Membrane Protein A from Escherichia Coli
Dissertation
zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften
an der Universität Konstanz
Mathematisch -Naturwissenschaftliche Sektion Fachbereich Biologie
vorgelegt von
Paula Vasilichia Bulieris
Tag der mündlichen Prüfung: 18.12.2007 Referent 1: PD Dr. Jörg H. Kleinschmidt Referent 2: Prof. Dr. Hans-Jürgen Apell
Konstanzer Online-Publikations-System (KOPS)
1 Introduction 1
1.1 The protein folding problem 1
1.2 Integral membrane proteins 2
1.3 Folding and insertion of outer membrane protein A OmpA 4 1.3.1 Structure and function of outer membrane protein A OmpA 4
1.3.2 OmpA folding and insertionin vitro 6
1.3.2.1 Requirements of OmpA folding and insertionin vitro 6 1.3.2.2 The mechanism of OmpA folding and insertion into
preformed lipid bilayers 9
1.3.3 OmpA folding and insertionin vivo 12
1.3.3.1 Requirements of OmpA folding and insertion in vivo 12
1.3.3.2 The periplasmic chaperone Skp 13
1.3.3.3 The periplasmic chaperone SurA 14
1.3.3.4 Lipopolysaccharides 16
1.4 Methods 18
1.4.1 Electrophorectic mobility measurements used to detect the
folding and insertion of OmpA 18
1.4.2 Fluorescence spectroscopy 20
2 The effect of phospholipids composition on the folding kinetics of outer membrane
protein A into model membranes 22
2.1 Introduction 22
2.2 Materials and methods 24
2.2.1 Materials 24
2.2.2 Preparation of small unilamellar vesicles (SUVs) 25
2.2.3 Purification of OmpA 25
2.2.4 Kinetics of tertiary structure formation of OmpA determined
by electrophoresis (KTSE) 25
2.2.5 OmpA refolding into lipid bilayers of various compositions 26 2.2.6 Kinetics densitometric analysis and rates of OmpA folding 26
2.3 Results 27
2.3.1 OmpA folding and insertion into model membranes mimicking
the phospholipids composition of the outer membrane 27 2.3.2 Folding and insertion of OmpA depend on the amount of
PG and PE included into model membranes 29
2.3.3 Efficient OmpA refolding into model membranes containing
DOPG and DOPE requires the presence of DOPC 32
2.4 Discussions 34
3 Folding and insertion of outer membrane protein A OmpA is assisted by the
chaperone Skp and by Lipopolysaccharide 36
3.3.1 Materials 38
3.3.2 Purification of Skp 39
3.3.3 Purification of OmpA and R-LPS 39
3.3.4 Preparation of lipid bilayers 40
3.3.5 Kinetics of OmpA insertion and folding into membranes detected
by electrophoresis 40
3.3.6 Folding monitored by CD spectroscopy 41
3.3.7 Skp and LPS-binding to OmpA monitored by fluorescence spectroscopy 41
3.4 Results 42
3.4.1 Effects of Skp and LPS on the folding of OmpA 42
3.4.2 LPS and Skp binding to denatured OmpA in solution 45 3.4.3 LPS concentration dependence of OmpA folding
into lipid bilayers 47
3.4.4 Skp binds to OmpA in an unfolded form in solution 49
3.5 Discussions 51
3.5.1 A pathway of assisted membrane insertion and folding of OmpA 51 3.5.2 OmpA can fold and insert in parallel folding pathways 53
3.5.3 Interactions of LPS with unfolded OmpA 54
3.5.4 Skp solubilizes unfolded OmpA by forming a positively
charged complex 55
3.5.5 Complex formation between OmpA, Skp and LPS is
thermodynamically controlled 57
3.5.6 Structure formation in OmpA requires the presence of the
phospholipid bilayer 58
3.6 Conclusions 58
4 The chaperone effect of SurA on the folding and insertion of outer membrane protein
A 60
4.1 Abstract 60
4.2 Introduction 60
4.3 Materials and methods 62
4.3.1 Materials 62
4.3.2 Purification of SurA and OmpA 62
4.3.3 Purification of R-LPS 62
4.3.4 Preparation of small unilamellar vesicles (SUV) 62 4.3.5 Kinetics of tertiary structure formation OmpA by electrophoresis (KTSE) 63
4.3.5.1 KTSE method 63
4.3.5.2 OmpA refolding in the presence of SurA 63
4.3.5.3 Refolding of aqueous form of OmpA in the presence of SurA 64 4.3.5.4 OmpA refolding at different SurA / OmpA ratios 64
4.3.5.5 OmpA refolding at different temperatures 65
4.3.5.6 OmpA refolding into lipid bilayers containing different ratios of PC and PG 65 4.3.5.7 OmpA refolding into lipid bilayers of various compositions 66
4.4 Results 67 4.4.1 SurA facilitates membrane insertion and folding of OmpA 67 4.4.2 SurA assists the folding and insertion of the aqueous form of OmpA 70 4.4.3 SurA chaperone effect on OmpA depends on the SurA / OmpA ratio 73 4.4.4 SurA presence modulates the temperature dependence of OmpA folding 74 4.4.5 DOPG included into lipid bilayers stimulates the SurA assisted
folding and insertion of OmpA 77
4.4.6 SurA facilitates the OmpA refolding into lipid bilayers of
various compositions 80
4.4.7 LPS interaction with OmpA during refolding is prevented by the SurA presence 83
4.5 Discussions 85
4.5.1 SurA assists the insertion and folding of OmpA 85
4.5.2 The chaperone effect of SurA on OmpA folding is temperature dependent 86 4.5.3 Lipid bilayers composition modulates the SurA chaperone effect
on OmpA folding 87
4.5.4 SurA assisted folding of OmpA does not require LPS 88
4.6 Conclusions 88
5 Kinetics of association of the transmembraneβ-strands 1,2,3, and 8 in outer
membrane protein A 90
5.1 Abstract 90
5.2 Introduction 90
5.3 Materials and methods 93
5.3.1 Materials 93
5.3.2 Single tryptophan mutants of OmpA 93
5.3.3 Single tryptophan, single cystein mutants of OmpA 94
5.3.4 OmpA purification from the strain BL21DE3 96
5.3.5 MTSL spin labeling of OmpA mutants 96
5.3.6 Preparation of small unilamellar vesicles (SUVs) of DOPC 96
5.3.7 Fluorescence Spectroscopy 97
5.3.8 Folding and membrane insertion detected by SDS-PAGE 97
5.4 Results 98
5.4.1 Mutants generated by site directed mutagenesis 98
5.4.2 Tryptophan fluorescence of unfolded, water collapsed
and refolded OmpA mutants 99
5.4.3 Folding and insertion of OmpA mutants into DOPC bilayers
detected by SDS-PAGE 102
5.4.4 Association of individual β-strands detected by kinetics
of tryptophan fluorescence quenching 103
5.4.5 Association ofβ-strands at 5°C detected by kinetics
of fluorescence quenching 107
5.4.8 Association of β- strands as a function of temperature 111 5.4.9 Intermediate stages in association ofβ-strands detected by
fluorescence quenching at 5, 20, 28 and 40°C 113
5.5 Discussions 114
5.5.1 Association on individualβ-strands in pairs during the
formation of β-barrel domain of OmpA is a concerted process 114 5.5.2 In comparison with the bottom-end, the assembly of β-strands
at the top-end of OmpAβ-barrel domain is markedly faster 115 5.5.3 The association and insertion of individualβ-strands into
the lipid bilayer are two highly concerted processes 116 5.5.4 The formation and insertion of OmpAβ-barrel domain into the
lipid bilayers take place by a concerted mechanism 117
5.6 Conclusions 120
Summary 121
Zusammenfassung 125
List of publications 129
References 130
Acknowledgements 139
BSA bovine serum albumine
CD circular dichroism
CL cardiolipin
Cys cysteine
DOPC 1,2-Dioleoyl-sn-glycero-3-phosphocholine DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine DOPG 1, 2-dioleoyl-sn-glycero-3-phosphoglycerol
DTNB Ellman reagent 5,5’-Dithiobis(2-nitrobenzoic acid) E. coli Escherichia coli
EDTA ethylenediaminetetraacetic acid
Eq. equation
FT-IR Fourier transform spectroscopy
HEPES 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid IMPs integral membrane proteins
IPTG isopropyl 1-thio-β-D-galactopyranoside
kDa kilo Dalton
KTSE kinetics of tertiary structure formation detected by electrophoresis
LB Luria-Bertani
LPS lipopolysaccharide
LUVs large unilamellar vesicles Mcps million counts per second
MTSSL 1-Oxyl-(2, 2, 5, 5-tetramethyl-pyrrolin-3-yl) methyl methanethiosulfonate
NMR nuclear magnetic resonance
OM outer membrane
Omp85 outer membrane protein 85
OmpA outer membrane protein A from Escherichia coli OMPs outer membrane proteins
PC 1,2-Dioleoyl-sn-glycero-3-phosphocholine PE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
PEG polyethylenglycol
SDS-PAGE SDS-polyacrylamide gel electrophoresis
Skp seventeen kDa protein
SurA survival factor A
SUVs small unilamellar vesicles
TCEP tris (2-carboxyethyl) phosphine HCl
TDFQ time-resolved distance determination by tryptophan fluorescence quenching
TM transmembrane
Tris tris(hydroxymethyl)aminomethane
Trp tryptophan
Hiermit erkläre ich, Paula Vasilichia Bulieris, dass alle Experimente von mir durchgeführt wurden, ausser folgenden:
Die Proteinen Skp und SurA wurden von Dr. Susanne Behrens zur Verfügung gestellt.
