Molecular dynamics simulations reveal that AEDANS is an inert fluorescent probe for the study of membrane proteins
Vos, W.L. ; Schor, M. ; Baumgaertner, A. ; Tieleman, D.P. ; Hemminga, M.A. - \ 2010
European Biophysics Journal 39 (2010)2. - ISSN 0175-7571 - p. 229 - 239.
major coat protein - transmembrane alpha-helix - energy-transfer - fret - orientation - conformation - spectroscopy - model - association - bilayers
Computer simulations were carried out of a number of AEDANS-labeled single cysteine mutants of a small reference membrane protein, M13 major coat protein, covering 60% of its primary sequence. M13 major coat protein is a single membrane-spanning, a-helical membrane protein with a relatively large water-exposed region in the N-terminus. In 10-ns molecular dynamics simulations, we analyze the behavior of the AEDANS label and the native tryptophan, which were used as acceptor and donor in previous FRET experiments. The results indicate that AEDANS is a relatively inert environmental probe that can move unhindered through the lipid membrane when attached to a membrane protein
Viruses: incredible nanomachines. New advances with filamentous phages
Hemminga, M.A. ; Vos, W.L. ; Nazarov, P.V. ; Koehorst, R.B.M. ; Wolfs, C.J.A.M. ; Spruijt, R.B. ; Stopar, D. - \ 2010
European Biophysics Journal 39 (2010)4. - ISSN 0175-7571 - p. 541 - 550.
major coat protein - transmembrane alpha-helix - membrane-protein - bacteriophage m13 - nmr-spectroscopy - ff fd - site - dynamics - display - domain
During recent decades, bacteriophages have been at the cutting edge of new developments in molecular biology, biophysics, and, more recently, bionanotechnology. In particular filamentous viruses, for example bacteriophage M13, have a virion architecture that enables precision building of ordered and defect-free two and three-dimensional structures on a nanometre scale. This could not have been possible without detailed knowledge of coat protein structure and dynamics during the virus reproduction cycle. The results of the spectroscopic studies conducted in our group compellingly demonstrate a critical role of membrane embedment of the protein both during infectious entry of the virus into the host cell and during assembly of the new virion in the host membrane. The protein is effectively embedded in the membrane by a strong C-terminal interfacial anchor, which together with a simple tilt mechanism and a subtle structural adjustment of the extreme end of its N terminus provides favourable thermodynamical association of the protein in the lipid bilayer. This basic physicochemical rule cannot be violated and any new bionanotechnology that will emerge from bacteriophage M13 should take this into accou
From "I" to "L" and back again: the odyssey of membrane-bound M13 protein
Vos, W.L. ; Nazarov, P.V. ; Koehorst, R.B.M. ; Spruijt, R.B. ; Hemminga, M.A. - \ 2009
Trends in Biochemical Sciences 34 (2009)5. - ISSN 0968-0004 - p. 249 - 255.
major coat protein - amino-acids - filamentous bacteriophages - nmr-spectroscopy - fd - dynamics - domain - helix - environments - resolution
The major coat protein of the filamentous bacteriophage M13 is a surprising protein because it exists both as a membrane protein and as part of the M13 phage coat during its life cycle. Early studies showed that the phage-bound structure of the coat protein was a continuous I-shaped ¿-helix. However, throughout the years various structural models, both I-shaped and L-shaped, have been proposed for the membrane-bound state of the coat protein. Recently, site-directed labelling approaches have enabled the study of the coat protein under conditions that more closely mimic the in vivo membrane-bound state. Interestingly, the structure that has emerged from this work is I-shaped and similar to the structure in the phage-bound state
Structure of membrane embedded M13 major coat protein is insensitive to hydrophobic stress
Vos, W.L. ; Schor, M. ; Nazarov, P.V. ; Koehorst, R.B.M. ; Spruijt, R.B. ; Hemminga, M.A. - \ 2007
Biophysical Journal 93 (2007)10. - ISSN 0006-3495 - p. 3541 - 3547.
