Carol R. Flach

Infrared reflection-absorption spectroscopy: principles and applications to lipid-protein interaction in Langmuir films.

Infrared reflection-absorption spectroscopy (IRRAS) of lipid/protein monolayer films in situ at the air/water interface provides unique molecular structure and orientation information from the film constituents. The technique is thus well suited for studies of lipid/protein interaction in a physiologically relevant environment. Initially, the nature of the IRRAS experiment is described and the molecular structure information that may be obtained is recapitulated. Subsequently, several types of applications, including the determination of lipid chain conformation and tilt as well as elucidation of protein secondary structure are reviewed. The current article attempts to provide the reader with an understanding of the current capabilities of IRRAS instrumentation and the type of results that have been achieved to date from IRRAS studies of lipids, proteins, and lipid/protein films of progressively increasing complexity. Finally, possible extensions of the technology are briefly considered.

A Quantitative Reconstruction of the Amide I Contour in the IR Spectra of Globular Proteins: From Structure to Spectrum

The Amide I contours of six globular proteins of varied secondary structure content along with a peptide model for collagen and pulmonary surfactant protein C have been simulated very closely by using a modified GF matrix method. The starting point for the method uses the three-dimensional structure as obtained from the Protein Data Bank. Elements of the interactions between peptide groups (e.g., transition dipole coupling) are very sensitive to tertiary structure, thus the current formalism demonstrates that the Amide I contour may be useful for a more detailed probe of 3-D conformation that goes beyond the traditional use of this band to probe the percentages of particular elements of secondary structure. For example, postulated changes to a known structure can be tested by comparing the new simulated band to the experimental band. A number of refinements to the transition dipole interaction calculation have been made. Most of the important interactions between the C=O oscillators that define the Amide I mode appear to have been identified, including through space transition dipole coupling, through valence bond and through hydrogen bond coupling. The eigenvector matrix produced by the method permits the contribution of each peptide group to the spectrum to be precisely determined. Analysis of the results shows that the often-used structure-frequency correlations are at best approximate and at worst misleading. The subbands from helices, sheets, turns, and loops are much broader and more overlapped than has been commonly assumed. Furthermore, the traditional alpha-helical marker band may be substantially distorted in short segments. Difference spectra based on isotope editing, a technique thought capable of revealing the spectral contributions of individual peptide groups, are shown to be prone to misinterpretation.

External reflection FTIR of peptide monolayer films in situ at the air/water interface: experimental design, spectra-structure correlations, and effects of hydrogen-deuterium exchange

A Fourier transform infrared spectrometer has been interfaced with a surface balance and a new external reflection infrared sampling accessory, which permits the acquisition of spectra from protein monolayers in situ at the air/water interface. The accessory, a sample shuttle that permits the collection of spectra in alternating fashion from sample and background troughs, reduces interference from water vapor rotation-vibration bands in the amide I and amide II regions of protein spectra (1520-1690 cm-1) by nearly an order of magnitude. Residual interference from water vapor absorbance ranges from 50 to 200 microabsorbance units. The performance of the device is demonstrated through spectra of synthetic peptides designed to adopt alpha-helical, antiparallel beta-sheet, mixed beta-sheet/beta-turn, and unordered conformations at the air/water interface. The extent of exchange on the surface can be monitored from the relative intensities of the amide II and amide I modes. Hydrogen-deuterium exchange may lower the amide I frequency by as much as 11-12 cm-1 for helical secondary structures. This shifts the vibrational mode into a region normally associated with unordered structures and leads to uncertainties in the application of algorithms commonly used for determination of secondary structure from amide I contours of proteins in D2O solution.

