Biomedical subjects

Rob B M Koehorst

Publications and source records attributed to Rob B M Koehorst.

8 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

Spectroscopy and photophysics of self-organized zinc porphyrin nanolayers. 1. Optical spectroscopy of excitonic interactions involving the soret band.

The photophysical properties of excited singlet states of zinc tetra-(p-octylphenyl)-porphyrin in 5-25-nm-thick films spin-coated onto quartz slides have been investigated by optical spectroscopy. Analysis of the polarized absorption spectra using a dipole-dipole exciton model with two mutually perpendicular transition dipole moments per molecule shows that the films are built from linear aggregates, i.e., stacks with a slipped-deck-of-cards configuration. The molecular planes of the porphyrins in the stacks are found to be perpendicularly oriented with respect to the substrate plane. Assuming a value of 2-3 for the dielectric constant of the film, from the excitonic shift, an angle of 44 degrees +/- 3 degrees and an interplanar distance of 0.35-0.36 nm between adjacent porphyrins are calculated, close to the ground-state geometry in solution. The ordering in these films was further investigated by the effects of various solvents and temperature annealing. Spin-coating from toluene as a solvent results in highly ordered films, and annealing of these films has little effect on their absorption spectra. However, spin-coating from chloroform or pyridine or exposure of the films to these solvents in their vapor phases changes their ordering presumably due to incorporation of residual solvent molecules. Annealing yields absorption spectra identical to those of films spin-coated from toluene. The absorption spectra are insensitive to atmospheric moisture, in contrast to those of zinc tetraphenylporphyrin films lacking octyl substituents.

Metalloporphyrins↗

PubMed2005

Spectroscopy and photophysics of self-organized zinc porphyrin nanolayers. 2. Transport properties of singlet excitation.

Exciton diffusion has been studied in 5-25-nm-thick films of zinc tetra-(p-octylphenyl)-porphyrin (ZnTOPP) spin-coated onto quartz slides by intentional doping with quenchers using steady-state as well as time-resolved fluorescence spectroscopy. The fluorescence spectra of the films are very similar to those of solutions, indicating emission from localized exciton states. From the dependence of the fluorescence quenching on the quencher concentration and fluorescence lifetime measurements, the exciton diffusion can be concluded to be quasi-one-dimensional with an exciton diffusion length of 9 +/- 3 nm and an intrastack energy-transfer rate constant of 10(11)-10(12) s(-1). From fluorescence anisotropy decay measurements, we conclude that neighboring stacks aggregate in a herringbone structure, forming ordered domains that are randomly oriented in the substrate plane. These measurements indicate an interstack energy-transfer rate constant of (7 +/- 2) x 10(10) s(-1).

Metalloporphyrins↗

PubMed2005

Membrane-bound conformation of M13 major coat protein: a structure validation through FRET-derived constraints.

M13 major coat protein, a 50-amino-acid-long protein, was incorporated into DOPC/DOPG (80/20 molar ratio) unilamellar vesicles. Over 60% of all amino acid residues was replaced with cysteine residues, and the single cysteine mutants were labeled with the fluorescent label I-AEDANS. The coat protein has a single tryptophan residue that is used as a donor in fluorescence (or Förster) resonance energy transfer (FRET) experiments, using AEDANS-labeled cysteines as acceptors. Based on FRET-derived constraints, a straight alpha-helix is proposed as the membrane-bound conformation of the coat protein. Different models were tested to represent the molecular conformations of the donor and acceptor moieties. The best model was used to make a quantitative comparison of the FRET data to the structures of M13 coat protein and related coat proteins in the Protein Data Bank. This shows that the membrane-bound conformation of the coat protein is similar to the structure of the coat protein in the bacteriophage that was obtained from x-ray diffraction. Coat protein embedded in stacked, oriented bilayers and in micelles turns out to be strongly affected by the environmental stress of these membrane-mimicking environments. Our findings emphasize the need to study membrane proteins in a suitable environment, such as in fully hydrated unilamellar vesicles. Although larger proteins than M13 major coat protein may be able to handle environmental stress in a different way, any membrane protein with water exposed parts in the C or N termini and hydrophilic loop regions should be treated with care.

Capsid Proteins↗

PubMed2004

Lipid bilayer topology of the transmembrane alpha-helix of M13 Major coat protein and bilayer polarity profile by site-directed fluorescence spectroscopy.

This article presents a new formalism to perform a quantitative fluorescence analysis using the Stokes shift of AEDANS-labeled cysteine mutants of M13 major coat protein incorporated in lipid bilayers. This site-directed fluorescence spectroscopy approach enables us to obtain the topology of the bilayer-embedded transmembrane alpha-helix from the orientation and tilt angles, and relative bilayer location. Both in pure dioleoylphosphatidylcholine and dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (4:1 mol/mol) bilayers, which have a similar bilayer thickness, the tilt angle of the transmembrane helix of the coat protein turns out to be 23 degrees +/- 4. Upon decreasing the hydrophobic thickness on going from dieicosenoylphosphatidylcholine to dimyristoylphosphatidylcholine, the tilt angle and orientation angle of the transmembrane alpha-helix change. The protein responds to an increase of hydrophobic stress by increasing the tilt angle so as to keep much of its hydrophobic part inside the bilayer. At the same time, the transmembrane helix rotates at its long axis so as to optimize the hydrophobic and electrostatic interactions of the C-terminal phenylalanines and lysines, respectively. The increase of tilt angle cannot completely keep the hydrophobic protein section within the bilayer, but the C-terminal part remains anchored at the acyl-chain/glycerol backbone interface at the cost of the N-terminal section. In addition, our analysis results in the profile of the dielectric constant of the hydrophobic domain of the bilayer. For all phospholipid bilayers studied the profile has a concave shape, with a value of the dielectric constant of 4.0 in the center of the bilayer. The dielectric constant increases on approaching the headgroup region with a value of 12.4 at the acyl-chain/glycerol backbone interface for the various phosphatidylcholines with different chain lengths. For dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (4:1 mol/mol) bilayers the value of the dielectric constant at the acyl-chain/glycerol backbone interface is 18.6. In conclusion, the consistency of our analysis shows that the applied cysteine-scanning mutagenesis method with AEDANS labeling of a helical transmembrane protein in combination with a quantitative formalism offers a reliable description of the lipid bilayer topology of the protein and bilayer properties. This also indicates that the spacer link between the protein and AEDANS label is long enough to monitor the local polarity of the lipid environment and not that of the amino-acid residues of the protein, and short enough to have the topology of the protein imposing on the fluorescence properties of the AEDANS label.

Capsid Proteins↗

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↗