lipid-bilayer - molecular-dynamics - alpha-helices - model - mismatch - conformation - modulation - fret - aggregation - association
The structure of a membrane-embedded -helical reference protein, the M13 major coat protein, is characterized under different conditions of hydrophobic mismatch using fluorescence resonance energy transfer in combination with high-throughput mutagenesis. We show that the structure is similar in both thin (14:1) and thick (20:1) phospholipid bilayers, indicating that the protein does not undergo large structural rearrangements in response to conditions of hydrophobic mismatch. We introduce a "helical fingerprint" analysis, showing that amino acid residues 1¿9 are unstructured in both phospholipid bilayers. Our findings indicate the presence of -helical domains in the transmembrane segment of the protein; however, no evidence is found for a structural adaptation to the degree of hydrophobic mismatch. In light of current literature, and based on our data, we conclude that aggregation (at high protein concentration) and adjustment of the tilt angle and the lipid structure are the dominant responses to conditions of hydrophobic mismatch.
Conformation of a peptide encompassing the proton translocation channel of vacuolar H+-ATPase
Vos, W.L. ; Vermeer, L.S. ; Hemminga, M.A. - \ 2007
Biophysical Journal 92 (2007). - ISSN 0006-3495 - p. 138 - 146.
yeast v-atpase - molecular-dynamics simulations - electron-spin-resonance - coiled-coils - alpha-helix - pi-helix - protein - subunit - domain - receptor
The structural properties of a crucial transmembrane helix for proton translocation in vacuolar ATPase are studied using double site-directed spin-labeling combined with electron spin resonance (ESR) (or electron paramagnetic resonance) and circular dichroism spectroscopy in sodium dodecyl sulfate micelles. For this purpose, we use a synthetic peptide derived from transmembrane helix 7 of subunit a from the yeast Saccharomyces cerevisiae vacuolar proton-translocating ATPase that contains two natural cysteine residues suitable for spin-labeling. The interspin distance is calculated using a second-moment analysis of the methanethiosulfonate spin-label ESR spectra at 150 K. Molecular dynamics simulation is used to study the effect of the side-chain dynamics and backbone dynamics on the interspin distance. Based on the combined results from ESR, circular dichroism, and molecular dynamics simulation we conclude that the peptide forms a dynamic -helix. We discuss this finding in the light of current models for proton translocation. A novel role for a buried charged residue (H729) is proposed.
FRET study of membrane proteins: determination of the tilt and orientation of the N-terminal domain of M13 major coat protein
Nazarov, P.V. ; Koehorst, R.B.M. ; Vos, W.L. ; Apanasovich, V.V. ; Hemminga, M.A. - \ 2007
Biophysical Journal 92 (2007)4. - ISSN 0006-3495 - p. 1296 - 1305.
bacteriophage m13 - transmembrane domain - energy-transfer - fluorescence - dynamics - conformation - spectroscopy - modulation - peptides - micelles
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 -helical protein conformation from amino acid residues 12¿46, close to the protein conformation in the intact phage.
