Engineering and Characterization of Peptides and Proteins at

May 18, 2016 - Biography. Joshua Jasensky is a postdoctoral researcher at the University of Michigan who is pursing the development of nonlinear optic...
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Engineering and Characterization of Peptides and Proteins at Surfaces and Interfaces: A Case Study in Surface-Sensitive Vibrational Spectroscopy Bei Ding, Joshua Jasensky, Yaoxin Li, and Zhan Chen* Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109, United States CONSPECTUS: 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.

1. INTRODUCTION Interactions of biomolecules at interfaces are important in many processes. For example, biocompatibility is determined by favorable interactions between biomolecules and biomaterial surfaces, more than half of current drug therapies target membrane proteins at cell boundaries, ship coatings are often fouled by the adhesive proteins secreted by marine organisms, and biosensor activity is often determined by the structure and conformation of surface immobilized biomolecules. Therefore, understanding and tailoring the interactions of biomolecules at © XXXX American Chemical Society

such interfaces is broadly important in development, application, and the like. The above examples are only a few of many interactions that are controlled by the molecular behavior of biomolecules at interfaces. However, it is technically challenging to study biomolecules at solid/liquid interfaces in situ at the molecular level. With the advent of surface-sensitive spectroscopies, Received: February 19, 2016

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Accounts of Chemical Research research into this field has begun to address many of these fundamental questions. Sum frequency generation (SFG) vibrational spectroscopy, a second-order nonlinear optical method, has recently been developed into a powerful tool to elucidate molecular structures at surfaces and interfaces,1−4 including interfacial peptides and proteins.5−10 Unlike other optical surface methods that use an evanescent wave, the submonolayer interface sensitivity of SFG originates from its selection rule: only a medium with no inversion symmetry can generate a signal (under the electric-dipole approximation). Most bulk materials possess inversion symmetry, while surfaces and interfaces do not. Therefore, SFG can specifically probe surfaces and interfaces. SFG spectroscopy is often performed using a frequency fixed visible laser and a frequency tunable mid-IR laser, where both beams are overlapped on a surface or interface, and the generated signal at the sum frequency is collected as a function of the input IR frequency. This provides a vibrational spectrum from which structural information on the surface or interface (e.g., coverage, orientation, and orientation distribution of various functional groups) can be deduced. Our research group has been making continuous and systematic efforts in the design, engineering, and characterization of peptides and proteins at interfaces using SFG along with other techniques such as attenuated total reflectance-FTIR (ATR-FTIR), isotope labeling, surface engineering, liquid crystal sensing, and molecular dynamics (MD) simulation.11−15 With these developments, we have successfully elucidated backbone structures globally and site-specifically for peptides and proteins at interfaces.16,17,12,13 This Account presents an executive summary and systematic introduction of our research results on interfacial peptides and proteins as well as their broader impact.

systematic methods to use SFG amide I signals to characterize such structural information.16,20,21 2.1. Orientation Analysis on Peptides and Proteins with Regular Secondary Structures

In general, the SFG susceptibility component ratios defined in the lab coordinate system, χzzz/χyyz and χzzz/χyzy, can be determined using SFG signals collected with different laser polarization combinations.22 For small chemical groups such as the methyl group, the calculated SFG hyperpolarizability defined in the molecular reference frame, and the above experimentally determined ratios are both needed to determine the orientation of that interfacial chemical group.22 We extended such SFG polarization analysis to determine orientations of interfacial secondary structures. With this developed methodology and the calculated hyperpolarizability using a bond additivity model, the tilt angle of an α-helical or 310-helical structure can be deduced from SFG polarization measurements.21 Similarly, the tilt and twist angles of an antiparallel β-sheet can also be determined.20 With these developed methods, we successfully determined the interfacial orientations of α-helical MSI-78,18 alamethicin (with an αhelical and a 310-helical segment),19 and β-sheet tachyplesin I,20 respectively (Figure 1). For more complicated proteins such as G proteins,23 two independent angles, tilt and twist, are needed to accurately define their orientation.24 Polarized SFG and ATR-FTIR can be used to study orientation of interfacial peptides and proteins independently, providing different orientation parameters.17 By combining complementary measurements from SFG and ATRFTIR spectroscopies, we determined the orientation of G proteins in lipid membranes more precisely with more independently measured parameters than using SFG or ATRFTIR alone.11 In order to easily deduce and visualize this orientation information, we developed computer software that calculates the most likely orientation of interfacial proteins determined by SFG and ATR-FTIR measurements in tandem with the protein structure from a given protein PDB file (Figure 2) and uses a heat map to visualize this information.24 Such measurements have also been successfully applied to study complex orientations of peptides such as melittin adopting dual orientations in a model cell membrane17 and β-sheet tachyplesin I, which needs the characterization of both tilt and twist angles.20

