Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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Exploring the Structure of the 100 Amino-Acid Residue Long N-Terminus of the Plant Antenna Protein CP29
Shabestari, M.H. ; Wolfs, C.J.A.M. ; Spruijt, R.B. ; Amerongen, H. van; Huber, M. - \ 2014
Biophysical Journal 106 (2014)6. - ISSN 0006-3495 - p. 1349 - 1358.
light-harvesting complex - electron-paramagnetic-resonance - comprehensive software package - labeled side-chains - photosystem-ii - distance measurements - conformational-changes - structure prediction - energy-transfer - t4 lysozyme
The structure of the unusually long (~100 amino-acid residues) N-terminal domain of the light-harvesting protein CP29 of plants is not defined in the crystal structure of this membrane protein. We studied the N-terminus using two electron paramagnetic resonance (EPR) approaches: the rotational diffusion of spin labels at 55 residues with continuous-wave EPR, and three sets of distances with a pulsed EPR method. The N-terminus is relatively structured. Five regions that differ considerably in their dynamics are identified. Two regions have low rotational diffusion, one of which shows a-helical character suggesting contact with the protein surface. This immobile part is flanked by two highly dynamic, unstructured regions (loops) that cover residues 10-22 and 82-91. These loops may be important for the interaction with other light-harvesting proteins. The region around residue 4 also has low rotational diffusion, presumably because it attaches noncovalently to the protein. This section is close to a phosphorylation site (Thr-6) in related proteins, such as those encoded by the Lhcb4.2 gene. Phosphorylation might influence the interaction with other antenna complexes, thereby regulating the supramolecular organization in the thylakoid membrane.
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
Site-directed spin labeling study of the light-harvesting complex CP29
Kavalenka, A.A. ; Spruijt, R.B. ; Wolfs, C.J.A.M. ; Strancar, J. ; Croce, R. ; Hemminga, M.A. ; Amerongen, H. van - \ 2009
Biophysical Journal 96 (2009)9. - ISSN 0006-3495 - p. 3620 - 3628.
chlorophyll-a/b protein - photosystem-ii subunit - plant antenna protein - n-terminal domain - energy-transfer - conformational-changes - biosystem complexity - membrane-proteins - escherichia-coli - binding protein
The topology of the long N-terminal domain (100 amino-acid residues) of the photosynthetic Lhc CP29 was studied using electron spin resonance. Wild-type protein containing a single cysteine at position 108 and nine single-cysteine mutants were produced, allowing to label different parts of the domain with a nitroxide spin label. In all cases, the apoproteins were either solubilized in detergent or they were reconstituted with their native pigments (holoproteins) in vitro. The spin-label electron spin resonance spectra were analyzed in terms of a multicomponent spectral simulation approach, based on hybrid evolutionary optimization and solution condensation. These results permit to trace the structural organization of the long N-terminal domain of CP29. Amino-acid residues 97 and 108 are located in the transmembrane pigment-containing protein body of the protein. Positions 65, 81, and 90 are located in a flexible loop that is proposed to extend out of the protein from the stromal surface. This loop also contains a phosphorylation site at Thr81, suggesting that the flexibility of this loop might play a role in the regulatory mechanisms of the light-harvesting process. Positions 4, 33, 40, and 56 are found to be located in a relatively rigid environment, close to the transmembrane protein body. On the other hand, position 15 is located in a flexible region, relatively far away from the transmembrane domain
Solubilization of V-ATPase transmembrane peptides by amphipol A8-35
Duarte, A.M. ; Wolfs, C.J.A.M. ; Koehorst, R.B.M. ; Popot, J.L. ; Hemminga, M.A. - \ 2008
Journal of Peptide Science 14 (2008)4. - ISSN 1075-2617 - p. 389 - 393.
