First Meeting
of the COST working group
« Principles of Membrane Protein Folding and Stability»
COST Chemistry, Action No.: D22, WG D22/0004/02
24.-27. March, 2004
Toulouse, France
Organisers
Alain Milon, Toulouse
(local organiser)
Jörg H. Kleinschmidt, Konstanz
(working group coordinator)
Support by COST Chemistry, University P. Sabatier, and Région Midi-Pyrénées is
gratefully acknowledged.
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Schedule of Meeting Presentations
WG D22/0004/02 “Principles of Membrane Protein Folding and Stability”
Toulouse
Institute of Pharmacology and Structural Biology
205 rte de Narbonne, F. Gallais amphithéâtre
WG Meeting 25th to 27th - MC meeting 27th to 28th
(All presentations will be 40 min talks with subsequent 5 min discussions)
Thu, March 25th, 1st day
13:15 to 13:30
Opening of the meeting and welcome of all participants
JF Sautereau, President of the University P. Sabatier, Toulouse
JH Kleinschmidt, Working Group organizer
13:30 — 14:15
Daniel Otzen, Aalborg University, Denmark
“Estimating membrane protein stability”
14:15 — 15:00
Jan-Willem deGier, Stockholm University, Sweden
“Assembly, overexpression and characterization of membrane
proteins in Escherichia coli”
15:00 —15:45
Fabrice Homblé, Université Libre de Bruxelles, Belgium
“Structure of the mitochondrial porin monitored by CD and FTIR
spectroscopy”
15:45 — 16 :00 Coffee Break
16:00 — 16:45
J. Antoinette Killian, University of Utrecht, Netherlands
“Membrane integration and folding of the K+-channel KcsA”
16:45 — 17:30
Anthony Lee, University of Southampton, United Kingdom
“How interactions with the lipid bilayer shape membrane
proteins.”
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17:30 —18:15
Flemming Poulsen, University of Copenhagen, Denmark
“Transient structures, ensembles of structures and transition
states in the protein folding process of a four helix bundle –
ACBP”
Evening
Free time in Toulouse
Fri, March 26th, 2nd day.
9:05 - 9:50
Erik Goormaghtigh, Université Libre de Bruxelles, Belgium
“Ligand induced modulation of the conformation of the gastric
H+, K+-ATPase monitored by FTIR spectroscopy.“
9:50 —10:35
Derek Marsh, Max-Planck-Institut für biophysikalische Chemie,
Germany
“Membrane protein assembly and folding by spin-label ESR.”
10:35 — 10:50
Coffee Break
10:50 — 11:35
Alain Milon, IPBS, Université P. Sabatier – CNRS, France:
“Efforts towards the refolding of the human -opiate receptor in a
synthetic lipid environment”
11:35 — 12:20
Ismael Mingarro, University of Valencia, Spain:
“Helix-helix packing between transmembrane fragments:
Glycophorin A as a model system”
12:20 — 13:15
Lunch Break
13:15 — 14:00
Tibor Páli, Biological Research Centre, Hungary:
“Membrane protein assembly and stability in photosynthetic
membranes”
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14:00 — 14:45
Teresa Pinheiro, University of Warwick, United Kingdom
“Aggregation and fibrillization of prion proteins in lipid
membranes”
14:45 —15:30
Andreas Plückthun, Universität Zürich, Switzerland
“Protein engineering for membrane proteins”
15:30 — 15:45
Coffee Break
15:45 — 16:30
Jean-Luc Popot, CNRS and Université Paris-7, France
“Amphipols and hemifluorinated surfactants : novel tools for
membrane biology research”
16:30 —17:15
Jörg H. Kleinschmidt, University of Konstanz, Germany
“Folding and Stability of -barrel membrane proteins.”
17:15 —18:00
Ruud Hovius, Ecole polytechnique fédérale de Lausanne,
Switzerland
“Investigating the structure and dynamics of ligand-gated ion
channels”
18:00 — 18:45
Laurence Salomé, Université P. Sabatier – CNRS, France
“Single molecule tracking as a tool to unravel the dynamic
membrane organization of the µ opioid receptor”
18:45 — 19:30
John Findlay, University of Leeds, United Kingdom
“Assembly of G-Protein coupled receptors”
19:30 —20:10
21:00
General discussion
Dinner “Les Caves de la Maréchale”, Toulouse
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Sat., March 27th, 3rd day.
9:00 — 10:45
Separate discussions on possible future collaborative projects
between 2 or more laboratories with common interests as
encouraged by COST.
10:45 — 11:15
Common discussion about current important topics and emerging
collaborations among all participating working group members.