2. CHARACTERIZATION AND METHODOLOGY DEVELOPMENT OF PROTEIN SECONDARY STRUCTURE As a vibrational spectroscopic technique, SFG has the capability of identifying unique secondary structural motifs important in peptides and proteins. Figure 1 highlights several characteristic spectra collected from peptides with different secondary structures including α-helix,18 310-helix,19 and β-sheet.20 Identification of each secondary structure independently allows for the characterization of structure (e.g., conformation and orientation) of interfacial peptides and proteins. We developed

2.2. Site-Specific Orientation Analysis

Inspired by our previous deuterated protein study25 and the orientation analysis of site-specifically labeled side chains by Castner et al.,7 we investigated isotope labeling of a peptide backbone using SFG, in collaboration with the Zanni group.12,13 By 13C16O isotope labeling of a single peptide unit in ovispirin-1, we successfully detected SFG signal from an isotope labeled unit at a polystyrene/solution interface13 and on a solid-supported lipid bilayer,12 providing more site-specific structural details of interfacial peptides.12,13 For example, we successfully probed the polarization response of a singleisotope-labeled residue backbone (Figure 3).12,13 We have also individually labeled ten residues of ovispirin-1 with 13C16O and investigated the site-specific orientation of ovispirin-1 when associated with a lipid bilayer. We showed that the line width of the isotope-labeled amide I peak is indicative of the microenvironment of a specific residue, namely, whether it is buried in the hydrophobic core or exposed to the aqueous solution.12

Figure 1. SFG spectra collected from membrane associated peptides: (a) α-helical MSI-78; (b) alamethicin with α-helical and 310-helical segments; (c) β-sheet tachyplesin I. Adapted with permission from refs18−20. Copyright 2010 and 2011 American Chemical Society. B

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Figure 2. (a) SFG spectra and (b) ATR-FTIR spectra collected from Gαiβ1γ2 associated with a 9:1 POPC/POPG bilayer. Heat maps showing the most likely orientations of Gαiβ1γ2 determined by (c) SFG and (d) ATR-FTIR. (e) The most likely orientation determined by combined SFG and ATR-FTIR data and (f) a schematic showing the most likely Gαiβ1γ2 orientation (orientation resulting from area indicated by an arrowhead in panel e). Adapted with permission from ref 11. Copyright 2013 American Chemical Society.

which adopts different bent and disrupted structures in different model cell membranes.26 Collaborating with the Zanni group, we have also applied a more general “Hamiltonian approach” where one exciton Hamiltonian is constructed with the amide I vibrational modes of each residue as the local oscillators to study interfacial peptides.13 The couplings between local modes were calculated according to the transition dipole coupling model. This approach does not require “ideal” secondary structure and is therefore more general and can be used to study any disrupted secondary structure with a PDB file. This Hamiltonian approach is also preferred when an isotope label was incorporated into different sites of an α-helix (section 2.2). In this case, the symmetry of the local modes was likely disrupted so that for an isotope labeled α-helix, the spectrum will change from two dominant exciton modes (namely, A mode and E1 mode) to more complicated line shapes,13 which can be fully addressed by the Hamiltonian approach.