R-LPS 3900 wurde von Dr. Otto Holst zur Verfügung gestellt.
Die Plasmiden pET1102, pET1115, pET185, pTB001, pTB004, pTB005, pTB008 und pTB003 wurden von Dr. Tanneke den Blaauwen zur Verfügung gestellt.
Paula Vasilichia Bulieris
1 Introduction
1.1 The protein folding problem
Proteins are essential for all biological processes. The specific role of each protein depends on its unique structure. Protein folding is the process by which the linear polypeptide chain synthesized on the ribosomes achieves the three-dimensional structure characterized by the lowest free energy level.
The principles governing the mechanism of protein folding continue to arise numerous questions and represent a major challenge for the modern biology.
The central dogma of protein folding or the principle of protein self-assembly was formulated initially by Anfisen (Anfisen 1973) and confirmed over the years by experimental evidence.
This principle states that the folding process is determined only by the amino acid sequence.
An important key question derived from the central dogma is how do the proteins reach the folded state?
In the case of a small protein with just 100 residues, the systematic search of all possible conformations in order to find the one having the lowest energy was calculated to take 1011 years (Dobson et al. 1998). Yet most proteins fold within a second. This incompatibility of facts is known as the Levinthal paradox (Levinthal 1968) and the initial proposal put forward to solve this paradox was that of specific pathways of folding.
A folding pathway is represented by a sequence of partially folded intermediates. By restricting the protein folding to a specific pathway, the Levinthal paradox is avoided. The theory of folding pathways is supported by experimental evidence and describes well the folding of many proteins.
In contrast, small proteins (specially those with less than 100 residues) fold by a two-state reaction meaning that, during the folding process the protein exists in just two states: either fully unfolded or fully folded (Jackson 1998). Partially folded intermediates are not involved in this kind of folding mechanism.
Various other models of protein folding have emerged over time trying to explain the protein folding process. Efforts are made towards establishing a universal mechanism of protein folding (Dobson and Karplus 1999). Decoding the mechanism of protein folding would lead not only to a deeper understanding of all biological processes but would also provide the necessary tools to predict the protein structure of a given amino-acid sequence.
As a contribution to the study of protein folding, my work did focus on significant aspects of the folding of an integral membrane protein: the outer membrane protein A (OmpA) from Escherichia coli.
1.2 Integral membrane proteins
Accumulating meaningful informations about the various ways adopted by different categories of proteins in order to find their native structure is essential for the progress of protein folding. For understanding how an unfolded polypeptide chain converts rapidly into the packed, cooperative, native structure it is essential to obtain data about the kinetic aspects of the folding process.
The experimental approach adopted for studying the kinetics of folding for a given protein is to monitor the structural changes taking place as the protein folds in a test tube. For this purpose, the pure protein is unfolded completely in a chemical denaturant and the refolding is initiated in specific conditions by strongly and rapidly reducing the concentration of denaturant. Afterwards the changes in the protein structure are monitored at given intervals of time. This procedure enabled the acumulation of a considerable amount of information regarding the folding of many globular water-soluble proteins.
In contrast, the folding of integral membrane proteins (IMPs) was less researched due to a series of difficulties related with the manipulation of this particular category of proteins. The hydrophobic nature of IMPs has been one of the major stumbling blocks for denaturing and refolding integral membrane proteins in vitro, and thus in studying the folding process.
Another difficulty is to manipulate the refolding solvent, in order to mimic the biological membranes and to meet the refolding requirements of the proteins. However, the field has an accumulating body of information (Popot and Engelman 2000; Kleinschmidt 2003).
The integral membrane proteins fall into two different classes that can be distinguished according with their secondary structure:α-helical andβ-barrel proteins (Figure 1.1). The α- helical transmembrane proteins are found in the cytoplasmic membranes. The class includes proteins that form very hydrophobic transmembrane α-helices. Best known is the bacteriorhodopsin, a seven-α-helix bundle membrane protein (Figure 1.1).
The integral membrane proteins with β-barrel structures are located in the outer membranes of bacteria, mitochondria, and chloroplasts. In contrast with α-helices, the β-barrel proteins have
a low hydrophobicity, therefore they can be completely solubilized in concentrated solutions of chemical denaturants (for example urea).
This class covers proteins with a transmembraneβ-sheet secondary structure (Figure 1.1). The β-strands constituting theβ-sheet form barrel structures in which at least eight neighbouring β-strands are connected by hydrogen bonds. The β-barrel proteins with a known crystal structure present always an even number of transmembrane β-strands ranging from 8 to 22 strands (Figure 1.2).
The protein which is the object of the present thesis, the bacterial outer membrane protein A, OmpA of Escherichia coli is well known and studied specially as a model in the folding studies of outer membrane proteins (OMPs). OmpA is constituted from 8 β-strands (Pautsch and Schulz 2000; Arora et al. 2001) (Figure 1.3).
Few other examples of outer membrane proteins (OMPs) from Gram-negative bacteria are:
OmpT with 10 β-strands (Vandeputte-Rutten et al. 2001), OmP1A with 12 β-strands (Snidjer et al.1999), OmpF and PhoE with 16 β-strands (Cowan et al.1995; Cowan et al.1992), maltoporin (LamB) with 18 β-strands (Schirmer et al.1995), FhuA with 22 β-strands (Ferguson et al.1998; Locher et al.1998). Several of the enumerated proteins are presented in Figure 1.2.
Figure 1.1 Examples of the two classes of membrane proteins. Left: anα-helical protein, the bacteriorhodopsin from Halobacterium salinarum (Faham et al. 2005). Right: a β-barrel protein, OpcA the integral membrane adhesin fromNeisseria meningitidis(Prince et al 2002)
Some β-barrel proteins form monomers: OmpA and FhuA. Others form dimers: OmPIA, or trimers: OmpF and PhoE. The following sections of the present chapter contain background information regading the folding and insertion of OmpA.
1.3 Folding and insertion of outer membrane protein A OmpA 1.3.1 Structure and function of outer membrane protein A OmpA
OmpA is expressed by Escherichia coli which belongs to the group of Gram-negative bacteria. This group of bacteria is characterized by the presence of two concentric membranes, which confine between them the periplasmic space.