proton translocation channel - integral membrane-proteins - amphipathic polymers - conformation - mimicking - segment - a8-35
Two transmembrane peptides encompassing the seventh transmembrane section of subunit a from V-ATPase from Saccharomyces cerevisiae were studied as complexes with APols A8-35 by CD and fluorescence spectroscopy, with the goal to use APols to provide a membrane-mimicking environment for the peptides. CD spectroscopy was used to obtain the overall secondary structure of the peptides, whereas fluorescence spectroscopy provided information about the local environment of their tryptophan residues. The fluorescence results indicate that both peptides are trapped by APols and the CD results that they adopt a beta-sheet conformation. This result is in contrast with previous work that showed that the same peptides are alpha-helical in SDS micelles and organic solvents. These observations are discussed in the context of APol physical-chemical properties and transmembrane peptide structural propensity.
Structure and localization of an essential transmembrane segment of the proton translocation channel of yeast H+-ATPase
Duarte, A.M. ; Wolfs, C.J.A.M. ; Nuland, N.A.J. van; Harrison, M.A. ; Findlay, J.B.C. ; Mierlo, C.P.M. van; Hemminga, M.A. - \ 2007
Biochimica et Biophysica Acta. Biomembranes 1768 (2007)2. - ISSN 0005-2736 - p. 218 - 227.
nuclear-magnetic-resonance - sarcoplasmic-reticulum ca2+-atpase - protein secondary structure - circular-dichroism spectra - sodium dodecyl-sulfate - m13 coat protein - v-atpase - vacuolar (h+)-atpases - membrane-proteins - nmr-spectroscopy
Vacuolar (H+)-ATPase (V-ATPase) is a proton pump present in several compartments of eukaryotic cells to regulate physiological processes. From biochemical studies it is known that the interaction between arginine 735 present in the seventh transmembrane (TM7) segment from subunit a and specific glutamic acid residues in the subunit c assembly plays an essential role in proton translocation. To provide more detailed structural information about this protein domain, a peptide resembling TM7 (denoted peptide MTM7) from Saccharomyces cerevisiae (yeast) V-ATPase was synthesized and dissolved in two membrane-mimicking solvents: DMSO and SDS. For the first time the secondary structure of the putative TM7 segment from subunit a is obtained by the combined use of CD and NMR spectroscopy. SDS micelles reveal an ¿-helical conformation for peptide MTM7 and in DMSO three ¿-helical regions are identified by 2D 1H-NMR. Based on these conformational findings a new structural model is proposed for the putative TM7 in its natural environment. It is composed of 32 amino acid residues that span the membrane in an ¿-helical conformation. It starts at the cytoplasmic side at residue T719 and ends at the luminal side at residue W751. Both the luminal and cytoplasmatic regions of TM7 are stabilized by the neighboring hydrophobic transmembrane segments of subunit a and the subunit c assembly from V-ATPase
Diffusion labeling time dependent correlated D-T2 NMR on water dynamics and permeability in plant tissue
Sibgatullin, T. ; Vergeldt, F.J. ; Wolfs, C.J.A.M. ; As, H. van - \ 2006
Membrane assembly of M13 major coat protein: evidence for a structural adaptation in the hinge region and a titled transmembrane domain
Spruijt, R.B. ; Wolfs, C.J.A.M. ; Hemminga, M.A. - \ 2004
Biochemistry 43 (2004)9. - ISSN 0006-2960 - p. 13972 - 13980.