Figure 3. (a) Schematic of an α-helix with one 13C label. (b) SFG spectrum collected from ovisprin-1 (with I7 labeled) associated with a lipid bilayer. The small peak originates from the isotope labeled segment (inset). Adapted with permission from ref 12. Copyright 2015 American Chemical Society.

Isotope labeling breaks local symmetry and has the potential to generate and enhance SFG signal from a specific site. The signal is spectrally distinct and easily resolved from peptide or protein side chains.12,13 The combination of SFG and isotope labeling has great potential in elucidating detailed structures of interfacial peptides and proteins and the effect of site-specific microenvironments.

3. MODEL CELL MEMBRANE ASSOCIATED PEPTIDES AND PROTEINS Interactions between cell membranes and biomolecules play crucial roles in many biological processes and medical applications. We have studied a variety of membrane-related peptides and proteins such as antimicrobial peptides (AMPs),18,19,21,26 cell penetrating peptides,27 and membrane proteins.11,24,28,29 Using methods developed to determine peptide or protein orientation via SFG, we elucidated molecular interaction mechanisms between various peptides and proteins and lipid bilayers, serving as model cell membranes.

2.3. Orientation Analysis on Peptides and Proteins with Disrupted Secondary Structure

Many proteins and peptides do not contain only ideal secondary structures. In order to address this issue, we successfully developed an SFG orientation analysis method for bent (a continuous helical structure that is bent) and disrupted (a structure with two helical segments separated by a short nonhelical structure) α-helices using a bond additivity model.26 The phase of the E1 vibrational mode should be continuous in a bent helix, but such phases for the two segments in a disrupted structure are unrelated.26 We successfully applied such a method to study peptide LL-37,

3.1. Effect of Lipid Charge

Bacterial cell membranes are more negatively charged than mammalian cell membranes. It is believed that AMPs can selectively kill bacteria due to their preferential interaction with negatively charged lipids. In order to understand antimicrobial activity and selectivity, we investigated the effect of lipid charge. C

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in fluid-phase but not gel-phase lipid bilayers, which agrees well with their amphipathic properties. This is the first time the detailed secondary structures of a cell penetrating peptide interacting with different model cell membranes have been elucidated. Such information is important for developing these peptides into drug delivery vehicles.

Our combined SFG and ATR-FTIR results indicated that magainin 2, a widely studied AMP, inserts into 1-palmitoyl-2oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) bilayers (a model bacterial cell membrane) and adopts a transmembrane orientation of ∼20° from the bilayer normal at a peptide concentration of 800 nM. For a 1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine (POPC) bilayer (a model mammalian cell membrane), SFG results indicated that magainin 2 molecules do not insert into the bilayer and are nearly parallel on the bilayer surface even up to 2.0 μM.30 SFG data indicated that MSI-78, a synthetic analogue of magainin 2, has a strong interaction with negatively charged POPG and 1,2-dipalmitoylsn-glycero-3-phospho-(1′-rac-glycerol) (DPPG) bilayers (see details below), but there is no interaction with zwitterionic bilayers 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or POPC at the same peptide concentration.18 LL-37, the only cathelicidin member in humans, was shown to have the same orientation (parallel to the membrane surface) while associated with a POPC lipid bilayer at different peptide concentrations (0.46 or 1.60 μM) measured by SFG. On the other hand, when associated with a POPG or POPC/POPG mixed lipid bilayer, LL-37 exhibits a reorientation upon an increase in peptide concentration, suggesting peptide aggregation.26 The above case studies suggest a common mechanism that AMPs generally interact with negatively charged lipid bilayers more strongly than with zwitterionic lipid bilayers.

