Another monolayer-related technique to study lipidprotein interactions is infrared re-ectionabsorption spectroscopy (IRRAS) on monolayers. Information on the molecular conformation, orientation and thickness of the monolayer at the airwater interface can be obtained. Furthermore, changes in the secondary structure of the proteins that occur upon binding to a lipid monolayer can be determined using IRRAS.234, 235 To calculate a reectance absorbance spectrum RAaccording to
RA=−log I
I0
(4.9) with single beam reectivity of pure water or pure buer surface (I0), respectively, and of the water or buer surface covered by a lipid monolayer (I) is recorded using a shuttle technique with a reference trough and a sample trough. This approach minimises the inuence of rotationalvibrational bands of water vapour in the light beam on the IRRA spectra.236 Recording IRRA spectra at dierent angles of incidence with parallel (p) and perpendicular (s) polarised light may enable the determination of the orientation of sec-ondary structure elements.237 The analysis of the spectra focusses on band intensities and wavenumbers of the dierent vibrational modes of the sample at the airwater interface.
Spectra recorded at dierent angles of incidence can be simulated and tted as described by Schwieger et al.237 If the adsorption of proteins to monolayers is investigated, the amide I and amide II bands are of particular interest.
For this thesis, IRRA spectra were measured to compare the adsorption of MBP C1 and MBP C8 to a monolayer of the normal myelin-like lipid composition (Figure 4.12), to a monolayer of PS:PI = 13:2 (Figure B.25) and to a monolayer of the normal composition without SM (Figure B.26). The respective protein solution was injected below a mono-layer at a surface pressure of 30 mN/m due to similar physical characteristics to a bimono-layer at this surface pressure.238 Figure 4.12 shows the reectance absorption spectrum of a lipid monolayer of the normal composition (green curve), as well as of MBP C1 (black curve) and MBP C8 (red curve) adsorbed to this lipid layer. The amide I and amide II bands of the adsorbed protein are overlapped by the lipid spectrum. Subtracting the lipid IRRA spectrum from the spectrum of the protein adsorbed to the monolayer visualises the amide bands of MBP C1 (blue curve) and MBP C8 (purple curve). The wavenumbers of these bands (amide I: 1662 cm−1, amide II: 1541 cm−1) are identical for both MBP
78
4.4 Outlook
ν(C=O) amide I amide II
Figure 4.12: IRRAS spectrum of (A) a monolayer of normal composition at 30 mN/m (green curve), (B) MBP C1 adsorbed to a monolayer of normal composition at 30 mN/m (black curve), (C) MBP C8 adsorbed to a monolayer of normal composition at 30 mN/m (red curve), (D) dierential spectrum of (B) and (A) (blue curve) and (E) dierential spectrum of (C) and (A) (purple curve).
Spectra were recorded with p-polarised IR radiation at an angle of incidence of 40◦.
Subphase: 20◦C, 20 mM HEPES and 10 mM NaCl at pH 7.4.
variants and indicate a α-helical structure (amide I: 16481657 cm−1, amide II: 1545 1550 cm−1)239 and the absence of β-sheets (amide I: 16231641 cm−1, 16741695 cm−1, amide II: 15251532 cm−1).239 The fact that the wavenumbers of the amide bands are slightly out of the typical intervals speak for contributions of disordered protein parts.
The same applies for MBP adsorbed to a monolayer of PS:PI = 13:2 and to a monolayer of the normal composition without SM. In the case of MBP adsorbed to the negatively charged lipids PS and PI (Figure B.25), the amide bands are more prominent than in the case of monolayers of a lower surface charge density due to the stronger interaction between MBP and monolayer resulting from electrostatic interactions.
Angle-dependent measurements of MBP C1 and MBP C8 adsorbed to the respective monolayers have been recorded for angles of incidenceϕ={26◦,30◦,34◦,38◦,42◦,46◦,62◦, 66◦,70◦}.∗ Despite all eorts, simulation and tting of the angle-dependent spectra to extract the tilt angle of the α-helix of MBP inside the monolayer are not likely to lead to success since MBP adsorbed to lipids forms three α-helices. The extracted tilt angle would be an error-prone average of the tilt angle of all helices. IRRAS measurement and
∗Angles near the Brewster angle ϕB= 53,1◦ (airwater interface) have not been taken into account due to a minimum of the reectivity of p-polarised light atϕBleading to a low signal-to-noise ratio.
4 Myelin basic protein
Table 4.9: Maximum insertion pressures for adsorption of bMBP to monolayers of the normal composition after incubation with FeCl3 and H2O2.
Incubation MIP in mN/m
time in h
bMBP bMBP bMBP + FeCl3
bMBP + FeCl3 + H2O2 + H2O2
0 42.3 42.0 42.5 49.9
±1.4 ±0.9 ±1.2 ±1.7
25 44.9 43.7 42.4 49.1
±1.4 ±2.3 ±1.9 ±1.5
analysis would be more reasonable if only the central segment of MBP comprising one α-helix (so-called immunodominant epitope145147) is investigated as it has already been done with NMR spectroscopy240, 241 and molecular dynamics simulation.241, 242 A com-parison of rmMBP C1 and rmMBP C8 is still not possible with this approach since none of the arginine to glutamine deiminations are located near the central segment. However, the tilt angle of this α-helix could be determined for monolayers of the several lipids of the cytoplasmic leaet of the myelin sheath using IRRAS to investigate the inuence of every lipid on the tilt angle. Prior to this, one has to make certain with monolayer adsorption measurements that these lipids aect the interaction between monolayers and the MBP segment in a similar manner as in case of the whole protein since a segment of a protein may behave completely dierent than the whole protein.