phospholipid-bilayers - hydrophobic mismatch - cytoplasmic membrane - bacteriophage m13 - escherichia-coli - lipid-bilayer - spectroscopy - residues - fd - environments
New insights into the low-resolution Structure of the hinge region and the transmembrane domain of the membrane-bound major coat protein of the bacteriophage M13 are deduced from a single cysteine-scanning approach using fluorescence spectroscopy. New mutant coat proteins are labeled and reconstituted into phospholipid bilayers with varying headgroup compositions (PC, PE, and PG) and thicknesses (14:1PC, 18:1PC, and 22:1PC). Information about the polarity of the local environment around the labeled sites is deduced from the wavelength of maximum emission using AEDANS attached to the SH groups of the cysteines as a fluorescent probe. It is found that the protein is almost entirely embedded in the membrane, whereas the phospholipid headgroup composition of the membrane hardly affects the overall embedment of the protein in the membrane. From the assessment of a hydrophobic and hydrophilic face of the transmembrane helix, it is concluded that the helix is tilted with respect to the membrane normal. As compared to the thicker 18:1PC and 22:1PC membranes, reconstitution of the protein in the thin 14:1PC membranes results in a loss of helical structure and in the formation of a stretched conformation of the hinge region. It is suggested that the hinge region acts as a flexible spring between the N-terminal amphipathic arm and transmembrane hydrophobic helix. On average, the membrane-bound state of the coat protein can be seen as a gently curved and tilted, "banana-shaped" molecule, which is strongly anchored in the membrane-water interface at the C-terminus. From our experiments, we propose a rather small conformational adaptation of the major coat protein as the most likely reversible mechanism for responding to environmental changes during the bacteriophage disassembly and assembly process.
Protein-lipid interactions of bacteriophage M13 major coat protein
Stopar, D. ; Spruijt, R.B. ; Wolfs, C.J.A.M. ; Hemminga, M.A. - \ 2003
Biochimica et Biophysica Acta. Biomembranes 1611 (2003). - ISSN 0005-2736 - p. 5 - 15.
magnetic-resonance spectroscopy - filamentous bacterial viruses - fd phage penetration - escherichia-coli - membrane-protein - detergent micelles - nmr-spectroscopy - structural characterization - molecular architecture - conformational states
During the past years, remarkable progress has been made in our understanding of the replication cycle of bacteriophage M13 and the molecular details that enable phage proteins to navigate in the complex environment of the host cell. With new developments in molecular membrane biology in combination with spectroscopic techniques, we are now in a position to ask how phages carry out this delicate process on a molecular level, and what sort of protein-lipid and protein-protein interactions are involved. In this review we will focus on the molecular details of the protein-protein and protein-lipid interactions of the major coat protein (gp8) that may play a role during the infection of Escherichia coli by bacteriophage M13. (C) 2003 Elsevier Science B.V. All rights reserved.
Structural characterization of bacteriophage M13 solubilization by amphiphiles
Stopar, D. ; Spruijt, R.B. ; Wolfs, C.J.A.M. ; Hemminga, M.A. - \ 2002
Biochimica et biophysica acta-protein structure and molecular enzymology 1594 (2002)1. - ISSN 0167-4838 - p. 54 - 63.
The structural properties of bacteriophage M13 during disassembly were studied in different membrane model systems, composed of a homologue series of the detergents sodium octyl sulfate, sodium decyl sulfate, and sodium dodecyl sulfate. The structural changes during phage disruption were monitored by spin-labeled electron spin resonance (ESR) and circular dichroism spectroscopy. For the purpose of ESR spectroscopy the major coat protein mutants V31C and G38C were site-directed spin labeled in the intact phage particle. These mutants were selected because the mutated sites are located in the hydrophobic part of the protein, and provide good reporting locations for phage integrity. All amphiphiles studied were capable of phage disruption. However, no significant phage disruption was detected below the critical micelle concentration of the amphiphile used. Based on this finding and the linear dependence of phage disruption by amphiphiles on the phage concentration, it is suggested that the solubilization of the proteins of the phage coat by amphiphiles starts with an attachment to and penetration of amphiphile molecules into the phage particle. The amphiphile concentration in the phage increases in proportion to the amphiphile concentration in the aqueous phase. Incorporation of the amphiphile in the phage particle is accompanied with a change in local mobility of the spin-labeled part of the coat protein and its secondary structure. With increasing the amphiphile concentration in the phage particle, a concentration is reached where the concentration of the amphiphile in the aqueous phase is around its critical micelle concentration. A further increase in amphiphile concentration results in massive phage disruption. Phage disruption by amphiphiles appears to be dependent on the phage coat mutations. It is concluded that phage disruption is dependent on a hydrophobic effect, since phage solubilization could significantly be increased by keeping the hydrophilic part of the amphiphile constant, while increasing its hydrophobic part.