The inner (cytoplasmic) membrane delimitates the cytoplasm and is constituted from two symmetric leaflets containing the phospholipids: phosphatidylethanolamine (70-80%),
Figure 1.2 Structures of β-barrel membrane proteins Integral membrane proteins with β- barrel structures are known from outer membranes of bacteria, mitochondria, and chloroplasts (Figure taken from Kleinschmidt 2005).
In contrast, the outer membrane which, comes in contact with the external medium is highly asymmetric: the inner leaflet showing the same lipid composition as the cytoplasmic membrane and the outer leaflet contains lipopolysaccharide (LPS). The role of the outer membrane is to protect the Gram-negative bacteria against a harsh environment. OMPs represent a considerable part from the total mass of the outer membrane (Koebnik et al.
2000). The OMPs are related with essential functions such as: maintaining the integrity of the outer membrane, translocation of proteins and signal transduction.
OmpA is one of the major outer membrane proteins of E .coli, being expressed at about 105 copies per cell (Koebnik et al. 2000). Topologically OmpA presents two domains: a N- terminal transmembrane domain of 171 amino acid residues which spans the inner leaflet of the outer membrane and a C-terminal periplasmic domain of 154 amino acid residues which resides in the periplamic space (Figure 1.3).
Figure 1.3 Outer membrane protein A (OmpA) of E.coli Left: Crystal structure of the OmpA fragment consisting of residues 1-171 (Pautsch and Schulz 1998). The protein forms an 8-stranded β-barrel in the outer membrane of E.coli. Right: Topological scheme of OmpA in the outer membrane of E.coli. The 8β-strands span the hydrophobic core of the lipid bilayer and strands 4, 5 and 6 also protrude from the lipid bilayer into the polysaccharide region of the outer leaflet of the bacterial cell wall.
The crystal structure of the transmembrane domain is comprised from an eight-stranded anti- parallel β-barrel as presented in Figure 1.3 (Pautsch and Schulz 2000; Arora et al. 2001).
OmpA has a significant structural role in maintaining the integrity of the outer membrane.
The physiological function of the OmpA protein is that of non-specific diffusion pore (Bond et al. 2002; Zakharian and Reusch 2005; Arora et al. 2000).
1.3.2 OmpA folding and insertion in vitro
1.3.2.1 Requirements of OmpA folding and insertion in vitro
Initial studies on OmpA demonstrate that the protein can be refolded in vitro from a completely denatured (i.e. unfolded) state. Schweizer et al. (Schweizer et al.1978) showed that OmpA can partially refold in presence of LPS and detergent Triton-X-100, after the dilution of denaturants: sodium dodecyl sulfate (SDS) or urea. Dornmair et al. (Dornmair et al.1990) demonstrated that after heat denaturation in SDS micelles, OmpA refolded into micelles of detergent octylglucoside, even in absence of LPS.
Figure 1.4 The shape of the amphiphilic molecules determines the morphology of aggregated structures. A, amphiphiles that have an overall inverted conical shape, such as detergent molecules, form structures such as micelles.B, cylindrical-shaped amphiphiles such as lipid molecules preferentially form bilayer structures.C, lipid molecules with an overall conical shape form structures with a negative curvature, such as the hexagonal phase (taken from van den
These initial studies on OmpA folding and insertion suggest that the amino acid sequence contain the whole information required for the formation of native structure. Thus, the principle of protein self-assembly formulated by Anfisen for soluble proteins is valid for outer membrane proteins as well.
In the studies described above, OmpA was refolded into micelles. The structure of a detergent micelle is presented in the figure 1.4 A. Surrey and Jähing (Surrey and Jähnig 1992) were the first to show that OmpA inserts and folds into phospholipid bilayers in the complete absence of detergents. The structure of a phospholipid bilayer is displayed in the figure 1.4 B.
Both the detergents and the phospholipids belong to the category of amphiphiles (Figure 1.4, A, B and C). An amphiphile presents a hydrophilic part (polar head) and a hydrophobic part (tail) (Figure 1.4, A, B and C) which, is constituted of hydrocarbon chains. With the exception of conical amphiphiles (Figure 1.4, C) the polar head orients toward the solvent, when in contact with a polar solvent like water (Figure 1.4, A and B), while the hydrophobic tail will turn in the opposite direction to avoid the contact with the solvent (Figure 1.4, A and B). The orientation of the amphiphile molecules in contact with solvent has as result a molecular alignment according to well-defined patterns and the formation of pluriform aggregates (Figure 1.4, A, B and C). The morphology of the aggregates is determined by the specifical shape of amphiphile (Figure 1.4, A, B and C), as well as by the nature of the solvent.
The phospholipid bilayers used for the initial studies of OmpA folding (Surrey and Jähnig 1992) were constituted of dimyristoylphosphatidylcholine (diC14:0PC) with hydrocarbon chains of 14 C atoms.
The initial experiments (Surrey and Jähnig 1992; Surrey and Jähnig 1995) have shown that the refolding of OmpA must meet several conditions in order to achieve completion. The conditions are:
(i) The 8 M urea solution used to solubilize the protein has to be strongly diluted upon initiating the refolding by adding the preformed phospholipid bilayers.
(ii) The bilayers have to be sonicated before being used for refolding experiments.
When vesicles prepared by extrusion were used, the folding was not successful (Surrey and Jähnig 1992; Surrey and Jähnig 1995). The sonication process produces small unilamellar vesicles (SUVs), while the extrusion yields large unilamellar vesicles (LUVs) (Figure 1.5). The OmpA insertion into SUVs is facilitated by the high surface curvature that increases the hydrophobic surface exposed to the protein. In contrast the LUVs have a much lower surface curvature which appears to hinder the folding and insertion.
(iii) The lipids must be in liquid-crystalline (lamellar disordered) phase for a complete refolding of OmpA. The insertion and refolding were not complete when the lipids were in gel-phase (lamellar ordered) (Surrey and Jähnig 1992; Surrey and Jähnig 1995). The transition between the two phases: liquid-crystalline phase and gel phase takes place at a specific melting temperature (Tm). Below Tmthe lipids exist as a solid like gel in which the acyl chains constituting the hydrophobic tail are tightly packed. Above the Tm the lipids are in a liquid like crystal phase in which the acyl chains are disordered. FordiC14:0PC the Tmis 23 °C.
(iv) (iv) The pH values influence also the outcome of the refolding experiments. At pH 10 almost 100% of OmpA will refold in comparison with only around 70% at pH 7. This pH effect is most probably a consequence of the increased negative charge of OmpA (pI 5.9) at pH 10, that enhance the solubility of the protein (Surrey and Jähnig 1995).
.
The OmpA refolding studies described above, were followed by the investigations of Kleinschmidt et.al (Kleinschmidt et al.1999b), who have refolded OmpA into a large set of detergents and phospholipids. OmpA was able to refold into 64 different detergents and phospholipids. The main characteristics of the compounds used for OmpA refolding are: a polar head group which do not carry a net charge and a length of the hydrocarbon chains ranging from 7 to 14 carbon atoms.
Figure 1.5 Schematic illustration of the size difference between SUVs and LUVs. Small unilamellar vesicles (SUVs) and large unilamellar vesicles (LUVs) are constituted of lipid bilayers. In comparison with LUVs, the SUVs have a smaller size and a high surface curvature.
Kleinschmidt et.al (Kleinschmidt et al.1999b) have shown that OmpA refolds only if a preformed hydrophobic core of detergent micelles or lipid bilayers is present before the addition of the unfolded protein. In all the OmpA folding experiments described above the folding is not observed in the absence of the micelles or lipid bilayers.
The studies described in this section demonstrate clearly that the minimal requirement for the formation of OmpA native structure is the presence of an amphiphilic supramolecular assembly represented by detergent micelles or lipid bilayers.
For the studies mentioned in this section, OmpA refolding was detected by fluorescence spectroscopy, circular dichroism (CD) spectroscopy and electrophoretic mobility measurements (SDS-PAGE).