11:15
concluding remarks and scheduling of the next meeting of the
working group (tentatively in Dec. 2004/Jan-2005)
11:30 to 12:30
Lunch
The management committee meetings will start in the afternoon on March 27th
(for management committee members) and end on Sunday 28th 12h00
12:30
Departure for Albi by Bus
13:45 – 14:30
Wellcome in Albi, sale des états, Albi town hall
14:30 — 17:00
Visit of the Albi-cathedral and the Toulouse Lautrec museum in
the old town, with a professional guide
17:30 — 19:30
Management committee meeting;
free in Albi for non MC members
19:30 — 22:30
Dinner in a Restaurant in Albi
22:30
Return to Toulouse by Bus
Sun., March 28th, 4th day.
9:00 — 12:00
Management committee meeting
12:00
Departure to hotel or airport
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Abstracts
(sorted alphabetically by the last names of the authors)
Assembly, overexpression and characterization of membrane
proteins in Escherichia coli
Jan-Willem deGier
Department of Biochemistry and Biophysics , Stockholm University, S-106 91
Stockholm, Sweden, Tel: +46-8-164389 (lab)/162420 (office), Fax +46-8-153679,
Email: degier@dbb.su.se
The first half of my seminar deals with the assembly of membrane proteins in E. coli.
Using a combined in vivo and in vitro approach it has been shown that there are at
least three different membrane protein assembly pathways in E. coli. The second half
of my seminar deals with how Green Fluorescent Protein can be used as a tool in
membrane protein research.
Assembly of G-Protein coupled receptors
John B. C. Findlay
School of Biochemistry and Molecular Biology, University of Leeds,Leeds, LS2 9JT
United Kingdom, Telephone: +44 113 233 3140/3029, Telefax: +44 113 233 3167
E-Mail: J.B.C.Findlay@leeds.ac.uk
G-Protein Coupled Receptors are the biggest and most prolific membrane proteins in
eukaryotic biology. We know considerable amounts about their structure and function
but very little about their folding and assembly mechanisms. This is true for most
helical systems. By dividing up the structure of the protein into a series of domains, it
is possible to show that, in the intact cell, a variety of fragments
are able to
spontaneously anneal into receptors able both the bind their respective ligands and to
undergo activation. This
seem to imply that each of the separate domains is able to insert and fold
6
independently and then to assemble and be processed accurately. Some elements that
might be important in this recognition phenomenon will be discussed.
Ligand induced modulation of the conformation of the gastric H+,K+ATPase monitored by FTIR spectroscopy.
Erik Goormaghtigh
Free University of Brussels, Campus Plaine Bld du Triomphe 2, CP206/2, Brussels,
B1050, Belgium, Telephone: +32-2-650-53-86, Telefax: +32-2-650-53-82, E-Mail:
egoor@ulb.ac.be
As more and more high-resolution structures of proteins become available, the new
challenge is the understanding of these small conformational changes that are
responsible for protein activity. Specialized difference FTIR techniques allow the
recording of side chain modifications or minute secondary structure changes. In the
setup we have designed for the study of the gastric ATPase in a buffer flow system,
the membranes are immobilized on a germanium internal reflection element and the
buffer composition is modulated in the course of the experiment. Spectral differences
arising from only a few residues can be put forward. Drug binding, enzyme
phosphorylation and ligand binding were investigated.
Large domain movements remain usually unsuspected. FTIR spectroscopy also
provides a unique opportunity to record H/D exchange kinetics at the level of the
amide proton. This approach is extremely sensitive to tertiary structure changes and
yields quantitative data on domain/domain interactions. It reveals that very small
conformational changes at the level of the secondary structure can result in major
rearrangements of the tertiary structure.
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Structure of the mitochondrial porin monitored by CD and FTIR
spectroscopy
Fabrice Homblé
Dept. of Plant Physiology, Université Libre de Bruxelles, Campus Plaine C.P. 206/2
Boulevard du Triomphe, B-1050 Brussels, Telephone: +32-2-650-5383, Telefax:
+32-2-650-5382, E-Mail: fhomble@ulb.ac.be
A simple protocol was used to purify two isoforms of the voltage-dependent anionselective channel from the outer mitochondrial membrane without loss of channel
activity. The gene encoding one isoform was cloned. Computer analysis suggests the
presence of a short alpha-helix at the N-terminal region and 18 transmembrane betastrands. Computer CD an ATR-FTIR were performed on both detergent-solubilized
proteins and reconstituted proteins to get experimental information about their
secondary structure.
Proton-deuterium exchange was used to probe the dynamics of
the protein. The method used to analyze the data was validated on the OmpF for
which the structure is known at the atomic level.