Spontaneous insertion of gene 9 minor coat protein of bacteriophage M13 in model membranes
Houbiers, M.C. ; Spruijt, R.B. ; Demel, R.A. ; Hemminga, M.A. ; Wolfs, C.J.A.M. - \ 2001
Biochimica et Biophysica Acta. Biomembranes 1511 (2001). - ISSN 0005-2736 - p. 309 - 316.
Conformation and orientation of the gene 9 minor coat protein of bacteriophage M13 in phospholipid bilayers
Houbiers, M.C. ; Wolfs, C.J.A.M. ; Spruijt, R.B. ; Bollen, Y.J.M. ; Hemminga, M.A. ; Goormachtigh, E. - \ 2001
Biochimica et Biophysica Acta. Biomembranes 1511 (2001). - ISSN 0005-2736 - p. 224 - 235.
Configurations of the N-terminal amphipathic domain of the membrane-bound M13 major coat protein
Meijer, A.B. ; Spruijt, R.B. ; Wolfs, C.J.A.M. ; Hemminga, M.A. - \ 2001
Biochemistry 40 (2001). - ISSN 0006-2960 - p. 5081 - 5086.
Membrane-anchoring interactions of M13 major coat protein
Meijer, A.B. ; Spruijt, R.B. ; Wolfs, C.J.A.M. ; Hemminga, M.A. - \ 2001
Biochemistry 40 (2001). - ISSN 0006-2960 - p. 8815 - 8820.
Localization and rearrangement modulation of the N-terminal arm of the membrane-bound major coat protein of bacteriophage M13
Spruijt, R.B. ; Meijer, A.B. ; Wolfs, C.J.A.M. ; Hemminga, M.A. - \ 2000
Biochimica et Biophysica Acta. Biomembranes 1509 (2000). - ISSN 0005-2736 - p. 311 - 323.
During infection the major coat protein of the filamentous bacteriophage M13 is in the cytoplasmic membrane of the host Escherichia coli. This study focuses on the configurational properties of the N-terminal part of the coat protein in the membrane-bound state. For this purpose X-Cys substitutions are generated at coat protein positions 3, 7, 9, 10, 11, 12, 13, 14, 15, 17, 19, 21, 22, 23 and 24, covering the N-terminal protein part. All coat protein mutants used are successfully produced in mg quantities by overexpression in E. coli. Mutant coat proteins are labeled and reconstituted into mixed bilayers of phospholipids. Information about the polarity of the local environment around the labeled sites is deduced from the wavelength of maximum emission using AEDANS attached to the SH groups of the cysteines as a fluorescent probe. Additional information is obtained by determining the accessibility of the fluorescence quenchers acrylamide and 5-doxyl stearic acid. By employing uniform coat protein surroundings provided by TFE and SDS, local effects of the backbone of the coat proteins or polarity of the residues could be excluded. Our data suggest that at a lipid to protein ratio around 100, the N-terminal arm of the protein gradually enters the membrane from residue 3 towards residue 19. The hinge region (residues 17-24), connecting the helical parts of the coat protein, is found to be more embedded in the membrane. Substitution of one or more of the membrane-anchoring amino acid residues lysine 8, phenylalanine 11 and leucine 14, results in a rearrangement of the N-terminal protein part into a more extended conformation. The N-terminal arm can also be forced in this conformation by allowing less space per coat protein at the membrane surface by decreasing the lipid to protein ratio. The influence of the phospholipid headgroup composition on the rearrangement of the N-terminal part of the protein is found to be negligible within the range thought to be relevant in vivo. From our experiments we conclude that membrane-anchoring and space-limiting effects are key factors for the structural rearrangement of the N-terminal protein part of the coat protein in the membrane.