Fluorescence spectroscopy was used to monitor the adsorption and insertion of tryptophan residues of OmpA into the hydrophobic core of micelles and lipid bilayers. OmpA has five tryptophans in the transmembrane domain (see Figure 1.3). The fluorescence emission maximum shifts to lower wavelengths and the quantum yield increases when the tryptophans are transferred from a polar into an apolar environment (i.e. when unfolded OmpA in contact with water interacts with the hydrophobic core of the micelles or lipid bilayer).
CD spectroscopy was used to observe the formation of secondary structure and electrophoresis was employed to monitor the development of the tertiary structure.
The mechanism of OmpA folding and insertion into a supramolecular assembly (i.e lipid bilayers) is discussed in the next section (1.3.2.2).
1.3.2.2 The mechanism of OmpA folding and insertion into preformed lipid bilayers
Kleinschmidt and Tamm (Kleinschmidt and Tamm 1996; Kleinschmidt and Tamm 2002) have investigated in detail the mechanism of OmpA folding and insertion into preformed lipid bilayers. The process of OmpA folding was kinetically characterized and the corresponding rate constants were calculated. Fluorescence spectroscopy was used to monitor the absorbtion of OmpA to the lipid bilayers. CD spectroscopy was used to determine the kinetics of secondary structure formation during OmpA insertion into bilayers. The rate constants of tertiary structure formation were analyzed using a SDS-PAGE based method: KTSE (see section 1.4.1).
Kleinschmidt and Tamm (Kleinschmidt and Tamm 2002) have shown that, the secondary and the tertiary structure formation of OmpA take place in parallel and are a consequence of
protein insertion into the lipid bilayers. In contrast, the interaction of OmpA with the lipid bilayer take place with much faster rates than the secondary and tertiary structure formation (Kleinschmidt and Tamm 2002).
A further important finding concerning the mechanism of OmpA folding is the presence of several folding intermediates during the process (Kleinschmidt and Tamm 1996;
Kleinschmidt and Tamm 2002). Fluorescence spectroscopy and KTSE were employed to detect these intermediate forms.
Thus, three kinetic phases were detected during OmpA refolding into SUVs made of diC18:1 PC. The first step is not temperature dependent (k1 = 0.16 min–1, at 0.5 mM lipid) (Kleinschmidt and Tamm 1996). The second step is slower and temperature dependent (~ 0.0058 min–1 at 2 °C and ~ 0.048 to 0.14 min–1 at 40 °C, for 0.5 mM lipid) (Kleinschmidt and Tamm 1996). The rate constant of the second step approaches the rate constant of the first step at higher temperatures. These kinetic steps indicate that at least two membrane-bound folding intermediates will form during the OmpA folding and insertion into lipid bilayers.
These intermediates could be trapped at temperatures between 2 and 25°C in lipid bilayers of diC18:1PC. A third step has a rate constant of k3= 0.9 x 10–2min–1 (at 3.6 mM lipid, at 40 °C) (Kleinschmidt and Tamm 1996). This last step correspond with the formation of OmpA tertiary structure which, as the value of the rate constant shows, takes place slower than the protein association with the lipid bilayer.
The mechanism of OmpA folding was further studied due to the method of time-resolved distance determination by tryptophan fluorescence quenching (TDFQ) (Kleinschmidt and Tamm 1999; Kleinschmidt et al.1999). This revolutionary technique allows to establish the average positions of OmpA´ tryptophans during protein refolding, with reference to the center of phospholipid bilayer (Kleinschmidt and Tamm 1999; Kleinschmidt et al.1999). The parameter used for this purpose was the quenching of tryptophans fluorescence by brominated phospholipids integrated in the membrane (Abrams and London 1992; Bolen and Holloway 1990; Chattopadhyay and London 1987; Everett et al.1986; Ladokhin 1999;
Ladokhin and Holloway 1995; McIntosh and Holloway 1987; Wiener and White 1991).
Consistently with the previous data (Kleinschmidt and Tamm 1996) three membrane-bound intermediates of OmpA were identified by TDFQ. The average distances of tryptophans from the bilayer center were for the three intermediates (in Å) : 14-16, 10-11, and 0-5, respectively (Kleinschmidt and Tamm 1999).
The first folding intermediate is represented by OmpA absorbed to membrane surface and is
initial stage of insertion into the bilayer, was identified at temperatures between 7 and 20 °C.
Finally, the third intermediate, consisting of protein in contact with the bilayer´ center, was identified at 26-28 °C. The native structure of OmpA that forms above 28°C presents a distribution of all the five tryptophans at 9 to 10 Å from the bilayer center, with the Trp-7 in the periplasmic leaflet and the other 4 tryptophans in the outer leaflet of the outer membrane (see also Figure 1.3).
The intermediate forms of OmpA folding, identified by TDFQ, were further dissected using 5 single tryptophan OmpA mutants (Kleinschmidt et al.1999). Below 30°C each of the 5 tryptophans approached a distance of 10-11 Å from the bilayer center. At temperatures above 30°C, all tryptophans were detected very close to the center of lipid bilayer during the first minutes of refolding, except Trp-7 that did not migrate any closer to the bilayer center than
~10 Å. Trp-7 remain in the same position in the final step of refolding, while the other four tryptophans crossed the center of bilayer and subsequently approached distances of ~10 Å from bilayer center. Similar distances of tryptophan residues in report with the center of lipid bilayer indicate a simultaneous translocation of all β-strands across the bilayer during OmpA folding and insertion.
The mechanism of OmpA folding and insertion, resulted from the data presented above, is represented in Figure 1.6 (Kleinschmidt et al.1999). According with this model, in a first phase OmpA adsorbs to the water-membrane interface (IM1). The next phase is slower and reflects the simultaneous migration of the tryptophans from the water-membrane interface into the hydrophobic core of the lipid bilayer (IM2). At this stage the tryptophans move at a distance of about 10 Å from the bilayer center. In the case of the third membrane-bound intermediate (IM3) all tryptophans, except Trp-7, are located at a distance of 0-5 Å from the bilayers center. Trp-7 remains at the same location as in intermediate (IM2). Finally the native OmpA structure is formed and the tryptophans: Trp-15, Trp-57, Trp-102, and Trp-143 move away from the center of the bilayer to a distance of about 10 Å. This distance of Trp residues compares well with the X-ray and NMR structures (Pautsch and Schulz 2000; Arora et al.
2001).
Figure X
1.3.3 OmpA folding and insertion in vivo
1.3.3.1 Requirements of OmpA folding and insertion in vivo
Two categories of requirements have been identified: those concerning the amino acid sequence and those related with the folding in a crowded cellular environment.
Koebnik showed that the amino acid sequence of β-barrels has to respect certain criteria (Koebnik 1999). Minimum four or five random chosen residues oriented toward the membrane must be hydrophobic. The residues pointing inwards the barrel must be small and proline residues are not tolerated. In addition three central residues of β-strand must not be charged.
Beside the constrains imposed by the amino acid sequence, the efficiency of OmpA folding in vivo depends largely on the periplasmic chaperons and other factors related with protein folding in a crowded cellular environment.
Figure 1.6 Scheme for the concerted folding and insertion mechanism of OmpA. The β- strands (boxed) are on average ~ 45inclined from the membrane normal (Pautsch and Schulz 1998). Hydrophobic residues believed to be in contact with the lipid bilayer are shown in boldface.
The folding model shows the nearly synchronous insertion and translocation of Trp-15, Trp-57, Trp-102, and Trp-143 across the lipid bilayer. After adsorption to the membrane surface (intermediate IM1, not shown), a “molten disk” intermediate (IM2) forms with all Trps at 10 Å in the cis monolayer, followed by an “inside-out molten globule” intermediate (IM3) with Trps at an average location in the bilayer center and finally by the native state with the four Trps translocated to a position at 10 Å in the transmonolayer. Trp-7 stays at 10 Å in the cis monolayer throughout these late stages of folding (from Kleinschmidt et al.1999)
The chaperones are defined as a large group of unrelated protein families whose general roles are to: (i) stabilize unfolded proteins, (ii) unfold them for translocation across membranes or for degradation, (iii) assist the correct protein folding and assembly.