Investigating the structure and dynamics of ligand-gated ion
channels
Ruud Hovius
Laboratory of Physical Chemistry of Polymers and Membranes, Institute of Chemical
Sciences and Engineering, Swiss Federal Institute of Technology, CH-1015
Lausanne, Switzerland, Telephone, +41 21 693 3134, Telefax +41 21 693 6190, EMail: ruud.hovius@epfl.ch
Ligand-gated ion channels (LGICs) are involved in the rapid signal transduction
between neurons and other cells like myocytes. Upon the binding of an agonist to
LGICs an intrinsic transmembrane channel opens allowing the flow of ions over the
membrane according to their electrochemical gradient. Even in the prolonged
presence of agonist the channel closes; a process called desensitisation. The structure
of the LGIC proteins has to change to allow channel gating. The ligand binding site
has been shown to be located at about 4 nm above the membrane surface whereas the
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actual channel gate is present in the middle of the membrane; i.e. agonist binding
provokes a long range structural change.
Here, we present our recent results obtained on the ionotropic receptors for serotonin
(5HT3R) and acetylcholine (nAchR).
• The overall secondary structure of purified 5HT3R and nAchR was studied by
infrared micro-spectroscopy using only several nanograms of protein. H/D-exchange
experiments yielded insight in the structure of the water accessible parts of these
proteins.
• A new labeling method exploiting the affinity of chelated metal-ions for oligohistidine sequences allowed site-specific introduction of fluorophores into the 5HT3R
within living cells. The topology and location of oligo-histidine sequences at inserted
at different positions into the receptor proteins was determined.
• Ligands bound to the 5HT3R sense acidic microenvironment, caused by single
glutamate residue identified by combining a pH-sensitive fluorescently labelled ligand
and mutagenesis.
• The structural dynamics of LGICs is being pursued by combining single channel
patch clamp and single molecule fluorescence measurements. For this approach,
fluorescently labelled ligands and mutant receptor proteins are being constructed.
Moreover, to facilitate combined fluorescence and electric measurements, planar
patch clamp on a chip is developed.
Membrane integration and folding of the K+-channel KcsA
J. Antoinette Killian
Department of Biochemistry of Membranes, Centre for Biomembranes and Lipid
Enzymology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands,
Telephone: +31 30-2533442, Telefax: +31 30-2522478,
E-Mail: j.a.killian@chem.uu.nl
KcsA is a relatively simple bacterial K+-channel that has many of the structural and
functional characteristics of larger K+-channels, including its assembly in tetramers as
9
functional conducting units in membranes. We investigated the role of protein-lipid
interactions on insertion, assembly and stability of KcsA in membranes. The results
show (i) that the protein can insert and assemble into tetramers in pure lipid bilayer,
without the help of other membrane proteins, (ii) that specific properties of lipids are
required for efficient insertion and assembly, (iii) that the protein has a special
preference for anionic lipids, and (iv) that the membrane lateral pressure profile plays
an important role in determining the stability of the tetrameric complex.
Folding and Stability of -barrel membrane proteins
Jörg H. Kleinschmidt
Department of Biology, University of Konstanz, Fach M694, Universitätsstraße 10,
D-78464 Konstanz, Germany, Telephone: +49-7531 88 33 60, Telefax: +49-7531 88
31 83, E-Mail: joerg.helmut.kleinschmidt@uni-konstanz.de
The thermodynamic stability (the free energy of folding and membrane insertion) of
integral membrane proteins strongly depends on the environment of the protein. To
compare the stabilities of an integral membrane protein in detergent micelles and in
lipid bilayers, we have performed equilibrium unfolding studies using the integral
membrane protein FhuA (ferric hydroxamate uptake protein A) as an example. In
contrast to -helical bundle membrane proteins,-barrel membrane proteins like
FhuA have a low average hydrophobicity and are soluble when denatured in 8 M
urea. Unfolding of the -barrels FhuA and OmpA (outer membrane protein A) was
reversible in detergent micelles and also in lipid bilayers. It was therefore possible to
monitor the equilibrium unfolding of FhuA by fluorescence spectroscopy. The free
energies of unfolding of FhuA from lipid bilayers and from LDAO micelles were
determined.
We also studied possible folding pathways of -barrel membrane proteins
using outer membrane protein A (OmpA) of Escherichia coli as an example. The
deletion of the gene of periplasmic Skp impairs the assembly of outer membrane
proteins of bacteria. We investigated, how Skp facilitates the insertion and folding of
completely unfolded OmpA into phospholipid membranes. In refolding experiments,
Skp alone was not sufficient to facilitate membrane insertion and folding of OmpA.
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In addition, lipopolysaccharide (LPS) was required. OmpA remained unfolded when
bound to Skp and LPS in solution. From this complex, OmpA folded spontaneously
into lipid bilayers as determined by electrophoretic mobility measurements,
fluorescence spectroscopy, and circular dichroism spectroscopy. OmpA folded into
lipid bilayers by a concerted mechanism2,3.