Membrane Assembly of the Bacteriophage Pf3 Major Coat Protein
Meijer, A.B. ; Spruijt, R.B. ; Wolfs, C.J.A.M. ; Hemminga, M.A. - \ 2000
Biochemistry 39 (2000)20. - ISSN 0006-2960 - p. 6157 - 6163.
The Pf3 major coat protein of the Pf3 bacteriophage is stored in the inner membrane of the infected cell during the reproductive cycle. The protein consists of 44 amino acids, and contains an acidic amphipathic N-terminal domain, a hydrophobic domain, and a short basic C-terminal domain. The mainly -helical membrane-bound protein traverses the membrane once, leaving the C-terminus in the cytoplasm and the N-terminus in the periplasm. A cysteine-scanning approach was followed to measure which part of the membrane-bound Pf3 protein is inside or outside the membrane. In this approach, the fluorescence probe N-[(iodoacetyl)amino]ethyl-1-sulfonaphthylamine (IAEDANS) was attached to single-cysteine mutants of the Pf3 coat protein. The labeled mutant coat proteins were reconstituted into the phospholipid DOPC/DOPG (80/20 molar ratio) and DOPE/DOPG (80/20 molar ratio) model membranes. We subsequently studied the fluorescence characteristics at the different positions in the protein. We measured the local polarity of the environment of the probe, as well as the accessibility of the probe to the fluorescence quencher acrylamide. The results of this study show a single membrane-spanning protein with both the C- and N-termini remaining close to the surface of the membrane. A nearly identical result was seen previously for the membrane-bound M13 coat protein. On the basis of a comparison between the results from both studies, we suggest an "L-shaped" membrane-bound model for the Pf3 coat protein. DOPE-containing model membranes revealed a higher polarity, and quenching efficiency at the membrane/water interface. Furthermore, from the outside to the inside of the membrane, a steeper polarity gradient was measured at the PE/PG interface as compared to the PC/PG interface. These results suggest a thinner interface for DOPE/DOPG than for DOPC/DOPG membranes.
Disassembly of bacteriophage M13 studied by ESR spectroscopy
Stopar, D. ; Spruijt, R.B. ; Wolfs, C.J.A.M. ; Hemminga, M.A. - \ 1999
In: Spectroscopy in Theory and Practice : Proceedings of the 11th International Symposium of the Slovenian Chemical Society, Bled, April 11-15, 1999. - [S.l.] : [s.n.], 1999
Spruijt, R.B. ; Wolfs, C.J.A.M. ; Hemminga, M.A. - \ 1999
In: Encyclopedia of Molecular Biology / Creighton, T.E., - p. 1425 - 1429.
Characterization of the brome mosaic virus movement protein expressed in E. coli.
Jansen, K.A.J. ; Wolfs, C.J.A.M. ; Lohuis, H. ; Goldbach, R.W. ; Verduin, B.J.M. - \ 1998
Virology 242 (1998). - ISSN 0042-6822 - p. 387 - 394.
Characterization of the gene VII and gene IX minor coat proteins from bacteriophage M13.
Wolfs, C.J.A.M. ; Houbiers, M.C. ; Spruijt, R.B. ; Hemminga, M.A. - \ 1998
In: NATO ASI Series: lipid and protein traffic pathways and molecular mechanisms. J.A.F. op den Kamp (ed.) Springer-Verlag, Berlin, Germany H106 - p. 105 - 114.
Conformational and aggregational properties of the gene 9 minor coat protein of bacteriophage M13 in membrane-mimicking systems.
Houbiers, M.C. ; Spruijt, R.B. ; Wolfs, C.J.A.M. ; Hemminga, M.A. - \ 1998
Biochemistry 38 (1998). - ISSN 0006-2960 - p. 1128 - 1135.
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