 in the range of 37.0–44.5 °C. We present a theoretical framework and supporting calculations that attribute this unusual temperature-dependent kinetics of rhodopsin to a lowering of the reaction barrier at higher temperatures due to entropy-driven partial breakup of the rigid hydrogen-bonding network that hinders the reaction at lower temperatures.

Ultraviolet (UV) cone pigments can provide insights into the molecular evolution of vertebrate vision since they are nearer to ancestral pigments than the dim-light rod photoreceptor rhodopsin. While visible-absorbing pigments contain an 11-cis retinyl chromophore with a protonated Schiff-base (PSB11), UV pigments uniquely contain an unprotonated Schiff-base (USB11). Upon F86Y mutation in model UV pigments, both the USB11 and PSB11 forms of the chromophore are found to coexist at physiological pH. The origin of this intriguing equilibrium remains to be understood at the molecular level. Here, we address this phenomenon and the role of the USB11 environment in spectral tuning by combining mutagenesis studies with spectroscopic (UV–vis) and theoretical [DFT-QM/MM (SORCI+Q//B3LYP/6-31G(d): Amber96)] analysis. We compare structural models of the wild-type (WT), F86Y, S90A and S90C mutants of Siberian hamster ultraviolet (SHUV) cone pigment to explore structural rearrangements that stabilize USB11 over PSB11. We find that the PSB11 forms upon F86Y mutation and is stabilized by an “inter-helical lock” (IHL) established by hydrogen-bonding networks between transmembrane (TM) helices TM6, TM2, and TM3 (including water w2c and amino acid residues Y265, F86Y, G117, S118, A114, and E113). The findings implicate the involvement of the IHL in constraining the displacement of TM6, an essential component of the activation of rhodopsin, in the spectral tuning of UV pigments.

Studying hydrogen/deuterium (H/D) exchange in proteins can provide valuable insight on protein structure and dynamics. Several techniques are available for probing H/D exchange in the bulk solution, including NMR, mass spectroscopy, and Fourier transform infrared spectroscopy. However, probing H/D exchange at interfaces is challenging because it requires surface-selective methods. Here, we introduce the combination of in situ chiral sum frequency generation (cSFG) spectroscopy and ab initio simulations of cSFG spectra as a powerful methodology to probe the dynamics of H/D exchange at interfaces. This method is applied to characterize H/D exchange in the antiparallel β-sheet peptide LK7β. We report here for the first time that the rate of D-to-H exchange is about 1 order of magnitude faster than H-to-D exchange in the antiparallel structure at the air/water interface, which is consistent with the existing knowledge that O–H/D dissociation in water is the rate-limiting step, and breaking the O–D bond is slower than breaking the O–H bond. The reported analysis also provides fundamental understanding of several vibrational modes and their couplings in peptide backbones that have been difficult to characterize by conventional methods, including Fermi resonances of various combinations of peptide vibrational modes such as amide I and amide II, C–N stretch, and N–H/N–D bending. These results demonstrate cSFG as a sensitive technique for probing the kinetics of H/D exchange in proteins at interfaces, with high signal-to-noise N–H/N–D stretch bands that are free of background from the water O–H/O–D stretch.

GPCRs mediate intracellular signaling upon external stimuli, making them ideal drug targets. However, little is known about their activation mechanisms due to the difficulty in purification. Here, we introduce a method to purify GPCRs in nanodiscs, which incorporates GPCRs into lipid bilayers immediately after membrane solubilization, followed by single-step purification. Using this approach, we purified a family B GPCR, parathyroid hormone 1 receptor (PTH1R), which regulates calcium and phosphate homeostasis and is a drug target for osteoporosis. We demonstrated that the purified PTH1R in nanodiscs can bind to PTH(1-34) and activate G protein. We also observed that Ca2+ is a weak agonist of PTH1R, and Ca2+ in millimolar concentration can switch PTH(1-34) from an inverse agonist to an agonist. Hence, our results show that nanodiscs are a viable vehicle for GPCR purification, enabling studies of GPCRs under precise experimental conditions without interference from other cellular or membrane components.

Cell membranes are crucial to many biological processes. Because of their complexity, however, lipid bilayers are often used as model systems. Lipid structures influence the physical properties of bilayers, but their interplay, especially in multiple-component lipid bilayers, has not been fully explored. Here, we used the Langmuir–Blodgett method to make mono- and bilayers of 1,2-dihexadecanoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), and 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phospho-l-serine (POPS) as well as their 1:1 binary mixtures. We studied the fluidity, stability, and rigidity of these structures using sum frequency generation (SFG) spectroscopy combined with analyses of surface pressure–area isotherms, compression modulus, and stability. Our results show that single-component bilayers, both saturated and unsaturated, may not be ideal membrane mimics because of their low fluidity and/or stability. However, the binary saturated and unsaturated DPPG/POPG and DPPG/POPS systems show not only high stability and fluidity but also high resistance to changes in surface pressure, especially in the range of 25–35 mN/m, the range typical of cell membranes. Because the ratio of saturated to unsaturated lipids is highly regulated in cells, our results underline the possibility of modulating biological properties using lipid compositions. Also, our use of flat optical windows as solid substrates in SFG experiments should make the SFG method more compatible with other techniques, enabling more comprehensive future surface characterizations of bilayers.

