Andrew P. Boughton

Observing a model ion channel gating action in model cell membranes in real time in situ: membrane potential change induced alamethicin orientation change.

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 α/3(10)-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 was 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 3(10)-helix at the C-terminus tilt at about 72° (θ(1)) and 50° (θ(2)) versus the surface normal, respectively. When pH increases to 11.9, θ(1) and θ(2) 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 θ(1) 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 the 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.

Heterotrimeric G protein β1γ2 subunits change orientation upon complex formation with G protein-coupled receptor kinase 2 (GRK2) on a model membrane

Few experimental techniques can assess the orientation of peripheral membrane proteins in their native environment. Sum Frequency Generation (SFG) vibrational spectroscopy was applied to study the formation of the complex between G protein-coupled receptor (GPCR) kinase 2 (GRK2) and heterotrimeric G protein β1γ2 subunits (Gβγ) at a lipid bilayer, without any exogenous labels. The most likely membrane orientation of the GRK2-Gβγ complex differs from that predicted from the known protein crystal structure, and positions the predicted receptor docking site of GRK2 such that it would more optimally interact with GPCRs. Gβγ also appears to change its orientation after binding to GRK2. The developed methodology is widely applicable for the study of other membrane proteins in situ.

Orientation difference of chemically immobilized and physically adsorbed biological molecules on polymers detected at the solid/liquid interfaces in situ

A surface sensitive second order nonlinear optical technique, sum frequency generation vibrational spectroscopy, was applied to study peptide orientation on polymer surfaces, supplemented by a linear vibrational spectroscopy, attenuated total reflectance Fourier transform infrared spectroscopy. Using the antimicrobial peptide Cecropin P1 as a model system, we have quantitatively demonstrated that chemically immobilized peptides on polymers adopt a more ordered orientation than less tightly bound physically adsorbed peptides. These differences were also observed in different chemical environments, for example, air versus water. Although numerous studies have reported a direct correlation between the choice of immobilization method and the performance of an attached biological molecule, the lack of direct biomolecular structure and orientation data has made it difficult to elucidate the relationship between structure, orientation, and function at a surface. In this work, we directly studied the effect of chemical immobilization method on biomolecular orientation/ordering, an important step for future studies of biomolecular activity. The methods for orientation analysis described within are also of relevance to understanding biosensors, biocompatibility, marine-antifouling, membrane protein functions, and antimicrobial peptide activities.

Surface Orientation of Magainin 2: Molecular Dynamics Simulation and Sum Frequency Generation Vibrational Spectroscopic Studies

We combined molecular dynamics based free energy calculations with sum frequency generation (SFG) spectroscopy to study the orientational distribution of solvated peptides near hydrophobic surfaces. Using a simplified atomistic model of the polystyrene (PS) surface, molecular dynamics simulations have been applied to compute the orientational probability of an α-helical peptide, magainin 2, with respect to the PS/water interface. Free energy calculations revealed that the preferred (horizontal) peptide orientation was driven by the favorable interactions between the hydrophobic PS surface and the hydrophobic residues on the helix, and additional simulations examined the importance of small aggregate formation. Concentration-dependent measurements obtained via SFG vibrational spectroscopy suggest that, at very low peptide concentrations, magainin molecules tend to lie down at the PS/solution interface, which correlates well with the simulation results. When the concentration is increased, peptides exhibit behavior not captured by MD simulations using single helical peptides. A combination of simulations and experiments was shown to yield more reliable results with molecular-level insights into interaction between peptides and polymer surfaces.

Interfacial Orientation and Secondary Structure Change in Tachyplesin I: Molecular Dynamics and Sum Frequency Generation Spectroscopy Studies

Recent advances in the collection and interpretation of surface-sensitive vibrational spectroscopic measurements have made it possible to study the orientation of peptides and proteins in situ in a biologically relevant environment. However, interpretation of sum frequency generation (SFG) and attenuated total reflectance Fourier transform infrared (ATR-FTIR) vibrational spectroscopy is hindered by the fact that orientation cannot be inferred without some prior knowledge of the protein structure. In this work, molecular dynamics simulations were used to study the interfacial orientation and structural deformation of the short β-sheet peptide tachyplesin I at the polystyrene/water interface. By combining these results with ATR-FTIR and SFG measurements, reasonable agreement was found with the simulation results, suggesting that tachyplesin I lies parallel to the surface, although the simulation results imply a broader distribution of peptide twist angles than could be characterized using available experimental measurements. The interfacial structure was found to be deformable even when disulfide bonds were preserved, and these local deviations from a purely extended β-sheet conformation may be of importance to future developments in the interpretation of SFG and ATR-FTIR spectra.

Hyperspectral Imaging and Characterization of Live Cells by Broadband Coherent Anti-Stokes Raman Scattering (CARS) Microscopy with Singular Value Decomposition (SVD) Analysis

Coherent anti-Stokes Raman scattering (CARS) microscopy can be used as a powerful imaging technique to identify chemical compositions of complex samples in biology, biophysics, medicine, and materials science. In this work we developed a CARS microscopic system capable of hyperspectral imaging. By employing an ultrafast laser source, a photonic crystal fiber, and a scanning laser microscope together with spectral detection by a highly sensitive back-illuminated cooled charge-coupled device (CCD) camera, we were able to rapidly acquire and process hyperspectral images of live cells with chemical selectivity. We discuss various aspects of hyperspectral CARS image analysis and demonstrate the use of singular value decomposition methods to characterize the cellular lipid content.

Live-cell quantification and comparison of mammalian oocyte cytosolic lipid content between species, during development, and in relation to body composition using nonlinear vibrational microscopy

Cytosolic lipids participate in the growth, development, and overall health of mammalian oocytes including many roles in cellular homeostasis. Significant emphasis has been placed on the study of lipids as a dynamic organelle, which in turn requires the development of tools and techniques to quantitate and compare how lipid content relates to cellular structure, function, and normalcy. Objectives of this study were to determine if nonlinear vibrational microscopy (e.g., coherent anti-Stokes Raman scattering or CARS microscopy) could be used for live-cell imaging to quantify and compare lipid content in mammalian oocytes during development and in relation to body composition; and compare its efficacy to methods involving cellular fixation and staining protocols. Results of this study demonstrate that CARS is able to identify lipids in live mammalian oocytes, and there exists quantifiable and consistent differences in percent lipid composition across ooctyes of different species, developmental stages, and in relation to body composition. Such a method of live-cell lipid quantification has (i) experimental power in basic cell biology, (ii) practical utility for identifying developmental predictive biomarkers while advancing biology-based oocyte/embryo selection, and (iii) ability to yield rationally supporting technology for decision-making in rodents, domestic species, and human assisted reproduction and/or fertility preservation.

Sum Frequency Generation Vibrational Spectroscopy: A Sensitive Technique for the Study of Biological Molecules at Interfaces

The behavior of biological molecules in interfacial environments is critical to understanding a broad range of phenomena, from biocompatibility to the functions of membrane-associated peptides and proteins. Sum frequency generation (SFG) vibrational spectroscopy is a nonlinear optical vibrational spectroscopic technique with an excellent sensitivity to interfacial molecules and molecular ordering and is well suited to probing biomolecules in a native interfacial environment. Using this technique, one can obtain unique information on the biomolecular orientation and conformation at interfaces. SFG also provides additional measurements that are complementary to other vibrational studies; more complicated protein structures and orientation distributions may be studied in greater detail when SFG is combined with other vibrational techniques.