Biomedical subjects

Petr V Nazarov

Publications and source records attributed to Petr V Nazarov.

5 recordsLinked to original sources

PubMed2006

FRET study of membrane proteins: determination of the tilt and orientation of the N-terminal domain of M13 major coat protein.

A formalism for membrane protein structure determination was developed. This method is based on steady-state FRET data and information about the position of the fluorescence maxima on site-directed fluorescent labeled proteins in combination with global data analysis utilizing simulation-based fitting. The methodology was applied to determine the structural properties of the N-terminal domain of the major coat protein from bacteriophage M13 reconstituted into unilamellar DOPC/DOPG (4:1 mol/mol) vesicles. For our purpose, the cysteine mutants A7C, A9C, N12C, S13C, Q15C, A16C, S17C, and A18C in the N-terminal domain of this protein were produced and specifically labeled with the fluorescence probe AEDANS. The energy transfer data from the natural Trp-26 to AEDANS were analyzed assuming a two-helix protein model. Furthermore, the polarity Stokes shift of the AEDANS fluorescence maxima is taken into account. As a result the orientation and tilt of the N-terminal protein domain with respect to the bilayer interface were obtained, showing for the first time, to our knowledge, an overall alpha-helical protein conformation from amino acid residues 12-46, close to the protein conformation in the intact phage.

Bacteriophage M13↗

PubMed2006

FRET study of membrane proteins: simulation-based fitting for analysis of membrane protein embedment and association.

A new formalism for the simultaneous determination of the membrane embedment and aggregation of membrane proteins is developed. This method is based on steady-state Förster (or fluorescence) resonance energy transfer (FRET) experiments on site-directed fluorescence labeled proteins in combination with global data analysis utilizing simulation-based fitting. The simulation of FRET was validated by a comparison with a known analytical solution for energy transfer in idealized membrane systems. The applicability of the simulation-based fitting approach was verified on simulated FRET data and then applied to determine the structural properties of the well-known major coat protein from bacteriophage M13 reconstituted into unilamellar DOPC/DOPG (4:1 mol/mol) vesicles. For our purpose, the cysteine mutants Y24C, G38C, and T46C of this protein were produced and specifically labeled with the fluorescence label AEDANS. The energy transfer data from the natural tryptophan at position 26, which is used as a donor, to AEDANS were analyzed assuming a helix model for the transmembrane domain of the protein. As a result of the FRET data analysis, the topology and bilayer embedment of this domain were quantitatively characterized. The resulting tilt of the transmembrane helix of the protein is 18 +/- 2 degrees. The tryptophan is located at a distance of 8.5 +/- 0.5 A from the membrane center. No specific aggregation of the protein was found. The methodology developed here is not limited to M13 major coat protein and can be used in principle to study the bilayer embedment of any small protein with a single transmembrane domain.

Amino Acids↗

PubMed2005

Membrane-bound peptides mimicking transmembrane Vph1p helix 7 of yeast V-ATPase: a spectroscopic and polarity mismatch study.

The V-ATPases are a family of ATP-dependent proton pumps, involved in a variety of cellular processes, including bone breakdown. V-ATPase enzymes that are too active in the latter process can result in osteoporosis, and inhibitors of the enzyme could be used to treat this disease. As a first step in studying the structure and function of the membrane-embedded interface at which proton translocation takes place, and its role in V-ATPase inhibition, synthetic peptides P1 and P2 consisting of 25 amino acid residues are presented here that mimic Vph1p helix 7 of yeast V-ATPase. A single mutation R10A between peptide P1 and P2 makes it possible to focus on the role of the essential arginine residue R735 in proton translocation. In the present work, we use a novel combination of spectroscopic techniques, such as CD spectroscopy, tryptophan emission spectra, acrylamide quenching and parallax analysis, and polarity mismatch modeling to characterize the peptides P1 and P2 in lipid bilayer systems. Based on both the spectroscopic experiments and the polarity mismatch modeling, P1 and P2 adopt a similar transmembrane conformation, with a mainly alpha-helical structure in the central part, placing the tryptophan residue at position 12 at a location 4+/-2 A from the centre of the lipid bilayer. Furthermore, the arginine at position 10 in P1 does not have an effect on the bilayer topology of the peptide, showing that the long, flexible side chain of this residue is able to snorkel towards the lipid headgroup region. This large flexibility of R735 might be important for its function in proton translocation in the V-ATPase enzyme.

Acrylamide↗

PubMed2005

Self-association of transmembrane alpha-helices in model membranes: importance of helix orientation and role of hydrophobic mismatch.

Interactions between transmembrane helices play a key role in almost all cellular processes involving membrane proteins. We have investigated helix-helix interactions in lipid bilayers with synthetic tryptophan-flanked peptides that mimic the membrane spanning parts of membrane proteins. The peptides were functionalized with pyrene to allow the self-association of the helices to be monitored by pyrene fluorescence and Trp-pyrene fluorescence resonance energy transfer (FRET). Specific labeling of peptides at either their N or C terminus has shown that helix-helix association occurs almost exclusively between antiparallel helices. Furthermore, computer modeling suggested that antiparallel association arises primarily from the electrostatic interactions between alpha-helix backbone atoms. We propose that such interactions may provide a force for the preferentially antiparallel association of helices in polytopic membrane proteins. Helix-helix association was also found to depend on the lipid environment. In bilayers of dioleoylphosphatidylcholine, in which the hydrophobic length of the peptides approximately matched the bilayer thickness, association between the helices was found to require peptide/lipid ratios exceeding 1/25. Self-association of the helices was promoted by either increasing or decreasing the bilayer thickness, and by adding cholesterol. These results indicate that helix-helix association in membrane proteins can be promoted by unfavorable protein-lipid interactions.

Computer Simulation↗

PubMedDate not supplied

Artificial neural network modification of simulation-based fitting: application to a protein-lipid system.

Simulation-based fitting has been applied to data analysis and parameter determination of complex experimental systems in many areas of chemistry and biophysics. However, this method is limited because of the time costs of the calculations. In this paper it is proposed to approximate and substitute a simulation model by an artificial neural network during the fitting procedure. Such a substitution significantly speeds up the parameter determination. This approach is tested on a model of fluorescence resonance energy transfer (FRET) within a system of site-directed fluorescence labeled M13 major coat protein mutants incorporated into a lipid bilayer. It is demonstrated that in our case the application of a trained artificial neural network for the substitution of the simulation model results in a significant gain in computing time by a factor of 5 x 10(4). Moreover, an artificial neural network produces a smooth approximation of the noisy results of a stochastic simulation.

Algorithms↗