Periplasmic chaperones prevent the inappropriate association or aggregation of exposed hydrophobic surfaces and direct their substrates into productive folding. It is known that in vivomolecular chaperones keep the OMPs soluble in the periplasm before they insert and fold into the outer membrane (Schäfer et al.1999; Müller et al.2001).
Following the synthesis in cytoplasm, the unfolded OmpA is translocated across the cytoplasmic membrane to the periplasmic space by SecA/E/Y/G machinery (Manting and Driessen 2000; Danese and Silhavy 1999). In the periplasmic space a signal peptidase cleaves off the signal sequence and OmpA inserts and folds into the outer membrane.
Genetic and biophysical studies were used to assess the role of periplasmic chaperones in the assembly of OmpA. It appears that indeed periplasmic chaperones like Skp and SurA play a major role in thein vivoassembly of OMPs, OmpA included (Chen and Henning 1996; Lazar and Kolter 1996; Rouvière and Gross 1996; Behrens et al.2001; Missiakas et al.1996; Ramm and Plückthun 2000; Bothmann and Plückthun 2000; Liu and Walsh 1990).
Other studies suggested that LPS, the major component of the outer membrane of E.coli, is also involved in the folding of OMPs (Schweizer et al.1978; Freudl et al.1986).
1.3.3.2 The periplasmic chaperone Skp
Skp or Seventeen kDa Protein, (141 residues, 15.7 kDa) is a periplasmic protein that was shown to bound unfolded OMPs (Chen and Henning 1996). The deletion of skp gene in E.
coliresulted in reduced levels of OmpA, OmpC, OmpF, and LamB (Chen and Henning 1996;
Missiakas et al.1996). Skp binds the amino-terminal of unfolded OmpA and is required for OmpA release into periplasm (Schäfer et al. 1999).
Skp recognizes non-native structures of OMPs (Skp does neither bind to folded OmpA nor to the periplasmic domain) (Chen and Henning 1996).
The properties of periplasmic chaperone are directly related with Skp structure and biochemical characteristics. The protein has a pI between 9.6 and 10.3 (according to different algorithms). In solution, Skp forms trimers (Schlapschy et al. 2004). The Skp trimer (Korndörfer et al. 2004; Walton and Sousa 2004) presents periferic α-helices protruding from
a central β-barrel (see Figure 1.7 A) and defining a central cavity. The entire Skp trimer is about 80 Å long and 50 Å wide.
The Skp monomer has two domains. The small association domain (residues 1-21 and 113- 141 of the mature sequence) is composed of three β-strands and two short α-helices. This domain mediates the trimerization of Skp and forms the core of the trimer. The second domain is constituted of the α-helix (amino acids 22-112). This domain is conformationally flexible. The charge distribution on the Skp surface gives the trimer a dipole moment of ~3 700 Debye (Korndörfer et al. 2004).
The positive charges are on the periferic domain, while negative charges are on the central (core) domain. The cavity surface of the periferic domain contains hydrophobic patches inside. It may be that Skp binds its substrates in this central cavity (Korndörfer et al. 2004;
Walton and Sousa 2004). Skp has also a putative LPS binding site (Walton and Sousa 2004) that was found using a previously identified LPS binding motif (Ferguson et al. 2000). The binding site is formed by the residues K77, R87, and R88 (Figure 1.7 A). In addition, Q99 may also form a hydrogen bond to an LPS phosphate (Figure 1.7 A).
The skp gene maps at the 4-min region on the chromosome and is located upstream of the genes that encode proteins involved in lipid A biosynthesis (Dicker and Seetharam 1991; Roy and Coleman 1994; Thome et al. 1990), an essential component of LPS of the OM. The gene firA, which codes for UDP-3-O-[3-hydroxymyristoyl]-glucosamine-N-acyltransferase starts only 4 bases downstream of the skp stop codon (Bothmann and Plückthun 1998). The presence of a putative binding site for LPS in Skp (Walton and Sousa 2004) could be related to the location of skpclose tofirA.
1.3.3.3 The periplasmic chaperone SurA
The survival factor A, SurA was shown initially to be necessary for stationary phase survival of E. coli cells (Tormo et al. 1990). The deletion of surA gene resulted in reduced concentrations of OmpA and LamB in the outer membrane (Lazar and Kolter, 1996; Rouvière and Gross, 1996). The crystal structure of SurA is shown in Figure 1.7 B. SurA presents an N terminal domain (N), which is composed of 148 amino acids and contains the α-helices H1 to H6. This domain is connected to the domain P1 (residues 149 to 260) and the domain P2
(residues 261 to 369). P2 connects the P1 domain to the C-terminal domain C (residues 370 to 428).
Together, the N and C domain constitute a compact core with a broad deep crevice of about 50 Å in length. The P2 domain is tethered to this core by two extended peptide segments. The
Figure 1.7 Crystal structure of the Skp trimer (PDB entry 1SG2, Korndörferet al.,2004). The Skp trimer consists of a tightly packed 9-stranded β-barrel that is surrounded by C-terminal α- helices of the three subunits that point away from the barrel in form of tentacles that are about 65 Å long. These tentacles form a cavity that may take up the unfolded OMP. The outside surface of the helical domain of Skp is highly basic. Each monomer of the trimeric Skp has a putative LPS binding site (Walton and Sousa, 2004) (Skp structure entry 1UM2 in the PDB). The LPS binding site was found using a previously identified LPS binding motif (Ferguson et al., 2000) and consists of K77, R87, R88. This motif matches the LPS binding motif in FhuA with residues K306, K351, and R382 and a root mean square (rms) deviation of 1.75 Å for the Cα– Cγatoms was calculated (Walton and Sousa, 2004). Q99 in Skp may also form a hydrogen bond to an LPS phosphate, completing the four-residue LPS binding motif. B Crystal structure of Survival Factor A, SurA (PDB entry 1M5Y, (Bitto and McKay, 2002). The N-terminal domain (N) is composed of theα-helices H1 to H6 (residues 1 to 148) and connected to peptidyl-prolycis/trans isomerise (PPI) domain P1 (residues 149 to 260). The P2 domain (residues 261 to 369) connects the P1 domain to the C-terminal domain C (residues 370 to 428, colored in red). Thus, the N and C domain together constitute a compact core, which is traversed by a broad deep crevice of about 50 Å in length, suggesting a polypeptide binding-site. The active PPIase domain 2 (P2) is tethered to this core by two extended peptide segments. It has been demonstrated that a mutant, SurAN(- Ct), which does not contain the two PPIase domains and is composed of the N and C domains only,functions like a chaperone (Behrens et al., 2001). This SurA “core domain” has been proposed to bind the tripeptide motif aromatic-random-aromatic, which is prevalent in the aromatic girdles ofβ-barrel membrane proteins (Bitto and McKay, 2003). Images of the structures were created with Pymol (Delano, 2002).
P1 and P2 domains have sequence similarity to parvulin, a cytoplasmic peptidyl-prolyl cis/transisomerise (PPIase) (Rahfeld et al.1994).
SurA function of assisting the folding of outer membrane proteins (OMPs) was first attributed to the two parvulin-like domains, P1 and P2 (Figure 1.7 B) (Lazar and Kolter 1996; Missiakas et al. 1996; Rouvière and Gross 1996).
Then, by using a mutant form of SurA, from which the PPIase domains P1 and P2 were removed, it was show that the N domain containing helices H1 to H6, functions as chaperone when linked together with the C helix (Behrens et al. 2001).
