Insights into the role of substrates on the interaction between cytochrome b5 and cytochrome P450 2B4 by NMR.

Mammalian cytochrome b5 (cyt b5) is a membrane-bound protein capable of donating an electron to cytochrome P450 (P450) in the P450 catalytic cycle. The interaction between cyt b5 and P450 has been reported to be affected by the substrates of P450; however, the mechanism of substrate modulation on the cyt b5-P450 complex formation is still unknown. In this study, the complexes between full-length rabbit cyt b5 and full-length substrate-free/substrate-bound cytochrome P450 2B4 (CYP2B4) are investigated using NMR techniques.

A model of the membrane-bound cytochrome b5-cytochrome P450 complex from NMR and mutagenesis data.

Microsomal cytochrome b5 (cytb5) is a membrane-bound protein that modulates the catalytic activity of its redox partner, cytochrome P4502B4 (cytP450). Here, we report the first structure of full-length rabbit ferric microsomal cytb5 (16 kDa), incorporated in two different membrane mimetics (detergent micelles and lipid bicelles). Differential line broadening of the cytb5 NMR resonances and site-directed mutagenesis data were used to characterize the cytb5 interaction epitope recognized by ferric microsomal cytP450 (56 kDa).

Molecular interactions between magainin 2 and model membranes in situ.

In this paper, we investigated the molecular interactions of magainin 2 with model cell membranes using sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). Symmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-[Phospho-rac-(1-glycerol)] (POPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers, which model the bacterial and mammalian cell membranes, respectively, were used in the studies. It was observed by SFG that magainin 2 orients relatively parallel to the POPG lipid bilayer surface at low solution concentrations, around 200 nM.

Orientation determination of protein helical secondary structures using linear and nonlinear vibrational spectroscopy.

In this paper, we systematically presented the orientation determination of protein helical secondary structures using vibrational spectroscopic methods, particularly, nonlinear sum frequency generation (SFG) vibrational spectroscopy, along with linear vibrational spectroscopic techniques such as infrared spectroscopy and Raman scattering. SFG amide I signals can be collected using different polarization combinations of the input laser beams and output signal beam to measure the second-order nonlinear optical susceptibility components of the helical amide I modes, which are related to their molecular hyperpolarizability elements through the orientation distribution of these helices. The molecular hyperpolarizability elements of amide I modes of a helix can be calculated based on the infrared transition dipole moment and Raman polarizability tensor of the helix; these quantities are determined by using the bond additivity model to sum over the individual infrared transition dipole moments and Raman polarizability tensors, respectively, of the peptide units (or the amino acid residues).

In situ molecular level studies on membrane related peptides and proteins in real time using sum frequency generation vibrational spectroscopy.

Sum frequency generation (SFG) vibrational spectroscopy has been demonstrated to be a powerful technique to study the molecular structures of surfaces and interfaces in different chemical environments. This review summarizes recent SFG studies on hybrid bilayer membranes and substrate-supported lipid monolayers and bilayers, the interaction between peptides/proteins and lipid monolayers/bilayers, and bilayer perturbation induced by peptides/proteins. To demonstrate the ability of SFG to determine the orientations of various secondary structures, studies on the interactions between different peptides/proteins (melittin, G proteins, alamethicin, and tachyplesin I) and lipid bilayers are discussed.

Effect of conformation and drop properties on surface-enhanced Raman spectroscopy of dried biopolymer drops.

Biofluids are complex solutions consisting of small ions and large biopolymers such as DNA, proteins, or proteoglycans. Biopolymers affect fluid properties but their effect on drop deposition has not been examined. Hyaluronic acid (HA), an important component in synovial fluid, was chosen as a model biopolymer, and examined using surface-enhanced Raman spectroscopy (SERS).