[1] Bulieris PV, Behrens S, Holst O, Kleinschmidt JH: Folding and insertion of the
outer membrane protein OmpA is assisted by the chaperone Skp and by
lipopolysaccharide. J. Biol. Chem., 278, 2003, 9092-9099.
[2] Kleinschmidt, JH: Membrane protein folding on the example of outer membrane
protein A of Escherichia coli. Cell Mol Life Sci 60, 2003, 1547-1558.
[3] Kleinschmidt, JH & Tamm, L.K.: Secondary and Tertiary Structure Formation of
the -Barrel Membrane Protein OmpA is Synchronized and Depends on
Membrane thickness. J. Mol. Biol. 324, 2002, 319-330.
How interactions with the lipid bilayer shape membrane proteins
Anthony Lee
Division of biochemistry and Molecular Biology, School of Biological Sciences,
University of Southampton, Southampton, SO16 7PX, United Kingdom, Telephone:
+44(0)23 8059 4331, Telefax: +44(0)23 8059 4459, E-Mail: agl@soton.ac.uk
Membrane proteins must have coevolved with the lipid component of the membrane
to form a functional membrane system. Membrane proteins cannot be considered as
rigid entities within a membrane around which the lipid bilayer distorts, leaving the
structure of the protein unaltered. Rather, changes in the structure of
the lipid
component of the membrane lead to changes in protein structure, with consequent
changes in protein function.
An example of the dependence of protein structure on lipid bilayer structure is
provided by studies of the effects of changing the fatty acyl chain length of a lipid
bilayer. In the potassium channel KcsA Trp residues are found at the ends of the
transmembrane -helices. In bilayers of phosphatidylcholine with fatty acyl chain
lengths between C12 and C24 these Trp residues remain close to the glycerol
backbone region of the bilayer, showing the efficiency of hydrophobic matching
11
between the protein and the lipid bilayer. Measurements of lipid binding constants
for membrane proteins show that binding constants vary little with fatty acyl chain
length, inconsistent with major distortions of the lipid bilayer around the protein to
provide hydrophobic matching. Hydrophobic matching therefore must involve
distortion of the protein structure, this being expected to lead to changes in function.
The activities of two intrinsic membrane proteins, the Ca2+-ATPase of sarcoplasmic
reticulum and diacylglycerol kinase from E. coli, have been shown to change with
changing fatty acyl chain length in bilayers of phosphatidylcholines. The nature of the
distortions on the membrane proteins is not yet clear, but studies with model
transmembrane -helices suggest that packing of helices changes with changing fatty
acyl chain length. The lipid headgroup region is also important in interactions
between lipids and intrinsic membrane proteins.
Trp residues are often found at the ends of transmembrane -helices, suggesting that
they could have a special role, although these Trp residues tend not to be conserved.
Studies with diacylglycerol kinase suggest that Trp residues are redundant when
helices are anchored into the membrane with charged residues. This means that Trp
fluorescence spectroscopy can be used to define the transmembrane region of a
membrane protein. An example is shown with the mechanosensitive channel MscL.
Membrane protein assembly and folding by spin-label ESR
Derek Marsh
Department of Spectroscopy, Max-Planck-Institut für biophysikalische Chemie,
Am Fassberg 11, D-37077 Göttingen, Germany, Telephone: +49-551 201 1285/1263,
Telefax: +49-551 201 1501, E-Mail: idreger@gwdg.de
Because of its optimum spectroscopic time window and inherent sensitivity to
molecular orientation, spin-label ESR is uniquely placed to study membrane structure
and dynamics. Conventional spectra are directly sensitive to lipid chain ordering, e.g.,
of sphingolipids and that induced by cholesterol. Lipid-protein interactions are
resolved directly in the ESR spectra, as are domains of different lipid mobility.
12
Saturation transfer spectroscopy extends the methodology to study rotational diffusion
of transmembrane proteins and hence to investigate protein-protein interactions and
assembly of protein oligomers. Site-directed spin labelling by cysteine mutagenesis
allows determination of 3-D structures of polytopic proteins. Spin-spin interactions
define accessibility of spin-labelled protein sites, lateral diffusion of spin-labelled
lipids and the formation of in-plane lipid domains. The field is both well established
and rapidly developing.
Efforts towards the refolding of the human -opiate receptor in a
synthetic lipid environment
Alain Milon
Institute of Pharmacology and Structural Biology, Université P. Sabatier – CNRS,
205 rte de Narbonne, 31077 Toulouse, France, Telephone: +33-561175423, Telefax:
+33-561175424, E-Mail: milon@ipbs.fr
High resolution NMR is an established method for the three dimensional structure
determination of medium size (50 – 300 aa) soluble proteins. Solid state NMR (MAS
and oriented bilayers) is a rapidly emerging for the characterization of peptide’s
conformation and orientation in membranes, and for the structure determination of the
structure and dynamics of membrane proteins.