3.3. Membrane Interactions of G Proteins

Membrane protein function is influenced by interfacial orientations which can be obtained via in situ measurement. Using SFG and ATR-FTIR, we showed that the geranylgeranyl group facilitates membrane binding of the Gβ1γ2 subunit and Gβ1γ2 does not change its orientation significantly when binding Gα i, suggesting that the geranylgeranyl group determines the orientation of Gαiβ1γ2.11 We also showed that lipid charge affects membrane protein orientation. For example, Gβ1γ2 adopts varied orientations when associated with a POPC, POPG, or mixed POPC−POPG lipid bilayer,28 while the lipid charge also influences the membrane orientation of G protein receptor kinase 5 (GRK5).29 By comparing the similar membrane orientation of GRK5 and its C-terminally truncated mutant GRK51−531, we found that likely the N-terminus plays the major role in GRK5−membrane interaction.29 In summary, the methodology we developed in section 2, as well as a multiple-technique approach, provides us unique insights to understand structure (e.g., orientation or secondary structure)−function relationships of membrane-associated peptides and proteins. By investigating a series of AMPs, we found a common mechanism that AMPs interact more strongly with negatively charged model membranes than zwitterionic ones in terms of adsorption amount and interfacial tilting angle. Certain anchor segments such as the geranylgeranyl group determine the assemblies and functions of G protein machinery. Lipid fluidity modulates peptide secondary structures and orientation distributions, which unveils the difference between interactions of peptides with “healthy” cell membranes and those with “injured” ones.

3.2. Effect of Peptide Concentration and Lipid Fluidity

Different from NMR and other biological methods, SFG is sensitive enough to probe AMP−membrane interactions at physiologically relevant peptide concentrations. At 400 nM, MSI-78 lies on the surface of a gel-phase DPPG bilayer with ∼70° deviation from the membrane surface normal.18 When that concentration increases to 600 nM, MSI-78 inserts into the membrane with a ∼25° tilt. For even higher peptide concentrations, multiple orientations were observed, possibly due to the formation of a toroidal pore. Interestingly, for a fluid-phase POPG bilayer, MSI-78 exhibits multiple orientations at the peptide concentration of 600 nM and no perpendicular orientation was observed, showing that lipid fluidity influences the peptide−bilayer interaction.18 Another AMP, alamethicin, which interacts with cell membranes through a barrel-stave mode, can form voltagegated ion channels in cell membranes.19 SFG has been applied to study alamethicin19,31−33 and SFG results showed that alamethicin lies down on gel-phase bilayers, but adopts a mixed α-helical/310-helical structure in fluid-phase bilayers. The αhelical and the 310-helical components tilt at ∼63° and ∼43°, respectively, versus the surface normal when interacting with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers.19 Different from AMPs, cell penetrating peptides are known to be able to translocate cell membranes nondisruptively. SFG results showed that on gel-phase lipid bilayers, cell penetrating peptide Pep-1 molecules are loosely adsorbed with random or β-sheet structures.27 On fluid-phase lipid bilayers, signals from α-helical and β-sheet structures were observed simultaneously. At the low and intermediate peptide concentrations, SFG results showed that Pep-1 helical component is nearly perpendicular to the lipid bilayer surface. At a high concentration of 7.0 μM, combined ATR-FTIR and SFG results show a random orientation of the helical component of Pep-1. Similar to alamethicin, Pep-1 selectively forms α-helices

4. PEPTIDES AND PROTEINS ON ABIOTIC SURFACES Surface immobilized peptides and proteins find wide applications in antimicrobial coatings, bacteria capturing, biosensing, biofuel production, and energy harvesting.34 We have successfully examined the secondary structures and orientations of surface immobilized peptides and proteins to achieve understanding on their structure−function relationships. 4.1. Physical Adsorption vs Chemical Immobilization

Physical adsorption and chemical immobilization are two major ways of attaching peptides and proteins onto a surface.14,35 Using SFG, we have shown that physically adsorbed peptides have a complex orientation distribution, while chemically immobilized peptides adopt a preferred orientation. For example, cecropin P1 (CP1) physically adsorbed on a polystyrene surface adopts multiple orientations, but Cterminal cysteine modified CP1 (CP1c) chemically immobilized on a polystyrene−maleimide surface through the maleimide−cysteine coupling tilts at 35° with a narrow distribution.35 Differences were also observed from chemically immobilized and physically adsorbed proteins. Chemical immobilization of 6-phospho-β-galactosidase on a maleimide terminated self-assembled monolayer (SAM) surface was achieved by reaction with a unique cysteine engineered onto each enzyme molecule. Combined SFG and ATR-FTIR D

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Figure 4. (a) SFG spectra from surface immobilized CP1c. (b) The resulting MD simulation results confirm that this peptide stands up. (c) SFG spectrum from surface immobilized cCP1. (d) MD simulations confirm SFG data for cCP1. Adapted with permission from refs 36 and 37. Copyright 2013 American Chemical Society.

