Interfacial dynamic adsorption and structure of molecular layers of peptide surfactants.

Short peptide surfactants have recently emerged as a new class of amphiphiles, with tremendous potential in improving surface biocompatibility and mediating interfacial DNA immobilization. To establish their basic interfacial adsorption properties, cationic peptide surfactants V(m)K(n) have been studied by combining the measurements of spectroscopic ellipsometry (SE), neutron reflection (NR) and atomic force microscopy (AFM). Our results showed that changes in peptide structure, concentration, solution pH and ionic strength all affected their interfacial assembly. Increases in m and decreases in n reduced the critical aggregation concentration (CAC), but increased the amount of adsorption, showing the strong influence of the amphiphilic balance between hydrophilic and hydrophobic moieties. While the surface adsorbed amount increased with time and peptide concentration, an increase in ionic strength decreased peptide adsorption due to surface charge neutralization. Changes in solution pH did not affect the equilibrium surface adsorbed amount on the weakly negative SiO(2) surface, but did alter the adsorption dynamics. Neutron reflection revealed that V(6)K readily formed a bilayer structure of 35 A thickness at the interface, with the main part of the V(6) fragments being packed back-to-back to form a 15 A hydrophobic core and the two outer K regions being incorporated with a minor amount of V fragments forming the headgroup layers of 9 A each. AFM imaging revealed a sheet-like membrane structure incorporating defects of holes but the thicknesses probed by AFM were consistent with neutron reflection. It was demonstrated that the V(6)K peptide bilayer was effective for immobilization of DNA. The amount of DNA immobilized followed approximate 1:1 charge neutralization between the outer leaf peptide sublayer and the negatively charged DNA.

The Small Ribozymes: Common and Diverse Features Observed through the FRET Lens.

The hammerhead, hairpin, HDV, VS and glmS ribozymes are the five known, naturally occurring catalytic RNAs classified as the "small ribozymes". They share common reaction chemistry in cleaving their own backbone by phosphodiester transfer, but are diverse in their secondary and tertiary structures, indicating that Nature has found at least five independent solutions to a common chemical task. Fluorescence resonance energy transfer (FRET) has been extensively used to detect conformational changes in these ribozymes and dissect their reaction pathways. Common and diverse features are beginning to emerge that, by extension, highlight general biophysical properties of non-protein coding RNAs.

Candidate cell and matrix interaction domains on the collagen fibril, the predominant protein of vertebrates.

Type I collagen, the predominant protein of vertebrates, polymerizes with type III and V collagens and non-collagenous molecules into large cable-like fibrils, yet how the fibril interacts with cells and other binding partners remains poorly understood. To help reveal insights into the collagen structure-function relationship, a data base was assembled including hundreds of type I collagen ligand binding sites and mutations on a two-dimensional model of the fibril. Visual examination of the distribution of functional sites, and statistical analysis of mutation distributions on the fibril suggest it is organized into two domains. The "cell interaction domain" is proposed to regulate dynamic aspects of collagen biology, including integrin-mediated cell interactions and fibril remodeling. The "matrix interaction domain" may assume a structural role, mediating collagen cross-linking, proteoglycan interactions, and tissue mineralization. Molecular modeling was used to superimpose the positions of functional sites and mutations from the two-dimensional fibril map onto a three-dimensional x-ray diffraction structure of the collagen microfibril in situ, indicating the existence of domains in the native fibril. Sequence searches revealed that major fibril domain elements are conserved in type I collagens through evolution and in the type II/XI collagen fibril predominant in cartilage. Moreover, the fibril domain model provides potential insights into the genotype-phenotype relationship for several classes of human connective tissue diseases, mechanisms of integrin clustering by fibrils, the polarity of fibril assembly, heterotypic fibril function, and connective tissue pathology in diabetes and aging.

Collagen fibril architecture, domain organization, and triple-helical conformation govern its proteolysis.