The SurA “core domain” was found to bind the tripeptide motif aromatic-random-aromatic, (Ar-X-Ar ) which is prevalent in the aromatic girdles of β-barrel membrane proteins (Bitto and McKay 2003; Bitto and McKay 2004 ). Ar-X-Ar motifs, where X can be any amino acid residue, are found with high frequency in OMPs, in particular in two aromatic girdles close to the polar-apolar interfaces of the lipid bilayer.
For example, the numbers of Ar-X-Ar motifs in the β-barrel domains of OMPs are 7 for OmpF, 10 for LamB, 3 in OmpA, and 1 for TolC.
Genetic evidence suggests that SurA and Skp act as chaperones that are involved in parallel pathways of OMPs targeting to the OM (Rizzitello et al. 2001). Null mutations in skp and surA as well as in degP and surA resulted in synthetic phenotypes. The skp surA null combination had a bacteriostatic effect and led to filamentation, while the degP surA null combination was bactericidal.
It was suggested that the redundancy of Skp, SurA, and DegP is involved in the periplasmic chaperone activity, in which Skp and DegP are components of one pathway and SurA is a component of a parallel pathway. While the loss of either pathway was tolerated, the loss of both pathways was lethal (Rizzitello et al. 2001).
1.3.3.4 Lipopolysaccharides
Lipopolysaccharides (LPS) are unique and abundant glycolipids found in the outer leaflet of the gram-negative outer membrane. In Escherichia coli, there are approximately 106 LPS molecules per cell and these constitute 75% of outer membrane surface area. LPS is a glycolipid composed of two parts: Lipid A and the polysaccharide chain that reaches out into
Lipid A is a highly conserved region of the lipopolysaccharide consisting of a phosphorylated N-acetylglucosamine dimer with six or seven fatty acids attached. Lipid A is responsible for the endotoxic properties of LPS and is essential for formation of outer membrane (and for the viability) in most Gram-negative bacteria.
Attached to Lipid A is a conserved core polysaccharide that contains KDO, heptose, glucose and glucosamine sugars. The inner core oligosaccharide is attached to lipid A and participates in the resistance of the cell to polycationic peptides and hydrophobic antimicrobial compounds.
Initial studies suggested that LPS is required for efficient assembly of OMPs such as monomeric OmpA (Freudl et al. 1986; Schweizer et al. 1978) and trimeric PhoE (de Cock et al. 1999b). Together with divalent cations, LPS was reported to facilitate trimerization of PhoE in mixed micelles of Triton-X-100 detergentin vitro(de Cock et al. 1999b).
In these studies, experiments were performed with micelles of LPS and Triton-X-100 instead of phospholipid bilayers. It was later found that monomeric OmpA folds relatively fast into micelles but with rather slow kinetics into phospholipid bilayers (Surrey and Jähnig, 1995;
Surrey et al.1996).
Proteins that are already folded in micelles may be easily inserted into OMs, especially when the membranes are present in large excess, but would end up with a random orientation in the bilayer, i.e. loops could be exposed to the periplasmic space instead of their normal orientation to the outer space when OMP micelle complexes fuse with membranes.
Since OmpA assumed a random orientation after micelle-bilayer fusion (Surrey and Jähnig, 1992), it is unlikely that OmpA would first fold into LPS micelles in the periplasm, which then fuse with the OM as first proposed for PhoE based on the appearance of a folded monomer in mixed micelles of LPS and Triton-X-100 in vitro (de Cock and Tommassen, 1996).
However, a PhoE mutant was later shown to fold in vivo and also in vitro into LDAO micelles but not into mixed micelles of Triton-X-100 and LPS, also leading to doubts about the existence of a folded monomeric intermediate of PhoE in LPSin vivo(Jansen et al. 2000).
Genetic studies have shown that the absence of proper LPS resulted in decreased rate of rate of OMP synthesis (Ried et al., 1990). The absence of proper LPS affected the assembly of certain OMPs (Nikaido and Vaara, 1985; Schnaitman and Klena, 1993).
1.4 Methods
1.4.1 Electrophorectic mobility measurements used to detect the folding and insertion of OmpA
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is a useful tool to monitor the insertion and folding of OmpA into detergents and lipid bilayers provided that the samples are not boiled prior the electrophoresis.
Figure 1.8 Chemical structure of LPSLPS consists of the Lipid A, an inner core region and an outer core region. The heterogeneity of LPS species is given by composition of inner core. The LPS represented in this figure has a R2 core type and a M.W of approximate 3900 Da.
Schweizer et al. were the first to describe this application of SDS-PAGE in the case of OmpA folding (Schweizer et al.1978). Folded OmpA migrates at 30 kDa and unfolded OmpA migrates at 35 kDa (Figure 1.9).
30 kDa form was tested by a whole range of methods: Raman, FT-IR, CD-spectroscopy (Dornmair et al.1990; Surrey and Jähnig 1992; Surrey and Jähnig 1995; Kleinschmidt et.al 1999; Rodionova et al.1995; Vogel and Jähnig 1986) phage inactivation assays (Schweizer et al.1978) and by single-channel conductivity measurements (Arora et al.2000). All the tests have shown that 30 kDa form correspond to the fully folded form of OmpA.
The SDS compound used for SDS-PAGE for was shown to inhibit OmpA folding (Surrey and Jähnig 1995). The protein does not fold into SDS micelles (Dornmair et al.1990). The cause of SDS effect on OmpA folding is not completely clear. The repulsions between the negative surface potential of SDS and negative charges on OmpA might have been the reason why OmpA did not fold into SDS micelles. The relatively small headgroup of SDS causes a higher charge density on the surface of the SDS micelle, preventing insertion and folding of OmpA, which is negatively charged above pH 5.9.
The inhibitory effect of SDS on OmpA folding is used to monitor the development of the native structure of OmpA as a function of time (i.e. the kinetics of native structure formation) (Figure 1.9) (Kleinschmidt and Tamm 1996; Kleinschmidt and Tamm 2002).
Figure 1.9 SDS-PAGE gel showing the time course of OmpA folding and insertion into preformed lipid bilayers at 30 °C 24 µg of OmpA (137 µM in 8 M urea) were reacted with a 200-fold molar excess of preformed lipid bilayers. The final concentration of OmpA was 7.1 µM in a total volume of 96 µl per sample. Aliquots were taken after 4, 8, 16, 30, 60,90, 120 and 180 minutes and SDS-buffer was added to stop further OmpA folding.The samples were then analyzed by SDS-PAGE. The upper arrow in each gel indicates the membrane-adsorbed 35 kDa protein, the lower arrow indicates the native 30 kDa protein. Times are indicated in minutes.
This kinetic assay is performed in a simple way: the refolding is initiated by mixing OmpA with the micelles or lipid bilayers, then small volumes of the reaction mixture are taken out at certain intervals of times and mixed with SDS buffer. The SDS will bind quickly to the OmpA both folded and unfolded and it will stop the OmpA folding. The samples are not boiled after the experiment and a SDS-PAGE is performed (Figure 1.9). The resulted gel is analyzed by densitometry in order to determine the fractions of refolded OmpA at each interval of time. The method described above was named kinetics of tertiary structure formation by electrophoresis (KTSE).
1.4.2 Fluorescence spectroscopy
When a molecule absorbs electromagnetic radiation from the ultraviolet and visible region it enters in an electronically excited state, with an electron being transferred in a higher orbital energy. A significant part of the energy of excitation is dissipated as disordered thermal motion into the surroundings. In addition the molecule may lose excitation energy by radiative decay, with the emission of a photon as the electron transfers back into its lower energy orbital.
The radiative decay falls into two categories: fluorescence and phosphorescence.
Fluorescence and phosphorescence are observed when aromatic molecules are excited by ultraviolet or visible radiation.
Fluorescence is the emission of radiation that appears directly after the molecule is excited. In contrast, phosphorescence is the emission of radiation over much longer timescales (seconds or even hours) as a consequence of energy storage in an intermediate reservoir.