Figure 5. Heat maps (top) and orientations (bottom) of surface immobilized NfsB via different surface immobilization sites. (a) V424C; (b) H360C. (bottom) Schematics showing the most likely NfsB orientation. Adapted with permission from Ref 40. Copyright 2014, American Chemical Society.

terminus. For MSI-78, SFG results and MD simulation showed that similar to CP1, the C-terminal cysteine modified MSI-78 stands up and N-terminal modified MSI-78 lies down on a maleimide SAM surface.38 These studies showed clearly that the same peptide immobilized with different termini can have varied orientations on a surface. We also immobilized MSI-78 terminated with an azide group onto an alkyne terminated SAM surface through click chemistry.39 Surprisingly, the SFG results are opposite to those for maleimide−cysteine coupling. MSI-78 stands up via N-terminal immobilization and lies down via C-terminal immobilization on an alkyne SAM surface.39 MD simulations supported this SFG observation. Therefore, surface hydrophobicity is thought to affect peptide orientation, because alkyne terminated SAM surfaces are more hydrophobic than maleimide terminated SAM surfaces. Similar phenomena were observed on enzyme immobilization, using nitro-reductase (NfsB) as an example.40 NfsB is a dimeric protein and therefore has two active sites. In collaboration with the Marsh group, two mutants, V424C (with the cysteine close to one active site) and H360C (with the cysteine farther away from the active sites), were engineered.40 A combined SFG/ATR-FTIR study showed that both mutants adopt orientations as designed: V424C

measurements showed that the immobilized enzyme adopts an orientation with the cysteine next to the SAM surface and an outward-facing orientation of the active site (the cysteine is on the opposite side of the enzyme to the active site). This agrees with enzymatic activity testing, which indicated that chemically immobilized enzymes have comparable activities to free enzymes in solution, but physically adsorbed enzymes appeared to be partially denatured and exhibited significantly reduced activity.14 4.2. Effects of Surface Immobilization Sites and Immobilization Strategy

It is interesting to study whether the same peptide immobilized via different termini exhibits the same orientation and whether the orientation of surface immobilized enzymes can be controlled by choosing an immobilization site on the enzyme. SFG results showed that CP1c adopts an α-helical structure with a uniform orientation when tethered to a maleimide SAM surface via its C-terminus. In contrast, immobilization via the N-terminal cysteine of CP1 (cCP1) results in a helical structure that lies down (Figure 4).36 Subsequent MD simulation results from the Brooks group confirmed SFG observations, providing an atomistic level explanation.37 Because the hydrophobic Cterminus could not favorably interact with water, the whole peptide lies down on the surface if immobilized via its NE

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Figure 6. (a) SFG ppp spectrum of surface immobilized nMSI-78 in phosphate buffer; (b) time-dependent SFG signals (at 1650 cm−1) detected from immobilized peptide after removal from buffer and exposure to air; (c) SFG ppp spectra of surface immobilized nMSI-78 with spin-coated sucrose in air detected as a function of time. (d) Schematics showing peptide orientation in different environments. Adapted from ref 44 with permission from the Royal Society of Chemistry.