Self-assembly of molecules into chiral macromolecular and supramolecular structures at interfaces is important in various fields, such as biomedicine, polymer sciences, material sciences, and supramolecular chemistry. However, probing the kinetics at interfaces remains challenging because it requires a real-time method that has selectivity to both interface and chirality. Here, we introduce an in situ approach of using the C–H stretch as a vibrational probe detected by chiral sum frequency generation spectroscopy (cSFG). We showed that the C–H stretch cSFG signals of an amphiphilic peptide (LK7β) can reveal the kinetics of its self-assembly into chiral β-sheet structures at the air–water interface. The cSFG experiments in conjunction with measurements of surface pressure allow us to propose a mechanism of the self-assembly process, which involves an immediate adsorption of disordered structures followed by a lag phase before the self-assembly into chiral antiparallel β-sheet structures. Our method of using the C–H stretch signals implies a general application of cSFG to study the self-assembly of bioactive, simple organic, and polymeric molecules into chiral macromolecular and supramolecular structures at interfaces, which will be useful in tackling problems, such as protein aggregation, rational design of functional materials, and fabrication of molecular devices.

Emulsions are widely used in industrial and environmental remediation applications. The breaking and reformulation of emulsions, which occur during their use, lead to changes in their surface composition as well as their physical and chemical properties. Hence, a fundamental understanding of the transfer of surfactant molecules between emulsion particles is required for optimization of their applications. However, such an understanding remains elusive because of the lack of in situ and real-time surface-specific techniques. To address this, we designed and synthesized the surfactant probe molecules MG-butyl-1 (2) and MG-octyl-1 (3), which contain an n-butyl and an n-octyl chain, respectively, and a charged headgroup similar to that in malachite green (MG, 1). MG is known to be effective in generating second harmonic generation (SHG) signals when adsorbed onto surfaces of colloidal microparticles. Making use of the coherent nature of SHG, we monitored in real-time the transfer of 2 and 3 between oil-in-water emulsion particles with diameters of ∼220 nm. We found that 3 is transferred ∼600 times slower than 2, suggesting that an increase in the hydrophobic chain length decreases the transfer rate. Our results show that SHG combined with molecular design and synthesis of surfactant probe molecules can be used to measure the rate of surfactant transfer between emulsion particles. This method provides an experimental framework for examining the factors controlling the kinetics of surfactant transfer between emulsion particles, which cannot be readily investigated in situ and in real-time using conventional methods.

Molecular structure and function studies of vertebrate ultraviolet (UV) cone visual pigments are needed to understand the molecular evolution of these photoreceptors, which uniquely contain unprotonated Schiff base linkages between the 11-cis-retinal chromophore and the opsin proteins. In this study, the Siberian hamster ultraviolet cone pigment (SHUV) was expressed and purified in an n-dodecyl-β-d-maltoside suspension for optical characterization. Time-resolved absorbance measurements, over a spectral range from 300 to 700 nm, were taken for the purified pigment at time delays from 30 ns to 4.64 s after photoexcitation using 7 ns pulses of 355 nm light. The resulting data were fit globally to a sum of exponential functions after noise reduction using singular-value decomposition. Four exponentials best fit the data with lifetimes of 1.4 μs, 210 μs, 47 ms, and 1 s. The first photointermediate species characterized here is an equilibrated mixture similar to the one formed after rhodopsin’s Batho intermediate decays into equilibrium with its successor, BSI. The extremely large red shift of the SHUV Batho component relative to the pigment suggests that SHUV Batho has a protonated Schiff base and that the SHUV cone pigment itself has an unprotonated Schiff base. In contrast to SHUV Batho, the portion of the equilibrated mixture’s spectrum corresponding to SHUV BSI is well fit by a model spectrum with an unprotonated Schiff base. The spectra of the next two photointermediate species revealed that they both have unprotonated Schiff bases and suggest they are analogous to rhodopsin’s Lumi I and Lumi II species. After decay of SHUV Lumi II, the correspondence with rhodopsin photointermediates breaks down and the next photointermediate, presumably including the G protein-activating species, is a mixture of protonated and unprotonated Schiff base photointermediate species.

In situ and real-time characterization of protein secondary structures at interfaces is important in biological and bioengineering sciences, yet remains technically challenging. In this study, we used chiral sum frequency generation (SFG) spectroscopy to establish a set of vibrational optical markers for characterizing protein secondary structures at interfaces. We discovered that the N–H stretches along the peptide backbones of α-helices can be detected in chiral SFG spectra. We further observed that the chiral vibrational signatures of the N–H stretch together with the peptide amide I are unique to α-helix, β-sheet, and random coil at interfaces. Using these chiral vibrational signatures, we studied the aggregation of human islet amyloid polypeptide (hIAPP), which is implicated in type II diabetes. We observed in situ and in real time the misfolding of hIAPP from random coils to α-helices and then β-sheets upon interaction with a lipid–water interface. Our findings show that chiral SFG spectroscopy is a powerful tool to follow changes in protein conformations at interfaces and identify interfacial protein secondary structures that elude conventional techniques.