The Jablonski diagram (Figure 1.10) is a schematic representation of fluorescence and phosphorescence in terms of molecular electronic and vibrational energy levels. According with the Jablonski diagram an electron in the ground electronic state (S
0) jumps to the upper electronic state (S
1) as a consequence of radiation absorbtion. The electron in the upper electronic state (S
1) has accumulated vibrational energy. The S nomenclature stands for singlet state and refers to the fact that the ground states of most molecules contain paired electron spins (↑↓), which can adopt only one orientation with respect to an external magnetic
The excited molecule enters in collision with surrounding molecules and the excited state loses its vibrational energy in sequential steps down the ladder of vibrational levels. When the energy is lost in a radiative transition, a fluorescence spectrum is produced.
In comparison with the radiation responsible for excitation, the fluorescence spectrum has a longer wavelength corresponding to a smaller energy (Whittaker et al. 2000).
The contribution of fluorescence to the deactivation process of the excited state is defined as the quantum yield. The quantum yield is the quotient of the number of photons that are emitted and the number of photons that are absorbed. The fluorescence lifetime is the time the molecule remains on average in the excited state, before the emission takes place. The fluorescence spectroscopy is used at the study of protein structure, protein interactions, protein folding and stability.
Figure 1.10 Jablonski diagram of energy levels participating in electronic absorption, fluorescence and phosphorescence(Figure from Whittaker et al. 2000)
2 The effect of phospholipids composition on the folding kinetics of outer membrane protein A into model membranes
2.1 Introduction
Lipids play an important role in the folding process of outer membrane proteins (OMPs) because they are the medium in which the folding and insertion take place (Bogdanov and Dowhan 1999; Cantor 1999; Kleinschmidt and Tamm 2002; Kleinschmidt 2003; Tamm et al.
2004; Mogensen and Otzen 2005).
The outer membrane (OM) of Escherichia coli presents two lipid components:
lipopolysaccharide (LPS) and phospholipids (PLs), in the outer and inner leaflet, respectively.
After folding and insertion, the outer membrane proteins (OMPs) reside mainly in the inner leaflet of OM.
The phospholipids composition of the inner leaflet of OM is relatively simple: 75% of the zwitterionic lipid phosphatidylethanolamine (PE), 20% phosphatidylglycerol (PG) and 5%
cardiolipin (CL) (Raetz 1978). PE and PG, the two most abundant components have different chemical characteristics.
PE has a small head group, an overall conical shape and therefore the tendency to form non- bilayer structures (van den Brink-van der Laan 2004) (Figure 1.4 C from section 1.3.2.1). The head group of PE (Figure 2.1) is zwitterionic and electrically neutral.
In contrast, PG (Figure 2.1) is negatively charged at neutral pH, has a relatively large head group and thus the tendency to form bilayers (van den Brink-van der Laan, 2004) (Figure 1.4 B from section 1.3.2.1).
The question that arises is whether these two major components of the inner leaflet of OM, PE and PG, facilitate the kinetics of folding and insertion of OMPs.
Previous studies about the role of phospholipids on folding and insertion of OmpA have shown that, the properties of the lipid bilayers (such as membrane fluidity, lateral pressure, bilayer thickness and curvature) play a significant role in the assembly of OMPs (for recent reviews see Kleinschmidt 2003; Tamm et al. 2004). A significant part of these investigations
were carried out in vitro, using model membranes composed of phosphatidylcholines (PC) (Figure 2.1), which is not a natural component of the bacterial membrane.
The effect of PE and PG on OmpA folding and insertion was limited to studies of thermodynamic stability (Hong and Tamm 2004). Thus, thermodynamic stability of OmpA increases with the amount of POPE included in lipid bilayers (Hong and Tamm 2004). The presence of moderate amounts of PG had a similar effect (Tamm et al. 2004).
Beside the thermodynamic aspect of the interaction with lipid bilayers, the effect of PE and PG on the refolding kinetics of OmpA remained an open question.
Figure 2.1 Structures of DOPC, DOPG and DOPE employed to prepare the model membranes used in the present study (taken from Avanti Lipids).
Chemical structures from top to bottom: DOPC (1,2-Dioleoyl-sn-Glycero-3-Phosphocholine), DOPG (1,2-Dioleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)](Sodium Salt)) and DOPE (1,2- Dioleoyl-sn-Glycero-3-Phosphoethanolamine).
Therefore, the aim of the present chapter is to report the effect of PE and PG on OmpA refolding kinetics into model membranes. Three principal aspects were addressed in this respect:
1. Which are the kinetic characteristics of OmpA refolding into model membranes mimicking the phospholipids composition of the outer membrane?
2. Is it possible to efficiently use model membranes containing PE and PG for kinetics studies of OmpA refolding?
3. How do the kinetics of OmpA refolding depend on the amount of PE or PG included into the composition of model membranes?
In order to address these issues I have used the phospholipids DOPC, DOPG and DOPE to prepare the model membranes (Figure 2.1). DOPC was used because a significant part of the previous kinetic studies on OmpA folding and insertion were performed on model membranes containing DOPC (Kleinschmidt and Tamm 1996; Kleinschmidt and Tamm 1999;
Kleinschmidt et al. 1999). The structure of hydrophobic tail for DOPC, DOPG and DOPE is identical (Figure 2.1) and similar with the corresponding part of PLs composing the OM (Raetz 1978). PLs used for the present study (Figure 2.1) and the PLs composing the OM have identical polar head groups.
2.2 Materials and methods
2.2.1 Materials
1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-
phosphoethanolamine (DOPE) and 1, 2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) were from Avanti Polar Lipids (Alabaster, AL). Urea was from Serva (Heidelberg, Germany) and was of analytical grade. All the other chemicals were purchased from standard sources such as Sigma (Steinheim, Germany) and were of analytical grade.
2.2.2 Preparation of small unilamellar vesicles (SUV)
The solutions of lipids were prepared in chloroform at a concentration of 10µg/µl. The solutions were mixed at the molar ratios indicated for each experiment. The lipid mixtures were dried on the bottom of a glass test tube under a stream of nitrogen, and desiccated under high vacuum for at least 3 h to remove the residual solvent. Each lipid mixture was dispersed in 0.5 ml of 10 mM glycine buffer, pH 8.5, with 2 mM EDTA and 150 mM NaCl. SUVs were prepared by sonication of the lipid dispersion for 40 min using the microtip of a Branson ultrasonifier at 50% duty cycle in an ice/water bath.
Titanium dust was removed by centrifugation using an Eppendorf tabletop centrifuge.
Vesicles were equilibrated overnight at 4° C and used the next day for OmpA refolding experiments.
2.2.3 Purification of OmpA
OmpA was purified from Escherichia coli as described (Surrey and Jähnig 1992). OmpA concentrations of stock solutions were determined using the Lowry method (Lowry et al.1951).
2.2.4 Kinetics of tertiary structure formation of OmpA determined by electrophoresis (KTSE)
The KTSE method consists in monitoring the kinetics of tertiary structure formation of OmpA by SDS-PAGE. After purification OmpA is completely unfolded in 8 M urea. The refolding of OmpA is initiated by diluting the urea in buffer at least 12-fold, followed by the addition of preformed lipid bilayers.
After the initiation of refolding, samples are taken at different times from the refolding reaction and mixed with an equal volume SDS-buffer, pH 6.8. The SDS-buffer contains 0.125 M Tris-HCl, 4% SDS, 20% glycerol, and 10% 2-mercaptoethanol. SDS prevents further OmpA folding (Kleinschmidt and Tamm 1996).
SDS-PAGE is performed as described previously but without heat denaturation of the samples (Laemmli 1970). On SDS-PAGE gel the folded OmpA migrates at 30 kDa while the unfolded form of the protein migrates at 35 kDa.
The difference in the apparent molecular mass is used to determine the fraction of folded OmpA by densitometry (Kleinschmidt and Tamm 1996).
The fraction of folded OmpA resulted from the densitometric analysis is plotted as a function of time. I used the KTSE method to study the effect of PE and PG on OmpA folding and insertion into model membranes. The next section (2.2.5) describes the experiments which, are based on KTSE method, performed during this study,.