interfacial peptides and proteins in air with the help of hydromimetic functionalities.44 Using SFG and CD, we have observed that by the application of a thin sugar coating, surface immobilized MSI-78 retains its “native” secondary structure and tilted orientation in air (Figure 6).44 This research provides insight for future design and development of biosensors and biochips that can fully function in air using surface immobilized peptides and proteins with “native” structure. In summary, by combining SFG with surface engineering and MD simulation, we have unveiled the insights from various immobilized peptide or protein systems. We have found that chemical immobilization is more likely to introduce a narrower orientation distribution of peptides and proteins than physical adsorption on surfaces and thus is more likely to retain peptide or protein activities that are seen in bulk solution. By changing surface immobilization sites and strategies, we have found that when the same peptide is immobilized with different termini, the structure and orientation are determined by the interplay between surface hydrophobicity and peptide or protein hydrophobicity. When a more hydrophilic surface is used for peptide immobilization, peptides have more flexibility and adopt a broader orientation distribution. By investigating the substrate effect, we have found that besides surface hydrophobicity, surface ordering also influences the immobilized peptide or protein orientation.

adopts an orientation with only one active site available (the other one is in contact with the surface) and H360C exposes both active sites (Figure 5). This agrees with the enzymatic activity testing data that H360C has higher activity.40 This experiment indicates that the orientation of a surface immobilized enzyme can be controlled by choosing an optimal immobilization site via protein engineering. Recently we also showed that surface immobilized 6-phospho-β-galactosidase molecules via two neighboring cysteine sites, one on a coil and one on a helix, have a similar orientation at room temperature. However, enzymes immobilized with the cysteine on a helix may lead to better thermal stability,41 indicating that immobilization of an enzyme through a site in a rigid structural segment leads to better stability. 4.3. Effect of Substrate Surfaces for Peptide and Protein Immobilization

We have studied the structure of CP1c immobilized on a maleimide terminated SAM and a maleimide/hydroxyl terminated mixed SAM.42 It was found that the CP1c molecules on the two surfaces both possess a dominant helical structure. However, CP1c has a single orientation on maleimide SAM but exhibits multiple orientations on the mixed SAM. The mixed SAM surface is more hydrophilic, which reduces the interaction between the immobilized CP1c molecules and the surface, leading to more flexible CP1c molecules with a multiple-orientation distribution. We also studied orientations of NfsB on a maleimide SAM and a polymer surface with dibromomaleimide functionality.43 Interestingly, NfsB H360C exhibited similar orientational behavior on the SAM and polymer surfaces, but the NfsB V424C showed drastically different orientations on the two surfaces.43

5. PERSPECTIVE AND CONCLUSION The capability of SFG to elucidate interfacial peptide and protein structure globally and site-specifically has been fully demonstrated. It is necessary to state that SFG has its limitations and many problems related to peptide or protein interfacial structure remain unsolved. First, for current SFG studies, we need to assume that the protein at the interface under study adopts its native crystal structure. This assumption can be validated using MD simulations but is a limitation when protein confirmation changes at the interface. We have developed an isotope labeling technique, with which we can

4.4. Surface Immobilized Peptides and Proteins in Air

In the application of biosensors and biofuel cells, it is very important to maintain the function of biomolecules under harsh conditions, for example, in the absence of bulk water. We have investigated methods of structure and orientation retention of F

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Figure 7. SFG spectra of (a) CP1, (b) lactoferricin B, (c) alamethicin, and (d) cecropin A−melittin hybrid peptide in contact with a lipid monolayer on liquid crystal. Insets: images taken of each peptide at a liquid crystal interface. Adapted from ref 15 with permission from the Royal Society of Chemistry.

research in combined use of SFG with other techniques and methods such as ATR-FTIR, optical and fluorescence microscopies,45 multiple reflection geometry,46 isotope labeling, and beyond will lead to an in-depth understanding on the structure−function relationships, local structures, and heterogeneous samples of interfacial peptides and proteins. Live cells can be used in future SFG studies, in order to understand AMP−bacteria interactions. In addition, SFG can be applied to study membrane protein behavior on live cell membranes by overexpressing certain membrane proteins. Peptide aptamer has been widely used in many biological and therapeutic applications; their interfacial structure and behavior can certainly be probed using SFG. Development of instrumentation is expected in the future and will make SFG equipment more user-friendly with the fast advancement of ultrafast lasers, nonlinear optical devices, detection systems, and data collection software. The development of SFG vibrational probes may shed light on more dynamic information on biological systems. We hope that scientists and engineers in many different disciplines will benefit from the research progress reported in this Account on SFG studies of interfacial peptides and proteins and apply this powerful technique to study such biomolecules in many different applications.