However, both method (and
particularly the latter) require large amount of pure, doubly labelled (15N, 13C) protein,
functionally reconstituted into lipid bilayers.
In order to determine the receptor-bound conformation of neuropeptides by NMR
(TRNOE and MAS SSNMR), we have engaged extensive efforts for the large scale
expression and purification of the human -opiate receptor. We are now able to
produce in pichia pastoris, and purify, 10 mg/L of receptor (and to 2H, 13C, 15N label
it).
Our expression/purification strategy had to rely on non functional, urea
denaturated receptor in order to achieve this goal. The challenge is now to refold and
reconstitute this GPCR into lipid bilayers, a goal for which we expect stimulating
discussions and collaborations within our COST d22 working group.
In this talk, I will summarise our expression strategy and show preliminary Circular
Dichroism data of the receptor solubilized in detergents. I will also show how solid
13
state NMR can be used to characterize the structure and dynamics of “easier”
membrane proteins, namely bacteriorhodopsin and OmpA.
References
- Sarramegna V, Talmont F, Demange P, Milon A. 2003. GPCRs heterologous
expression: comparison of the main expression systems from the standpoint of largescale production and purification. Cell. Mol. Life Sci. 60(8):1529-1546.
- Soubias O, Reat V, Saurel O, Milon A. 2004.
15
N T2' relaxation times of
bacteriorhodopsin transmembrane amide nitrogens. Magn Reson Chem 42(2):212217.
Helix-helix packing between transmembrane fragments:
Glycophorin A as a model system
Ismael Mingarro
Department of Biochemistry and Molecular Biology, University of Valencia
Dr. Moliner 50, E-46100 Burjassot, Spain, Telephone: +34-96-386 4636, Telefax:
+34-96-386 4635, E-Mail: Ismael.Mingarro@uv.es
In the work that will be presented in the meeting we have collected a series of studies
devoted to understand the principles that govern the molecular mechanism of folding
and packing of membrane proteins. In particular, we have focussed our attention to
Glycophorin A (GpA), a single span membrane protein, which has been extensively
used as a model system to study helix-helix transmembrane (TM) packing. The
importance of the molecular distance between the critical oligomerization motif and
the polar residues found flanking TM fragments will be discussed. We will also report
on the influence of the length of the TM fragment (hydrophobic mismatch) as well as
the effect of proline residues on the packing of TM alpha helices.
14
Estimating membrane protein stability
Daniel Otzen
Department of Life Sciences, Aalborg University, DK - 9000 Aalborg, Denmark,
Telephone: +45 9635 8491, E-Mail: dao@bio.auc.dk
Compared to water-soluble proteins, little is known of the mechanisms of folding of
membrane proteins. Our group uses two membrane proteins from E. coli as model
folding systems, namely the inner membrane protein disulfide bond formation protein
B (DsbB, an -helical protein with 4 transmembrane segments) and the outer
membrane-bound -domain of the autotransporter AIDA.
DsbB: We modulate the folding of DsbB by varying the molar ratios of the non-ionic
detergent dodecyl maltoside (DM) and the denaturing detergent SDS. The rapid
kinetics of folding and unfolding are interpreted to indicate a folding scheme with a
single unfolding intermediate. The rate constants allow us to estimate the stability of
DsbB to be around 4 kcal/mol in DM. Protein engineering data suggest a gradual
accretion of structure during folding rather than a well-defined nucleus.
AIDA: Autotransporters are multi-domain proteins found in a variety of Gramnegative bacteria. They consist of a short signal sequence, a large (approx. 1000residue) passenger domain and a smaller 440-residue C-terminal membrane-bound domain. The passenger domain is displayed on the cell surface. The -domain is
believed to be a 14-strand -barrel. Autotransporters insert into the outer membrane
by a mechanism that apparently does not involve chaperones, but the detailed
mechanism of insertion of the -domain and extrusion of the passenger domain is
unknown. Tryptic digestion of full-length AIDA in vivo leaves the C-terminal 330residues as a membrane-bound core (AIDA-b2). Although the 110 residues upstream
of this sequence (the 1-domain) are not buried in the membrane, they significantly
stabilize the -domain. When the -domain is expressed as inclusion bodies and
purified in urea, it only refolds spontaneously when it contains the 1-domain as an
N-terminal extension. In the absence of this domain, AIDA-2 only refolds on solid
support. Simple dilution from urea into detergent or buffer in the absence of solid
support leads to a compact, beta-strand rich state. This state takes minutes to hours to
form; while soluble in water, it binds to vesicles and detergents and shows
15
cooperative thermal and chemical unfolding. However, it is protease-sensitive and
does not show the SDS-PAGE band-shift upon heating which is characteristic of
folded beta-barrel proteins. Nevertheless, once AIDA-2 is folded on a solid support
in the presence of non-ionic detergent and subsequently released into solution, it
shows extremely high thermal stability, and even in the presence of SDS only unfolds
at elevated temperatures. We present a simple method to determine the stability in the
absence of SDS, with the caveat that unfolding is an irreversible process.