2.2.5 OmpA refolding into lipid bilayers of various compositions
The OmpA stock was in 8 M urea (137 µM, pH 7). Separate samples of 5 µl (24 µg) were taken from the OmpA stock and each was diluted separately into 56 µl glycine buffer (10 mM, pH 8.5, with 2 mM EDTA and 150 mM NaCl). A series of 10 identical samples was prepared. The refolding of OmpA was initiated by adding to each solution a 200-fold molar excess of preformed lipid bilayers (35 µl).
The composition of the lipid bilayers added to each sample was different. I have used lipid bilayers constituted of PC, PG and PE at the molar ratios of: PC: 100, PC/PG: 80/20, PC/PG:
70/30, PC/PG: 50/50, PC/PG: 30/70, PC/PG/PE: 50/20/30, PC/PE: 90/10, PC/PE: 70/30, PC/PE: 60/40 and PE/PG: 80/20.
The final concentration of OmpA was 7.1 µM in a total volume of 96 µl per solution. The OmpA was refolded at 30°C. Aliquots of each reaction were taken after 2, 4, 8, 16, 30, 60, 120 and 180 minutes and SDS-buffer was added to stop further OmpA folding. The samples were then analyzed by SDS-PAGE.
2.2.6 Kinetics densitometric analysis and rates of OmpA folding
To determine the kinetic parameters of membrane folding and insertion of OmpA into lipid bilayers the SDS-gels were analyzed by densitometry to obtain the plots of the fractions of folded OmpA as a function of time. The point zero was added at each plot of the fractions of
The point zero represents the unfolded OmpA. For the present study I assumed that OmpA folding kinetics are accurately described by a two-step mechanism with two pseudo-first order components: a fast phase followed by a slow phase. Thus, I fitted my plots to double- exponential functions. The equation used to fit the plots of the fraction of folded OmpA as a function of time is:
[
PF] (
t)
=FK*[
AF*exp(
-kF*t)
+(
1-AF)
*exp(
kS*t)
- 1]
(Eq.2.1)In Eq.2.1
[
PF] (
t)
is the concentration of the folded protein at time t, kF andkS are the rate constants of the fast and the slow kinetic phases. The pre-exponential factorAFis the relative contribution of the fast phase to the folding kinetics, while (1- AF) represents the relative contribution of the slow process.2.3 Results
2.3.1 OmpA folding and insertion into model membranes mimicking the phospholipids composition of the outer membrane
The first goal of my study was to investigate the kinetics of OmpA folding and insertion into model membranes mimicking the phospholipids composition of the inner leaflet of OM. For this purpose I refolded OmpA in preformed lipid bilayers containing PE and PG at the molar ratio of 80/20.
First two separate samples of OmpA in 8 M urea were 12-fold diluted in buffer and then lipid bilayers were added immediately to each sample, at 30°C. For one sample were used bilayers constituted of PC and for the second, bilayers of PE and PG at the molar ratio of 80/20. The appearance of the folded form of OmpA was monitored for 180 minutes, as described in detail in the method sections: 2.2.4 and 2.2.5.
The results of the OmpA refolding experiments are displayed in Figure 2.2. The upper panel displays the SDS-PAGE gels and the lower panel the densitometric analysis of the respective SDS-gels.
The results indicate clearly that the OmpA folding into PE80PG20 bilayers, which mimic the inner leaflet of OM, is significantly less effective than the refolding into PC bilayers (Figure 2.2). The yield of folded OmpA after 180 minutes from the refolding initiation is only 0.36 when bilayers of PE80PG20 are used in comparison with 0.88 for the experiment using PC bilayers. These results show that model membrane mimicking the inner leaflet of the OM cannot be efficiently used forin vivoOmpA folding studies.
Figure 2.2 Kinetics of OmpA folding and insertion into model mebranes containing (i) PC and (ii) PE and PG at a molar ratio of 80/20.Two separate identical samples of OmpA in 8 M urea were first diluted 12-fold into buffer and then without delay 200-fold molar excess of preformed lipid bilayers containing PC and PE/PG at the molar ratios indicated in the figure, was added to each sample, at 30 °C. Aliquots of each reaction were taken after 2, 4, 8, 16, 30, 60, 120 and 180 minutes and SDS-buffer was added to stop further OmpA folding. The samples were then analyzed by SDS-PAGE. The gels are displayed in theupper panel. The arrows indicate unfolded (U) and folded (F) forms of OmpA. The lower panel display the densitometric analysis of the fraction of folded OmpA determined from SDS-gels shown in the upper panel; PC100 (●) and PE80PG20 (■).
2.3.2 Folding and insertion of OmpA depend on the amount of PG and PE included into model membranes
In the previous section 2.3.1, was shown that model membranes containing exclusively PE and PG cannot be efficiently employed to study the effect of these phospholipids on OmpA refolding. Therefore, binary lipid mixtures of PC/PE and PC/PG were used to investigate the effect of PE and PG on the kinetics of OmpA folding and insertion.
The experiments performed for this purpose had a common set-up: first separate samples of OmpA in 8 M urea were 12-fold diluted in buffer and then the lipid bilayers containing PC/PE or PC/PG respectively, were immediately added at 30°C. I have used lipid bilayers constituted of PC and PE at the molar ratios of: PC100, PC/PE: 90/10, PC/PE: 70/30, PC/PE: 60/40. For the bilayers containing PC and PG the molar ratios were: PC: 100, PC/PG: 80/20, PC/PG:
70/30, PC/PG: 50/50, PC/PG: 30/70.
The appereance of the folded form of OmpA was monitored for 180 minutes, as described in detail in the method sections: 2.2.4 and 2.2.5. The results of OmpA refolding experiments are displayed in Figure 2.3. The upper panel of Figure 2.3 displays the SDS-PAGE gels. The lower panels of Figure 2.3 (A and B) represent the densitometric analysis of SDS-gels from upper panel.
At least two plots obtained from separate experiments performed in identical conditions and different days were averaged in order to generate the graphs from panels A and B, Figure 2.3.
In Figure 2.4 the OmpA folding yields obtained after 180 minutes from OmpA refolding initiation were plotted as a function of the PE and respectively PG amount present in bilayers.
The OmpA folding yields at 180 minutes were obtained from the densitometric analysis of refolding kinetics (Figure 2.3, lower panels, A and B).
The kinetic parameters of OmpA refolding (Table 2.1) were derived from the plots of Figure 2.3 (lower panels A and B) and were calculated as described in the section 2.2.6.
Overall, the data from Figure 2.3, 2.4 and Table 2.1 show that the progressive increase of PE and PG amounts result, in average, in reduced OmpA folding yields and slower refolding kinetics.
In the case of PC/PE bilayers, the decrease of OmpA folding yields after 180 minutes from refolding initiation is more pronounced than for PC/PG bilayers. This difference is notably higher for amounts of PE and PG above 30 % (Figure 2.4 and Table 2.1). Thus, for a 40 % PE the folding yield is 0.46, while a very similar yield 0.47 is obtained for a significantly higher amount of PG: 70 % (Figure 2.4 and Table 2.1).
Figure 2.3 Kinetics of OmpA folding and insertion into model mebranes containing binary mixtures of PC/PE and PC/PG. Separate identical samples of OmpA in 8 M urea were first diluted 12-fold into buffer and then without delay 200-fold molar excess of preformed lipid bilayers containing PC/PE or PC/PG at the molar ratios indicated in the figure, was added to each sample, at 30 °C. Aliquots of each reaction were taken after 2, 4, 8, 16, 30, 60, 120 and 180 minutes and SDS-buffer was added to stop further OmpA folding. The samples were then analyzed by SDS-PAGE. The gels are displayed in theupper panel. The arrows indicate unfolded (U) and folded (F) forms of OmpA.Lower panelsAandBdisplay the densitometric analysis of the fraction of folded OmpA determined from SDS-gels shown in the upper panel.