obtain site-specific information without the knowledge of the global structure. In the future, the development of more SFG vibrational probes may shed light on peptide or protein interfacial structure and dynamics. Second, amide I vibrational modes are hard to characterize due to their delocalized nature. We have used a bond additivity model to address this delocalization and have proven that SFG results are reliable for relatively regular structures; such SFG results are well correlated to data obtained from previous FTIR, Raman, and NMR studies. More recently we have developed models to interpret SFG data detected from peptides with a kink and a Hamiltionian approach to more accurately calculate mode coupling to understand SFG spectra. In the future, by selectively isotope labeling model peptide backbone units in different positions, it will be possible to benchmark the nature of the amide mode coupling of various secondary structures. Third, peptides and proteins with complicated structures need more measured parameters for orientation analysis, for example, to determine twist angle, tilt angle, and orientation distribution width. One approach we used is to obtain more measured data from other techniques such as ATR-FTIR. In the future, we should measure more data using more polarization combinations of SFG beams (e.g., to collect chiral SFG spectra). Future combination with other higher order nonlinear optical techniques such as CARS will provide further additional measurements to get more structural information on peptides and proteins at interfaces. It is worth mentioning that other analytical tools may have advantages in membrane protein studies. For example, NMR and X-ray crystallography may be applied to deduce more detailed membrane protein structure despite the lower sensitivity, the need for more sample, and more complex sample preparation procedures. However, structure determination by NMR or X-ray crystallography is difficult at the solid/liquid interface, while SFG does not have this constraint. Recently there are a few new directions our group is pursuing. We observed using SFG that peptides can change orientation upon membrane potential change,33 which lays a foundation for future SFG studies on the gating mechanisms of ion-channel proteins. In collaboration with the Abbott group, we showed that liquid crystals respond to peptides with different secondary structures in different ways (Figure 7),15 demonstrating the feasibility to apply SFG to examine molecular interactions between liquid crystals and biomolecules. We believe that many such new research directions involving SFG will be developed, ranging from antimicrobial coatings, enzyme biosensors and biofuel cells, liquid crystals for biological molecular recognition, and ion channel gating mechanisms to membrane protein−drug interactions. Further

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AUTHOR INFORMATION

Corresponding Author

*Zhan Chen. E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biographies Bei Ding received her B.S. degree in Chemistry from Peking University and her Ph.D. degree in Chemistry along with a M.S.E. degree in Electrical Engineering (Optics/Photonics) from University of Michigan. Currently she is a postdoctoral researcher at the University of Pennsylvania. Joshua Jasensky is a postdoctoral researcher at the University of Michigan who is pursing the development of nonlinear optical methods for noninvasive, label-free analytics. He received his B.S. in Physics from Miami University and Ph.D. in Biophysics from the University of Michigan. Yaoxin Li is a graduate student in the Department of Chemistry at the University of Michigan. She received her B.S. degree in Chemistry from Xiamen University. Her research focuses on nonlinear spectroscopic studies of peptide and protein immobilization on abiotic surfaces. G

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Accounts of Chemical Research

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Zhan Chen is a professor of chemistry at the University of Michigan, specializing in molecular level studies on surfaces and interfaces using nonlinear optical spectroscopy. Dr. Chen received his B.S., M.S., and Ph.D. degrees from Peking University, Chinese Academy of Sciences, and UC-Berkeley, respectively. He was a postdoctoral fellow at the Lawrence Berkeley National Laboratory.

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ACKNOWLEDGMENTS This research is supported by Defense Threat Reduction Agency (Grant HDTRA1-11-1-0019), Army Research Office (Grant W911NF-11-1-0251), and National Science Foundation (Grant CHE-1505385).

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DOI: 10.1021/acs.accounts.6b00091 Acc. Chem. Res. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.accounts.6b00091 Acc. Chem. Res. XXXX, XXX, XXX−XXX