Calcium ion interactions with insoluble phospholipid monolayer films at the A/W interface. External reflection-absorption IR studies

External reflection Fourier transform infrared (FT-IR) experiments are reported for insoluble monomolecular films of an equimolar mixture of 1,2-dipalmitoylphosphatidylcholine (DPPC) and 1,2-dipalmitoylphosphatidylserine (DPPS) at the A/W interface as a function of surface pressure and Ca2+ ion presence. The separate components showed a surface pressure-induced conformational ordering of the acyl chains. The conformational ordering occurred more cooperatively for the DPPS. Acyl chain perdeuteration of the DPPC permitted the observation of the response of the individual components in the binary mixture to changes in surface tension and to the presence of Ca2+. Plots of surface pressure versus CH2 or CD2 stretching frequencies were analyzed with a two-state model. At each surface pressure within the two-state region, the fraction of disordered form was the same for each lipid component, suggesting that they are well mixed on the surface. Calcium ion (5 mM in the subphase) produces almost no effect on the pressure-induced acyl chain ordering of the DPPC in a single component film, whereas the same levels of Ca2+ induce acyl chain ordering at all surface pressures in both components of the binary mixture. Thus, unlike the bulk phase mixture of DPPC/DPPS, the binary lipids in this mixed monolayer film appear to retain their miscibility in the presence of Ca2+. Finally, Ca(2+)-induced dehydration of the phosphate group was observed through characteristic frequency shifts in the asymmetric PO2- stretching mode.

Graphene-Catalyzed Direct Friedel–Crafts Alkylation Reactions: Mechanism, Selectivity, and Synthetic Utility

Transition-metal-catalyzed alkylation reactions of arenes have become a central transformation in organic synthesis. Herein, we report the first general strategy for alkylation of arenes with styrenes and alcohols catalyzed by carbon-based materials, exploiting the unique property of graphenes to produce valuable diarylalkane products in high yields and excellent regioselectivity. The protocol is characterized by a wide substrate scope and excellent functional group tolerance. Notably, this process constitutes the first general application of graphenes to promote direct C-C bond formation utilizing polar functional groups anchored on the GO surface, thus opening the door for an array of functional group alkylations using benign and readily available graphene materials. Mechanistic studies suggest that the reaction proceeds via a tandem catalysis mechanism in which both of the coupling partners are activated by interaction with the GO surface.

Direct Production of Graphene Nanosheets for Near Infrared Photoacoustic Imaging

Hummers method is commonly used for the fabrication of graphene oxide (GO) from graphite particles. The oxidation process also leads to the cutting of graphene sheets into small pieces. From a thermodynamic perspective, it seems improbable that the aggressive, somewhat random oxidative cutting process could directly result in graphene nanosheets without destroying the intrinsic π-conjugated structures and the associated exotic properties of graphene. In Hummers method, both KMnO4 and NO2(+) (nitronium ions) in concentrated H2SO4 solutions act as oxidants via different oxidation mechanisms. From both experimental observations and theoretical calculations, it appears that KMnO4 plays a major role in the observed oxidative cutting and unzipping processes. We find that KMnO4 also limits nitronium oxidative etching of graphene basal planes, therefore slowing down graphene fracturing processes for nanosheet fabrication. By intentionally excluding KMnO4 and exploiting pure nitronium ion oxidation, aided by the unique thermal and kinetic effects induced by microwave heating, we find that graphite particles can be converted into graphene nanosheets with their π-conjugated aromatic structures and properties largely retained. Without the need of any postreduction processes to remove the high concentration of oxygenated groups that results from Hummers GO formation, the graphene nanosheets as-fabricated exhibit strong absorption, which is nearly wavelength-independent in the visible and near-infrared (NIR) regions, an optical property typical for intrinsic graphene sheets. For the first time, we demonstrate that strong photoacoustic signals can be generated from these graphene nanosheets with NIR excitation. The photo-to-acoustic conversion is weakly dependent on the wavelength of the NIR excitation, which is different from all other NIR photoacoustic contrast agents previously reported.

Monolayer–multilayer transitions in a lung surfactant model: IR reflection–absorption spectroscopy and atomic force microscopy

A hydrophobic pulmonary surfactant protein, SP-C, has been implicated in surface-associated activities thought to facilitate the work of breathing. Model surfactant films composed of lipids and SP-C display a reversible transition from a monolayer to surface-associated multilayers upon compression and expansion at the air/water (A/W) interface. The molecular-level mechanics of this process are not yet fully understood. The current work uses atomic force microscopy on Langmuir–Blodgett films to verify the formation of multilayers in a dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, cholesterol, and SP-C model system. Isotherms of SP-C-containing films are consistent with exclusion and essentially complete respreading during compression and expansion, respectively. Multilayer formation was not detected in the absence of SP-C. Most notable are the results from IR reflection–absorption spectroscopy (IRRAS) conducted at the A/W interface, where the position and intensity of the Amide I band of SP-C reveal that the predominantly helical structure changes its orientation in monolayers versus multilayers. IRRAS measurements indicate that the helix tilt angle changed from approximately 80° in monolayers to a transmembrane orientation in multilayers. The results constitute the first quantitative measure of helix orientation in mixed monolayer/multilamellar domains at the A/W interface and provide insight into the molecular mechanism for SP-C-facilitated respreading of surfactant.

