Secondary structure in lung surfactant SP-B peptides: IR and CD studies of bulk and monolayer phases

AbstractPulmonary surfactant protein SP-B is known to facilitate adsorption and spreading of surfactant components across the air/water interface. This property appears essential for in vivo function in the alveolar subphase and at the air/alveolar surface. Three peptides with amino acid sequences based on SP-B containing predicted α-helical regions (SP-B1-20, SP-B9-36A, SP-B40-60A) have been synthesized to probe structure-function relationships and protein-lipid interaction in bulk phase and monolayer environments. IR and CD studies are reported along with traditional surface pressure-molecular area (π-A) isotherms and IR reflection-absorption spectroscopy (IRRAS) investigations conducted at the air/water interface. In bulk phase, helix-promoting environments (methanol and aqueous dispersions of lipid vesicles), SP-B1-20 and SP-B9-36A contained significant amounts of α-helical structure, whereas varying degrees of α-helix, random coil, and β-sheet were observed in aqueous solutions and monolayers. The most striking behavior was observed for SP-B9-36A, which displayed reversible surface pressure-induced β-sheet formation. Bulk phase lipid melting curves and monolayer experiments with peptide-lipid mixtures showed subtle differences in the degree of bulk phase interaction and substantial differences in peptide surface activity. The uniqueness of IRRAS is emphasized as the importance of evaluating secondary structure in both bulk phase and monolayer environments for lung surfactant peptide mimics is demonstrated

Vibrational microscopy and imaging of skin: from single cells to intact tissue

Vibrational microscopy and imaging offer several advantages for a variety of dermatological applications, ranging from studies of isolated single cells (corneocytes) to characterization of endogenous components in intact tissue. Two applications are described to illustrate the power of these techniques for skin research. First, the feasibility of tracking structural alterations in the components of individual corneocytes is demonstrated. Two solvents, DMSO and chloroform/methanol, commonly used in dermatological research, are shown to induce large reversible alterations (α-helix to β-sheet) in the secondary structure of keratin in isolated corneocytes. Second, factor analysis of image planes acquired with confocal Raman microscopy to a depth of 70 μm in intact pigskin, demonstrates the delineation of specific skin regions. Two particular components that are difficult to identify by other means were observed in the epidermis. One small region was formed from a conformationally ordered lipid phase containing cholesterol. In addition, the presence of nucleated cells in the tissue (most likely keratinocytes) was revealed by the spectral signatures of the phosphodiester and cytosine moieties of cellular DNA

Vibrational Microspectroscopy and Imaging of Molecular Composition and Structure During Human Corneocyte Maturation

The outermost region of the epidermis, the stratum corneum (SC), provides an essential barrier to water loss and protects against exogenous substances. The functional integrity of the SC depends on a complex maturation and exfoliation process, which is often perturbed in skin diseases. The maturation of corneocytes isolated from different depths in healthy human SC was investigated using infrared (IR) spectroscopic imaging and Raman microscopy. Both IR and Raman spectral quality of individual corneocytes was high and revealed depth-dependent variations in molecular composition. Spectral changes were identified as arising from alterations in the concentration of the major constituents of natural moisturizing factor (NMF), important in maintaining SC hydration. A significant decrease in the concentration of NMF was observed for corneocytes isolated from superficial compared to deeper SC layers (layer 3 vs. layer 11, respectively). An IR parameter that measures the relative NMF concentration in corneocytes is introduced. The potential role of vibrational imaging to evaluate corneocyte composition and molecular structure in the treatment of NMF-related diseases is discussed

Infrared microscopic imaging of cutaneous wound healing: lipid conformation in the migrating epithelial tongue

Infrared microscopic imaging has been utilized to analyze for the first time the spatial distribution of lipid structure in an ex vivo human organ culture skin wound healing model. Infrared images were collected at zero, two, four, and six days following wounding. Analysis of lipid infrared spectral properties revealed the presence of a lipid class with disordered chains within and in the vicinity of the migrating epithelial tongue. The presence of lipid ester C=O bands colocalized with the disordered chains provided evidence for the presence of carbonyl-containing lipid species. Gene array data complemented the biophysical studies and provided a biological rationale for the generation of the disordered chain species. This is the first clear observation, to our knowledge, of disordered lipid involvement in cutaneous wound healing. Several possibilities are discussed for the biological relevance of these observations