16
Membrane protein assembly and stability in photosynthetic
membranes
Tibor Páli
Institute of Biophysics, Biological Research Centre, P. O. Box 521, 6701 Szeged
Hungary, Telephone: +36-62 432 232 143, Telefax: +36-62 433 133, E-Mail:
tpali@nucleus.szbk.u-szeged.hu or tpali@gwdg.de
The development of the thylakoid membrane was studied during illumination of darkgrown barley seedlings using biochemical methods, and FTIR and spin label EPR
spectroscopic techniques. Correlated, gross changes in the secondary structure of
membrane proteins, conformation, composition and dynamics of lipid acyl chains,
SDS-PAGE pattern and thermally-induced structural alterations show that greening is
accompanied with the reorganization of membrane protein assemblies and the
protein-lipid interface. The increase in the amount of protein-solvating immobile
lipids, which reaches a maximum at 12 hours, is caused by 40% decrease in the
membranous mean diameter of protein assemblies at constant protein/lipid mass ratio.
Alterations in the SDS-PAGE pattern are most significant between 6-24 hours. The
size of membrane protein assemblies increases ~4.5-fold over the 12-48 hours period.
The thermal stability of thylakoid membrane proteins increases monotonically as
detected by an increasing temperature of partial protein unfolding during greening.
Our data suggest that a structural coupling between major protein and lipid
components develops during greening. These results are discussed in view of recent
data on the structural and functional significance of lipid-protein interactions in
photosynthetic membranes.
17
Aggregation and fibrillization of prion proteins in lipid membranes
Teresa J. T. Pinheiro
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL,
United Kingdom, Telephone: +44 24 7652 8364 or 7652 2882, Telefax: +44 24 7652
3701 or 7652 3568, E-mail: t.pinheiro@warwick.ac.uk
A key molecular event in transmissible spongiform encephalopathies (TSEs), also
known as prion diseases, is the conversion of the benign cellular form of the prion
protein (PrPC) into an infectious scrapie isoform (PrPSc). The molecular details of this
conformational transition and its role in the development of disease are not fully
understood. Several experimental observations suggest that an interaction of PrP with
the membrane surface may play a role in the conversion of PrPC to PrPSc. In addition,
the subcellular site for the formation of PrPSc is unknown. Several lines of
experimental observations implicate the plasma membrane and endocytic organelles
as relevant sites, but it is unclear which provides a more favourable environment for
conversion and whether compartments along the secretory pathway might also be
involved. The majority of PrP is attached to the plasma membrane of cells via a
glycosyl phosphatidyl inositol (GPI) anchor, and like other GPI-anchored proteins is
segregated into cholesterol and sphingomyelin-rich domains, also known as lipid
rafts. In addition to fully secreted GPI-anchored PrP, anchorless, truncated forms of
PrP are also produced in the ER or released as soluble forms. In this talk, recent
results on studies of the interaction of truncated forms of the prion protein,
PrP(90231), with model lipid membranes will be presented along with early studies
on a membrane-anchored PrP form.
Using structural biology approaches comprising fluorescence methods, Fourier
transform infrared spectroscopy (FTIR) and electron microscopy (EM) we have
characterised the interaction of PrP isoforms of the truncated protein PrP(90231) to
model lipid membranes, both at pH 7 and 5, to model the pH environment of the
plasma membrane and within endosomes, respectively. We have shown that PrP binds
to lipid membranes in a pH-dependent manner and can lead to aggregation and
fibrillization, depending on the starting conformation of PrP and the lipid composition
of the membrane. Rafts appear to provide an environment that favours the -helical
conformation of PrP, whereas outside rafts interactions with negatively charged lipids
18
induce -sheet structure and lead to amorphous aggregation. However, if PrP is
primed with -sheet structure (-PrP form), this isoform has a higher affinity to lipid
membranes. Binding of -PrP to negatively charged lipid membranes results in
extensive amorphous aggregation and binding to rafts leads to fibrillization of PrP.
Vesicle leakage measurements revealed that increase in -sheet structure and
aggregation strongly destabilise the lipid membrane. These results suggest that in vivo
such destabilisation would lead to cell death, pointing to a possible mechanism for the
neurotoxic effect of PrP in brain cells. FTIR structural results revealed that
fibrillization appears to involve an early partial unfolding of PrP at the membrane
surface followed by slow refolding into fibrils via a loosely packed state with high
content of -turns and exposed side chains (Kazlauskaite et al., in preparation).
