Ion channels play crucial roles in transport and regulatory functions of living cells. Understanding the gating mechanisms of these channels is important to understanding and treating diseases that have been linked to ion channels. One potential model peptide for studying the mechanism of ion channel gating is alamethicin, which adopts a split alpha/310 helix structure and responds to changes in electric potential. In this study, sum frequency generation vibrational spectroscopy (SFG-VS), supplemented by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), has been applied to characterize interactions between alamethicin (a model for larger channel proteins) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayers in the presence of an electric potential across the membrane. The membrane potential difference was controlled by changing the pH of the solution in contact with the bilayer, and measured using fluorescence spectroscopy. The orientation angle of alamethicin in POPC lipid bilayers was then determined at different pH values using polarized SFG amide I spectra. Assuming that all molecules adopt the same orientation (a δ distribution), at pH=6.7 the α-helix at the N-terminus and the 310 helix at the C-terminus tilt at about 72° (θ1) and 50° (θ2) versus the surface normal, respectively. When pH increases to 11.9, θ1 and θ2 decrease to 56.5° and 45°, respectively. The δ distribution assumption was verified using a combination of SFG and ATR-FTIR measurements, which showed a quite narrow distribution in the angle of θ1 for both pH conditions. This indicates that all alamethicin molecules at the surface adopt a nearly identical orientation in POPC lipid bilayers. The localized pH change in proximity to the bilayer modulates the membrane potential and thus induces a decrease in both the tilt and bend angles of the two helices in alamethicin. This is the first reported application of SFG to the study of model ion channel gating mechanisms in model cell membranes.

Applications of graphene have extended into areas of nanobio-technology such as nanobio-medicine, nanobio-sensing, as well as nanoelectronics with biomolecules. These applications involve interactions between proteins, peptides, DNA, RNA etc. and graphene, therefore understanding such molecular interactions is essential. For example, many applications based on using graphene and peptides require peptides to interact with (e.g., noncovalently bind to) graphene at one end, while simultaneously exposing the other end to the surrounding medium (e.g., to detect analytes in solution). To control and characterize peptide behavior on a graphene surface in solution is difficult. Here we successfully probed the molecular interactions between two peptides (cecropin P1 and MSI-78(C1)) and graphene in situ and in real-time using sum frequency generation (SFG) vibrational spectroscopy and molecular dynamics (MD) simulation. We demonstrated that the distribution of various planar (including aromatic (Phe, Trp, Tyr, and His)/amide (Asn and Gln)/Guanidine (Arg)) side-chains and charged hydrophilic (such as Lys) side-chains in a peptide sequence determines the orientation of the peptide adsorbed on a graphene surface. It was found that peptide interactions with graphene depend on the competition between both planar and hydrophilic residues in the peptide. Our results indicated that part of cecropin P1 stands up on graphene due to an unbalanced distribution of planar and hydrophilic residues, whereas MSI-78(C1) lies down on graphene due to an even distribution of Phe residues and hydrophilic residues. With such knowledge, we could rationally design peptides with desired residues to manipulate peptide-graphene interactions, which allows peptides to adopt optimized structure and exhibit excellent activity for nanobio-technological applications. This research again demonstrates the power to combine SFG vibrational spectroscopy and MD simulation in studying interfacial biological molecules.

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 liqu...

A wide variety of charge carrier dynamics, such as transport, separation, and extraction, occur at the interfaces of planar heterojunction solar cells. Such factors can affect the overall device performance. Therefore, understanding the buried interfacial molecular structure in various devices and the correlation between interfacial structure and function has become increasingly important. Current characterization techniques for thin films such as X-ray diffraction, cross section scanning electronmicroscopy, and UV-visible absorption spectroscopy are unable to provide the needed molecular structural information at buried interfaces. In this study, by controlling the structure of the hole transport layer (HTL) in a perovskite solar cell and applying a surface/interface-sensitive nonlinear vibrational spectroscopic technique (sum frequency generation vibrational spectroscopy (SFG)), we successfully probed the molecular structure at the buried interface and correlated its structural characteristics to solar cell performance. Here, an edge-on (normal to the interface) polythiophene (PT) interfacial molecular orientation at the buried perovskite (photoactive layer)/PT (HTL) interface showed more than two times the power conversion efficiency (PCE) of a lying down (tangential) PT interfacial orientation. The difference in interfacial molecular structure was achieved by altering the alkyl side chain length of the PT derivatives, where PT with a shorter alkyl side chain showed an edge-on interfacial orientation with a higher PCE than that of PT with a longer alkyl side chain. With similar band gap alignment and bulk structure within the PT layer, it is believed that the interfacial molecular structural variation (i.e., the orientation difference) of the various PT derivatives is the underlying cause of the difference in perovskite solar cell PCE.

Antimicrobial peptides, because of their unique structural and chemical properties, hold a promising future for the development of a new class of bacterial-resistant antibiotics, effective antimicrobial coatings, and high performance biosensors. To understand the structure/function relationship of surface-bound peptides as they relate to such applications, sum frequency generation (SFG) vibrational spectroscopy, coarse grained molecular dynamics simulations, and antimicrobial activity tests were used to characterize both surface peptide structural information and peptide activity. Results from MSI-78, an antimicrobial peptide, chemically immobilized via the N-(nMSI-78) or C-terminus (MSI-78n), demonstrate that the attachment site influences the structure and behavior of surface-bound peptides. Although both immobilized peptides adopt an α-helical structure in aqueous buffer, nMSI-78 stands up and MSI-78n lies down on the surface, as indicated by both SFG and MD simulations. Antimicrobial activity tests indicated that peptides that stand up interact with bacterial cells much quicker than peptides that lie down. We believe that this study provides fundamental insights into how to rationally engineer peptides and substrate surfaces to produce optimized abiotic/biotic interfaces for antimicrobial applications and beyond.