Distance constraints from site-directed spectroscopy as a tool to study membrane protein structure
Vos, W.L. - \ 2007
Wageningen University. Promotor(en): Herbert van Amerongen, co-promotor(en): Marcus Hemminga. - [S.l.] : S.n. - ISBN 9789085046257 - 106
oppervlakte-eiwitten - moleculaire structuur - spectroscopie - surface proteins - molecular conformation - spectroscopy
Membrane proteins are involved in nearly every process in the living cell. Their scientific importance cannot be overstated, and they account for nearly 60% of all prescribed drugs. Despite being an abundant and important class of proteins, high-resolution structural data on membrane proteins are relatively scarce. X-ray diffraction and NMR spectroscopy are routinely applied nowadays for the determination of structures of water-soluble proteins. However, for membrane proteins that require an amphipathic environment, there is not yet a well-defined strategy for obtaining the structure. For this reason, techniques based on site-directed labeling are being developed to study membrane proteins in their natural environment. In this work, we use two techniques based on the dipole-dipole interaction between two labels, electron spin resonance (ESR) and fluorescence (or Förster) resonance energy transfer (FRET) to obtain low-resolution (0.3-3 nm) distance information on the structure of membrane peptides. FRET is used to study the conformation of a reference membrane protein, i.e. M13 major coat protein, in fully hydrated vesicles. The FRET-derived distance constraints are used to refine the set of high-resolution structures that is available in the protein databank. We show that the coat protein adopts an extended conformation that is not very different from the conformation in the phage particle. In a separate part of this work, we use the FRET approach to monitor the conformation of the coat protein under conditions of hydrophobic mismatch. Although it was suggested that transmembrane protein domains can adapt their backbone conformation to different conditions of hydrophobic stress and that M13 coat protein is a flexible protein that can adapt to a multitude of environments, we show that the conformation of the coat protein in fact is similar under different conditions of hydrophobic mismatch. A parallel approach, based on ESR spin labeling, is used to study the conformation of a peptide that is derived from the crucial proton translocating domain of vacuolar ATPase. First we present a method to enhance the analysis for the determination of distances between two spin labels based on matrix-assisted laser desorption/ionization - time of flight mass spectrometry. Secondly, we use the data from the ESR experiments to study the structure of the peptide. Based on the combined results from the ESR experiments, molecular dynamics simulations and circular dichroism studies we conclude that the peptide forms a dynamica-helix when bound to SDS micelles. We discuss these findings in the light of the current models for proton translocation in the vacuolar ATPase.
Decomposition of ESR spectra using MALDI-TOF mass spectrometry
Vos, W.L. ; Vermeer, L.S. ; Wolfs, C. ; Spruijt, R.B. ; Hemminga, M.A. - \ 2006
Analytical Chemistry 78 (2006)15. - ISSN 0003-2700 - p. 5296 - 5301.
electron-paramagnetic-resonance - major coat protein - spin labels - containing peptides - lipid-bilayer - tryptophan - distances - helix - ions
ESR ( or EPR) spectroscopy on spin-labeled site-directed cysteine mutants is ideally suited for structural studies of membrane proteins due to its high sensitivity and its low demands with respect to sample purity and preparation. Many features can be inferred from the spectral line shape of an ESR spectrum, but the analysis of ESR spectra is complicated when multiple sites with different line shapes are present. Here, we present a method to decompose the spectrum of a doubly labeled peptide that is composed of a singly labeled, noninteracting component and a doubly labeled, dipolar-broadened component using a combination of optical and matrix-assisted laser desorption/ ionization-time-of-flight mass spectrometry. The effect on the interspin distance calculation based on the dipolar broadening is quantified and discussed.
FRET study of membrane proteins: simulation-based fitting for analysis of membrane protein embedment and association
Nazarov, P.V. ; Koehorst, R.B.M. ; Vos, W.L. ; Apanasovich, V.V. ; Hemminga, M.A. - \ 2006
Biophysical Journal 91 (2006)2. - ISSN 0006-3495 - p. 454 - 466.
major coat protein - resonance energy-transfer - bacteriophage m13 - detergent micelles - fluorescence - model - spectroscopy - domain - conformation - orientation
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°. The tryptophan is located at a distance of 8.5 ± 0.5 Å 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.
Membrane-bound conformation of M13 major coat protein : a structure validation through FRET-derived constraints
Vos, W.L. ; Koehorst, R.B.M. ; Spruijt, R.B. ; Hemminga, M.A. - \ 2005
Journal of Biological Chemistry 280 (2005)46. - ISSN 0021-9258 - p. 38522 - 38527.
bacteriophage m13 - energy-transfer - lipid-bilayers - spectroscopy - tryptophan - domain - model - modulation - simulation - micelles
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 Forster) 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.