N. Sanghera, T.J.T. Pinheiro (2002) Binding of prion protein to lipid membranes and implications for
prion conversion. J. Mol. Biol. 315:12411256.
J. Kazlauskaite, N. Sanghera, I. Sylvester, C. Vénien-Bryan, T.J.T. Pinheiro (2003) Structural changes
of the prion protein in lipid membranes leading to aggregation and fibrillization. Biochemistry
42
P. Critchley, J. Kazlauskaite, R. Eason, T.J.T. Pinheiro (2004) Binding of prion proteins to lipid
membranes. Biochem. Biophys. Res. Commun. 313:
Protein engineering for membrane proteins
Andreas Plückthun
Department of Biochemistry, University of Zürich, Winterthurerstr. 190, CH-8057
Zürich, Switzerland, Tel:
(+41-1) 635 5570, FAX (+41-1) 635 5712, E-Mail:
plueckthun@bioc.unizh.ch
To facilitate the structural and biophysical investigation of G-protein coupled
receptors, we have employed protein engineering, of the receptors themselves and of
binding molecules which may stabilize them. We have constructed an optimized
mutant of the kappa opioid receptor (KOR), which is devoid of its ten free cysteines
(1). It was necessary to test different amino acid replacements at various positions,
and we used a structural model and homology with other receptor family members as
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a guide. This mutant binds ligands and couples to the cognate G-proteins in a very
similar fashion to wild-type KOR. This mutant, which now permits the insertion of
single cysteines, was designed for use in spectroscopic studies of ligand-induced
receptor conformational changes as well as to simplify folding studies. This protein
can be produced in inclusion bodies in very large amounts. We have devised methods
of solubilizing this protein from the membrane.
To help crystallize membrane (and soluble) proteins, we have developed new
antibody-libraries (2) and new antibody-like molecules (3-6), which greatly surpass
antibodies in stability, expression yield in E. coli and which are free of any cysteines.
They can thus not only be used for extracellular targets but also for intracellular
applications as stable and selective inhibitors in proteomics applications. They are
based on fully synthetic libraries of either ankyrin or leucine rich repeats, which are
naturally involved in tight protein-proteins interactions. A crystal structure
demonstrated the success of the synthetic library design and suggests how the
synthetic molecules also surpass the natural repeat proteins in biophysical properties.
Very rapid enrichment with ribosome display against a variety of protein targets was
observed, presumably due to their efficient folding, and functional inhibitors in the
low nanomolar range have been obtained. A crystal structure of the complex against
one protein was obtained.
1.
D. Ott, R. Frischknecht and A. Plückthun, Construction and characterization of a kappa opioid
receptor devoid of all free cysteines. Protein Engineering, Design and Selection 17, 37-48
(2004).
2.
D. Röthlisberger, K. M. Pos and A. Plückthun, An Antibody Library for Stabilizing and
Crystallizing Membrane Proteins - Selecting Binders to the Citrate Carrier CitS. FEBS Lett. in
press, (2004).
3.
H. K. Binz, P. Amstutz, A. Kohl, M. T. Stumpp, C. Briand, P. Forrer, M. G. Grütter and A.
Plückthun, High-Affinity Binders Selected from Designed Ankyrin Repeat Protein Libraries.
Nature Biotechnol. in press, (2004).
4.
P. Forrer, H. K. Binz, M. T. Stumpp and A. Plückthun, Consensus Design of Repeat Proteins.
ChemBioChem in press, (2004).
5.
P. Forrer, M. T. Stumpp, H. K. Binz and A. Plückthun, A novel strategy to design binding
molecules harnessing the modular nature of repeat proteins. FEBS Lett. 539, 2-6 (2003).
6.
M. T. Stumpp, P. Forrer, H. K. Binz and A. Plückthun, Designing Repeat Proteins: Modular
Leucine-rich Repeat Protein Libraries Based on the Mammalian Ribonuclease Inhibitor
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Family. J. Mol. Biol. 332, 471-487 (2003).
7.
H. K. Binz, M. T. Stumpp, P. Forrer, P. Amstutz and A. Plückthun, Designing Repeat Proteins:
Well-expressed, Soluble and Stable Proteins from Combinatorial Libraries of Consensus
Ankyrin Repeat Proteins. J. Mol. Biol. 332, 489-503 (2003).
Amphipols and hemifluorinated surfactants : novel tools for
membrane biology research
Jean-Luc Popot
Laboratoire de Physico-Chimie Moléculaire des Membranes Biologiques, UMR 7099,
CNRS and Université Paris-7, Institut de Biologie Physico-Chimique, CNRS IFR 550, 13,
rue Pierre-et-Marie Curie, F-75005 Paris, France, Telephone: +33 1.58.41.50.14 and
04 (office), Telefax: +33 1.58.41.50.24, E-Mail: jean-luc.popot@ibpc.fr
Membrane proteins classically are handled in aqueous solutions as complexes with
detergents. The dissociating character of detergents, combined with the need to maintain
an excess thereof, frequently results in more or less rapid inactivation of the protein under
study. Over the past decade, we have endeavored to develop two novel families of
surfactants, dubbed 'amphipols' (aps) and 'hemifluorinated surfactants' (hfss), respectively.