We describe the molecular structure of the collagen fibril and how it affects collagen proteolysis or "collagenolysis." The fibril-forming collagens are major components of all mammalian connective tissues, providing the structural and organizational framework for skin, blood vessels, bone, tendon, and other tissues. The triple helix of the collagen molecule is resistant to most proteinases, and the matrix metalloproteinases that do proteolyze collagen are affected by the architecture of collagen fibrils, which are notably more resistant to collagenolysis than lone collagen monomers. Until now, there has been no molecular explanation for this. Full or limited proteolysis of the collagen fibril is known to be a key process in normal growth, development, repair, and cell differentiation, and in cancerous tumor progression and heart disease. Peptide fragments generated by collagenolysis, and the conformation of exposed sites on the fibril as a result of limited proteolysis, regulate these processes and that of cellular attachment, but it is not known how or why. Using computational and molecular visualization methods, we found that the arrangement of collagen monomers in the fibril (its architecture) protects areas vulnerable to collagenolysis and strictly governs the process. This in turn affects the accessibility of a cell interaction site located near the cleavage region. Our observations suggest that the C-terminal telopeptide must be proteolyzed before collagenase can gain access to the cleavage site. Collagenase then binds to the substrate's "interaction domain," which facilitates the triple-helix unwinding/dissociation function of the enzyme before collagenolysis.

Interfacial adsorption of antifreeze proteins: a neutron reflection study.

Interfacial adsorption from two antifreeze proteins (AFP) from ocean pout (Macrozoarces americanus, type III AFP, AFP III, or maAFP) and spruce budworm (Choristoneura fumiferana, isoform 501, or cfAFP) were studied by neutron reflection. Hydrophilic silicon oxide was used as model substrate to facilitate the solid/liquid interfacial measurement so that the structural features from AFP adsorption can be examined. All adsorbed layers from AFP III could be modeled into uniform layer distribution assuming that the protein molecules were adsorbed with their ice-binding surface in direct contact with the SiO(2) substrate. The layer thickness of 32 A was consistent with the height of the molecule in its crystalline form. With the concentration decreasing from 2 mg/ml to 0.01 mg/ml, the volume fraction of the protein packed in the monolayer decreased steadily from 0.4 to 0.1, consistent with the concentration-dependent inhibition of ice growth observed over the range. In comparison, insect cfAFP showed stronger adsorption over the same concentration range. Below 0.1 mg/ml, uniform layers were formed. But above 1 mg/ml, the adsorbed layers were characterized by a dense middle layer and two outer diffuse layers, with a total thickness around 100 A. The structural transition indicated the responsive changes of conformational orientation to increasing surface packing density. As the higher interfacial adsorption of cfAFP was strongly correlated with the greater thermal hysteresis of spruce budworm, our results indicated the important relation between protein adsorption and antifreeze activity.

Surface-induced unfolding of human lactoferrin.

We have determined the structural conformations of human lactoferrin adsorbed at the air/water interface by neutron reflectivity (NR) and its solution structure by small angle neutron scattering (SANS). The neutron reflectivity measurements revealed a strong structural unfolding of the molecule when adsorbed at the interface from a pH 7 phosphate buffer solution (PBS with a total ionic strength at 4.5 mM) over a wide concentration range. Two distinct regions, a top dense layer of 15-20 angstroms on the air side and a bottom diffuse layer of some 50 angstroms into the aqueous subphase, characterized the unfolded interfacial layer. At a concentration around 1 g dm(-3), close to the physiological concentration of lactoferrin in biological fluids, the adsorbed amount was 5.5 x 10(-8) mol m(-2) in the absence of NaCl, but the addition of 0.3 M NaCl reduced protein adsorption to 3.5 x 10(-8) mol m(-2). Although the polypeptide distributions at the interface remained similar, quantitative analysis showed that the addition of NaCl reduced the layer thickness. Parallel measurements of lactoferrin adsorption in D2O instead of null reflecting water confirmed the unfolded structure at the interface. Furthermore, the D2O data indicated that the polypeptide in the top layer was predominantly protruded out of water, consistent with it being hydrophobic. In contrast, the scattering intensity profiles from SANS were well described by a cylindrical model with a diameter of 47 angstroms and a length of 105 angstroms in the presence of 0.3 M NaCl, indicating a retention of the globular framework in the bulk solution. In the absence of NaCl but with the same amount of phosphate buffer, the length of the cylinder increased to some 190 angstroms and the diameter remained constant. The length increase is indicative of changes in distance and orientation between the bilobal monomers due to the change in charge interactions. The results thus demonstrate that the surface structural unfolding was caused by the exposure of the protein molecule to the unsymmetrical energetic balance following surface adsorption.

