Molecular Orientation Distributions in Protein Films: III. Yeast Cytochrome c Immobilized on Pyridyl Disulfide-Capped Phospholipid Bilayers

Abstract

Molecular orientation in a hydrated monolayer film of yeast cytochrome c, immobilized via disulfide bonding between Cys-102 and a pyridyl disulfide-capped phospholipid bilayer deposited from an air-water interface onto glass substrates, was investigated. The orientation distribution of the heme groups in the protein film was determined using a combination of absorption linear dichroism, measured in a planarintegrated optical waveguide-attenuated total reflection geometry- and fluorescence anisotropy, measured in a total internal reflection geometry. A gaussian model for the orientation distribution was used to recover the mean heme tilt angle and angular distribution about the mean, which were 40 and 11 degrees, respectively. Additional experiments showed that a large fraction of the cytochrome c was disulfide bonded to the bilayer, which correlates with the high degree of macroscopic order in the protein film. However, a subpopulation of yeast cytochrome c molecules in the film (approximately 30% of the total) appeared to be nonspecifically adsorbed. The orientation distribution of this subpopulation was found to be much broader than the specifically bound fraction.

Details of the Partial Unfolding of T4 Lysozyme on Quartz Using Site‐Directed Spin Labeling

“…[35,36] In those studies, protein adsorption with conservation of the secondary structure was demonstrated, and methods to elucidate the orientation of the protein or of secondary structural elements were developed. In contrast to the zwitterionic lipid bilayers in those studies, the quartz surface is negatively charged at neutral pH [37] while T4L carries a net positive charge under these conditions. Therefore, the protein-surface interaction is expected to be increased on the quartz surface, and a more severe perturbation of the protein structure is expected.…”

Adsorption Behavior of Cytochrome c, Myoglobin and Hemoglobin in a Quartz Surface Probed Using Slab Optical Waveguide (SOWG) Spectroscopy

“…This is mainly due to the foreseen benefits that understanding the kinetics and mechanism of protein adsorption will have essential applications not only in protein analysis, but also on industrial processes, such as the purification and storage of pharmaceutical products, in the biocompatibility of surgical implants, new materials and coatings, in biosensor development as well as in explaining some fundamental biochemical Spectroscopic techniques based on internal total reflection (ITR) in an optical waveguide are one of the most effective methods for investigating molecular-adsorption processes onto surfaces, because they allow in situ measurements without influencing the adsorption process. 2,3 The reported nomenclature in the literature include slab optical waveguide (SOWG) spectroscopy, 4-17 the integrated optical waveguideattenuated total reflection technique (IOW-ATR) [18][19][20][21][22][23][24][25][26][27] and optical waveguide lightmode spectroscopy (OWLS). 1,[28][29][30][31][32][33][34][35][36][37][38] These techniques have been successfully used in real-time studies on the adsorption of dye molecules 11,12,[14][15][16] and proteins 4,8,[18][19][20][21][22][23][24][25][28]…”

“…2,3 The reported nomenclature in the literature include slab optical waveguide (SOWG) spectroscopy, 4-17 the integrated optical waveguideattenuated total reflection technique (IOW-ATR) [18][19][20][21][22][23][24][25][26][27] and optical waveguide lightmode spectroscopy (OWLS). 1,[28][29][30][31][32][33][34][35][36][37][38] These techniques have been successfully used in real-time studies on the adsorption of dye molecules 11,12,[14][15][16] and proteins 4,8,[18][19][20][21][22][23][24][25][28][29][30][31][32][33][34][35][36][37]…”

“…Since the interacting photons come from the waveguiding surface, one advantage of ITR-based spectroscopy is its ability to interact mostly with the adsorbed molecules and much less with those in the bulk solution. Some of the research groups that are actively involved in studying protein adsorption on surfaces based on ITR are Jeremy Ramsden's group in Basel, Switzerland, 28-37 Scott Saavedra's group in Arizona, [18][19][20][21][22][23][24][25][26][27] Paul R. Van Tassel's group in Wayne, USA 38,40 and some research groups in Japan [4][5][6][7][8][9][10][11][12][13][14][15][16][17]39 and elsewhere. 41,42 In this work we investigated, under various solution conditions, the adsorption behavior of hemoglobin, myoglobin and cytochrome c in a bare quartz surface.…”

Partielle Entfaltung von T4‐Lysozym auf einer Quarzoberfläche: Analyse der Strukturänderungen adsorbierter Proteine durch ESR‐Spektroskopie

“…[35,36] Dabei wurde gezeigt, dass die Sekundärstruktur von T4L bei Adsorption auf einer Doppelschicht eines zwitterionischen Lipids erhalten bleibt, und es wurden Methoden zur Bestimmung der Orientierung adsorbierter Proteine entwickelt. Im Unterschied zu den dort verwendeten Lipiddoppelschichten ist die in dieser Untersuchung betrachtete Quarzoberfläche bei neutralem pHWert [37] negativ geladen, während T4L positiv geladen ist. Daher sollte die Wechselwirkung zwischen Protein und Oberfläche bei der Adsorption auf einer Quarzoberfläche stärker sein als auf einer Lipiddoppelschicht; daher wird auch eine stärkere Veränderung der Proteinstruktur erwartet.…”