Kinetic analysis of conformational changes of proteins at interfaces is crucial for understanding many biological processes at membrane surfaces. In this study, we demonstrate that surface-selective sum frequency generation (SFG) spectroscopy can be used to investigate kinetics of conformational changes of proteins at interfaces. We focus on an intrinsically disordered protein, human islet amyloid polypeptide (hIAPP) that is known to misfold into the β-sheet structure upon interaction with membranes. Using the ssp polarization setting (s-polarized SFG, s-polarized visible, and p-polarized infrared), we observe changes in the amide I spectra of hIAPP at the air/water interface after addition of dipalmitoylphosphoglycerol (DPPG) that correspond to the lipid-induced changes in secondary structures. We also used the chiral-sensitive psp polarization setting to obtain amide I spectra and observed a gradual buildup of the chiral structures that display the vibrational characteristics of parallel β-sheets. We speculate that the second-order chiral-optical response at the antisymmetric stretch frequency of parallel β-sheet at 1622 cm−1 could be a highly characteristic optical property of the β-sheet aggregates not only for hIAPP, but possibly also for other amyloid proteins. Analyzing the achiral and chiral amide I spectra, we conclude that DPPG induces the misfolding of hIAPP from α-helical and random-coil structures to the parallel β-sheet structure at the air/water interface. We propose that SFG could complement existing techniques in obtaining kinetic and structural information for probing structures and functions of proteins at interfaces.

Although thermal stability of the G protein-coupled receptor rhodopsin is directly related to its extremely low dark noise level and has recently generated considerable interest, the chemistry behind the thermal decay process of rhodopsin has remained unclear. Using UV−vis spectroscopy and HPLC analysis, we have demonstrated that the thermal decay of rhodopsin involves both hydrolysis of the protonated Schiff base and thermal isomerization of 11-cis to all-trans retinal. Examining the unfolding of rhodopsin by circular dichroism spectroscopy and measuring the rate of thermal isomerization of 11-cis retinal in solution, we conclude that the observed thermal isomerization of 11-cis to all-trans retinal happens when 11-cis retinal is in the binding pocket of rhodopsin. Furthermore, we demonstrate that solvent deuterium isotope effects are involved in the thermal decay process by decreasing the rates of thermal isomerization and hydrolysis, suggesting that the rate-determining step of these processes involves breaking hydrogen bonds. These results provide insight into understanding the critical role of an extensive hydrogen-bonding network on stabilizing the inactive state of rhodopsin and contribute to our current understanding of the low dark noise level of rhodopsin, which enables this specialized protein to function as an extremely sensitive biological light detector. Because similar hydrogen-bonding networks have also been suggested by structural analysis of two other GPCRs, β1 and β2 adrenergic receptors, our results could reveal a general role of hydrogen bonds in facilitating GPCR function.

Many amyloid proteins misfold into β-sheet aggregates upon interacting with biomembranes at the onset of diseases, such as Parkinson's disease and type II diabetes. The molecular mechanisms triggering aggregation depend on the orientation of β-sheets at the cell membranes. However, understanding how β-sheets adsorb onto lipid/aqueous interfaces is challenging. Here, we combine chiral sum frequency generation (SFG) spectroscopy and ab initio quantum chemistry calculations based on a divide-and-conquer strategy to characterize the orientation of human islet amyloid polypeptides (hIAPPs) at lipid/aqueous interfaces. We show that the aggregates bind with β-strands oriented at 48° relative to the interface. This orientation reflects the amphiphilic properties of hIAPP β-sheet aggregates and suggests the potential disruptive effect on membrane integrity.Download high-res image (228KB)Download full-size imageHighlights► We obtain a high-resolution chiral SFG spectrum of hIAPP aggregates. ► In theory, we relate the ratio of two SFG peaks to the aggregates' orientation. ► We find four possible orientations for the hIAPP by the theory. ► We simulate the four possible SFG spectra by a new “divide-and-conquer” approach. ► By comparison, we find that the hIAPP orients at an angle of 48° at interfaces.

We characterized BslA, a bacterial biofilm protein, at the air/water interface using vibrational sum frequency generation spectroscopy and observed one of the sharpest amide I bands ever reported. Combining methods of surface pressure measurements, thin film X-ray reflectivity, and atomic force microscopy, we showed extremely ordered BslA at the interface.

Two peptide–linker–lipid constructs were designed and prepared which target the parathyroid hormone 1 receptor, a family B G protein-coupled receptor. Both show increased agonist activity in a cell-based assay. The lipid moiety enables the formation of micelle-like nanostructures, which is shown to hinder proteolytic digestion and is expected to reduce renal clearance.

Correction for ‘A narrow amide I vibrational band observed by sum frequency generation spectroscopy reveals highly ordered structures of a biofilm protein at the air/water interface’ by Zhuguang Wang et al., Chem. Commun., 2016, 52, 2956–2959.