Interfacial nano-structuring of designed peptides regulated by solution pH.

The in-situ conformations of peptide layers formed from the adsorption of two different synthetic 15-mer peptides at the hydrophilic silicon oxide/aqueous solution interface have been determined using neutron reflectivity (NR). The first peptide is based on the native sequence of a protein-binding domain within a heteromeric transcriptional activator, HAP2, identified from yeast Saccharomyces cerevisiae, with tyrosine (Y) present at the 1st, 8th and 15th amino acid positions, hence we denote this YYY15. Substitution of tryptophan (W) at the same locations gives WWW15. Both peptides have alpha-helical structure in phosphate buffer, as determined by circular dichroism (CD) spectra. D(2)O was used as solvent in the NR experiments to highlight structural heterogeneity across the hydrogenated peptide layers. At pH 7, YYY15 was found to form a weakly adsorbed interfacial monolayer. However, the mutant WWW15 showed strong interfacial adsorption, with the interfacial layer characterized by a middle hydrophobic sublayer of 7-8 A with lower scattering length density and two almost symmetrical hydrophilic outer sublayers of 6-8 A with higher scattering length density, suggesting the formation of a "sideways-on" helical conformation. An increase in pH to 9 resulted in the improved packing within the interfacial layer with similar structure. However, decrease in pH to 5 reduced the interfacial adsorption, mainly due to the enhanced solubility of the peptides associated with the protonation of arginine (R) and lysine (K) groups and the decreasing concentration of divalent HPO(4)(2-) in the phosphate buffer. Subsequent assessment of the reversibility of adsorption showed that once the peptide layers were formed they did not desorb. These interfacial structures may provide feasible routes to interfacial nano-templating.

Adsorption of beta-hairpin peptides on the surface of water: a neutron reflection study.

Neutron reflectivity has been used to determine the thickness and surface coverage of monolayers of two 14-residue beta-hairpin peptides adsorbed at the air/water interface. The peptides differed only in that one was labeled with a fluorophore, while the other was not. The neutron reflection measurements were mainly made in null reflecting water, NRW, containing 8.1% D(2)O. Under this isotopic contrast the water is invisible to neutrons and the specular signal was then only from the peptide layer. At the highest concentration of ca. 4 microg/mL studied, the area per peptide molecule (A) was found to be 230 +/- 10 and 210 +/- 10 A(2) for the peptides with and without a BODIPY-based fluorophore, respectively. The thickness of the peptide layers was about 10 A for a Gaussian distribution. With decreasing bulk peptide concentration, both surface excess and layer thickness showed a steady trend of decrease. While the neutron results clearly indicate structural changes within the peptide monolayers with increasing bulk concentration, the outstanding structural feature is the formation of rather uniform peptide layers, consistent with the structural characteristics typical of beta-strand peptide conformations. These structural features are well supported by the parallel measurements of the adsorbed layers in D(2)O. With this isotopic contrast the neutron reflectivity provides an estimate about the extent of immersion of the peptide layers into water. The results strongly suggest that the 14-mer peptide monolayers were fully afloat on the surface of water, with only the carboxy groups on Glu residues hydrated.