Aps are amphiphilic polymers that bind to the transmembrane surface of the protein in a
non-covalent but, in the absence of a competing surfactant, quasi-irreversible manner.
Membrane proteins complexed by aps are in their native state, stable, and they remain
water-soluble in the absence of detergent or free aps. The molecular structure of hfss
resembles that of classical detergents, but their hydrophobic moiety comprises a
perfluorinated section. Because of the poor miscibility of hydrocarbons and
perfluorocarbons, hfss behave as extremely mild detergents. An update will be presented
of the current knowledge about these two classes of compounds and their putative uses for
membrane protein renaturation and/or reconstitution will be discussed.
References
• Tribet, C., Audebert, R., & Popot, J.-L. (1996) Amphipols : polymers that keep membrane
proteins soluble in aqueous solutions. Proc. Natl. Acad. Sci. USA 93: 15047-15050.
• Gohon Y., & Popot, J.-L. (2003) Membrane protein-surfactant complexes. Curr. Opin. Colloid
Interface Sci 8:15-22.
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• Popot, J.-L., Berry, E. A., Charvolin, D., Creuzenet, C., Ebel, C., Engelman, D. M., Flötenmeyer,
M., Giusti, F., Gohon, Y., Hervé, P., Hong, Q., Lakey, J. H., Leonard, K., Shuman, H. A.,
Timmins, P., Warschawski, D. E., Zito, F., Zoonens, M., Pucci, B. & Tribet, C. (2003). Amphipols
: polymeric surfactants for membrane biology research. Cell. Mol. Life Sci. 60:1559-1574.
• Breyton, C., Chabaud, E., Chaudier, Y., Pucci, B. & Popot, J.-L. (2004). Hemifluorinated
surfactants: a non-dissociating environment for handling membrane proteins in aqueous solutions?
FEBS Lett., in the press.
Transient structures, ensembles of structures and transition states in
the protein folding process of a four helix bundle - ACBP
Flemming M. Poulsen
SBiN- Lab, Institute of Molecular Biology, University of Copenhagen, Institute of
Molecular Biology, Oster Farimagsgade 2A, DK-1353 Copenhagen, Denmark,
Telephone: +45 353 22077, Telefax: +45 353 22075, E-Mail: fmp@apk.molbio.ku.dk
We have been using NMR spectroscopy and mutation analysis to study both the acid
and the guanidine hydrochloride unfolded states of yeast and bovine ACBP. The
paper will describe the results of our analysis of long and short range interactions in
the unfolded states as obtained by chemical shift, residual dipolar coupling, relaxation
and spin label induced relaxation for bovine ACBP.
The results of the structural characterization in the unfolded state support the results
of a phi value analysis of yeast ACBP (Kragelund et al. 1999), which showed that
residues in the C- terminal and N- terminal part of the ACBP molecule get together to
form a transition state of folding.
A phi-value analysis of the homologous yeast ACBP shows that despite of the many
residues in the transition state are conserved in cow and yeast ACBP, the apparent
transition state in yeast involves less residues than the transition state of bovine
ACBP.
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Single molecule tracking as a tool to unravel the dynamic membrane
organization of the µ opioid receptor
Laurence Salomé
IPBS – CNRS, 205, route de Narbonne, 31077 TOULOUSE cedex, France, Tel : (33)
05 61 17 59 39, Fax : (33) 05 61 17 59 94, E-mail: laurence.salome@ipbs.fr
Monitoring of the movements of membrane proteins (or lipids) by single
particle tracking enables to get reliable insights into the complex dynamic
organisation of the plasma membrane constituents. We investigated by this technique
the diffusional behavior of the human µ opioid receptor (a G protein coupled
receptor) at the surface of living cells. The trajectories of the receptors revealed a
diffusion mode combining a short term confined diffusion with a long term random
walk. A detailed statistical analysis shows that the receptors have a diffusion confined
to a domain which itself diffuses, the confinement being due to long-range attractive
interprotein interactions. The existing models of the dynamic organisation of the cell
membrane cannot explain our results. We propose a theoretical Brownian model of
interacting proteins that is consistent with the experimental observations and accounts
for the variations found with the domain size of the short term and long term diffusion
coefficients. The changes in the dynamic membrane organization of the µ opioid
receptors accompagnying the binding of peptide agonist and antagonist will be
discussed.
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