Raman microspectroscopic and dynamic vapor sorption characterization of hydration in collagen and dermal tissue

Water is an integral part of collagens triple helical and higher order structure. Studies of model triple helical peptides have revealed the presence of repetitive intrachain, interchain, and intermolecular water bridges (Bella et al., Structure 1995, 15, 893-906). In addition, an extended cylinder of hydration is thought to be responsible for collagen fiber assembly. Confocal Raman spectroscopy and dynamic vapor sorption (DVS) measurements of human Type I collagen and pigskin dermis were performed to probe relative humidity (RH)-dependent differences in the nature and level of collagen hydration. Raman spectra were also acquired as a function of time for both Type I collagen and pigskin dermis samples upon exchange of a 100% RH H(2) O to deuterium oxide (D(2) O) environment. Alterations in Amide I and III modes were consistent with anticipated changes in hydrogen bonding strength as RH increased and upon H → D exchange. Of note is the identification of a Raman spectral marker (band at 938 cm(-1) ) which appears to be sensitive to alterations in collagen-bound water. Analysis of DVS isotherms provided a quantitative measure of adsorbed and absorbed water vapor consistent with the Raman results. The development of a Raman spectral marker of collagen hydration in intact tissue is relevant to diverse fields of study ranging from the evaluation of therapeutics for wound healing to hydration of aging skin.

Interactions of the HIV-1 fusion peptide with large unilamellar vesicles and monolayers. A cryo-TEM and spectroscopic study.

We have examined the interaction of the human immunodeficiency virustype 1 fusion peptide (23 amino acid residues) and of a Trp-containing analog with vesicles composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine and cholesterol (molar ratio, 1:1:1). Both the native and the Trp-substituted peptides bound the vesicles to the same extent and induced intervesicular lipid mixing with comparable efficiency. Infrared reflection-absorption spectroscopy data are compatible with the adoption by the peptide of a main beta-sheet structure in a cospread lipid/peptide monolayer. Cryo-transmission electron microscopy observations of peptide-treated vesicles reveal the existence of a peculiar morphology consisting of membrane tubular elongations protruding from single vesicles. Tryptophan fluorescence quenching by brominated phospholipids and by water-soluble acrylamide further indicated that the peptide penetrated into the acyl chain region closer to the interface rather than into the bilayer core. We conclude that the differential partition and shallow penetration of the fusion peptide into the outer monolayer of a surface-constrained bilayer may account for the detected morphological effects. Such single monolayer-restricted interaction and its structural consequences are compatible with specific predictions of current theories on viral fusion.

Palmitoylation of lung surfactant protein SP-C alters surface thermodynamics, but not protein secondary structure or orientation in 1,2-dipalmitoylphosphatidylcholine langmuir films.

Pulmonary surfactant-specific protein, SP-C, isolated from porcine lung lavage, has been deacylated to investigate the role of the two thioester linked palmitoyl chains located near the N-terminus. Surface thermodynamic properties, secondary structure, and orientation of native and deacylated SP-C in 1, 2-dipalmitoylphosphatidylcholine (DPPC) monolayers has been characterized by combined surface pressure-molecular area (pi-A) isotherms and infrared reflection-absorption spectroscopy (IRRAS) measurements. The isotherms indicate that deacylation of SP-C produces more fluid monolayers at pressures less than 30 mN m-1. The helical secondary structure and tilt angle (70-80 degrees relative to the surface normal) of SP-C remained essentially unchanged upon deacylation in DPPC monolayers at a surface pressure approximately 30 mN m-1. The results are consistent with a model that acylation of SP-C may influence the rapid protein-aided spreading of a surface-associated surfactant reservoir, but not the structure of DPPC or SP-C in the monolayer at higher surface pressures.