A potential role in the pathogenesis of MS is attributed to haemoglobin in combination with other factors, such as oxidative stress,160 due to abnormal accumulations of iron in brains of MS patients, e.g. near demyelinated plaques.158, 159, 243, 244 Bamm et al. have performed oxidation experiments with MBP, haemoglobin and H2O2 in the presence of cytoplasmic large unilamellar vesicles dependent on various times of incubation.177 In accordance to this study, preliminary experiments have been carried out to investigate the role of iron for the interaction of MBP and myelin-like lipid monolayers. Bovine MBP has been incubated with FeCl3 (74 nM), with H2O2 (18.5 nM) and with FeCl3and H2O2, respectively, for incubation times of 0 h and 25 h at 37◦. H2O2 was added to simulate oxidative stress. The MIPs of all bMBP-mixtures adsorbed to monolayers of the normal composition have been determined with monolayer adsorption experiments (Table 4.9) at 20◦C on 20 mM HEPES and 10 mM NaCl at pH 7.4 (for ∆π(πini) andπ(t) diagrams, see Figures B.33 and B.34). The incubation time, as well as the addition of FeCl3 or H2O2 has no signicant eect on the MIP of bMBP adsorbing to a monolayer of the normal composition. However, the addition of both substances, FeCl3 and H2O2, leads to a signicant increase of the MIP of 7.6 mN/m (0 h incubation) and 4.2 mN/m (25 h incubation time). These preliminary experiments already show an inuence of iron on the MBPlipid interaction in the presence of H2O2. It remains to examine the reason for the increase of the MIP. A possible increase of electrostatic interactions could be con-sidered with monolayer adsorption experiments with monolayers of negatively charged
80
4.4 Outlook lipids. Furthermore, the mixtures of bMBP, FeCl3 and H2O2 should be injected below monolayers of the normal composition without SM due to the important role of SM re-vealed in Chapter 4.3. Fluorescence microscopy may yield further insights.
The potential role of iron for the pathogenesis of MS may be related to three pathways: i) the reactivity of free iron, ii) the activity of haeme and iii) the activity of haemoglobin.177 As a consequence the above suggested experiments have to be compared to measurements using haemin chloride and haemoglobin, respectively, instead of FeCl3. In addition to that, measurements with protoporphyrin IX may help with the separation of eects on the MBPlipid interaction arising on one hand from the iron and on the other hand from the porphyrin part of the haemoglobin. Since haemoglobin is surface active,245 too, all experiments have to be repeated without bMBP.
To compare the inuence of iron and iron-containing substances on dierent charge vari-ants of MBP, recombinant produced proteins used in this thesis are not suitable since they contain a His-tag. This His-tag facilitates the purication of the protein after its expression in E. coli. However, its metal-anity would distort the investigation of the ironMBP interaction. For these measurement, untagged rmMBP should be produced.
CHAPTER 5
Experimental
5.1 Thermoresponsive peptide polymers
5.1.1 Materials
All diblock ELPs are composed of a hydrophobic block (VPGVG)x1 at the N-terminal end of the polypeptide and a hydrophilic block (VPGXG)x2 located at the C-terminal end. The so-called leader sequence MGCGWPG (for x2 = 60) or GCGWPG (for x2 = 30), respectively, was encoded at the N-terminus and the respective trailer sequence PGGS at the C-terminus (see Figure 5.1 a). In case of the hydrophilic block with guest residue X = A, G (1:1) and x2 = 60, the hydrophobic block lengths were varied within x1 ∈ {40,80,120,200}. Two additional diblocks were investigated with a hydrophobic block length ofx1 = 200and a hydrophilic block length ofx2 = 30: one with the previous guest residue X = A, G (1:1) and the second one with X = S. In summary, the investigated diblock ELP are composed of the following amino acid sequences:
i) ELP 4060: MGCGWPG(VGVPG)40(AGVPGGGVPG)30PGGS ii) ELP 8060: MGCGWPG(VGVPG)80(AGVPGGGVPG)30PGGS iii) ELP 12060: MGCGWPG(VGVPG)120(AGVPGGGVPG)30PGGS iv) ELP 20060: MGCGWPG(VGVPG)200(AGVPGGGVPG)30PGGS
v) ELP 20030: GCGWPG(VGVPG)200(AGVPGGGVPG)15PGGS vi) ELP 200S30: GCGWPG(VGVPG)200(SGVPG)30PGGS.
These diblock compositions lead to hydrophilic mass fractions between 0.12 and 0.56 (Table 3.3 in Section 3.2). Following the established nomenclature, it will be referred to the diblock ELPs as 'ELP x1x2' with the hydrophobic block length x1 and the hydrophilic block length x2. In case of serine as the guest residue of the hydrophilic block the polymer is called 'ELP 200S30'.
The diblock ELPs were cloned and produced in Escherichia coli by Sarah MacEwan (Chilkoti Lab, Duke University). They were received from Elisabeth Garanger (Université de Bordeaux), who already described their synthesis, expression and purication.103
The hysteretic peptide polymers were cloned and produced in Escherichia coli by Felipe García Quiroz (Chilkoti Lab, Duke University), who already investigated their hysteretic
5 Experimental
a)
N C
MGCGWPG PGGS
(VPGVG)x1 (VPGXG)x2
b)
O−N• O
COOH
Figure 5.1: a) Schematic depiction of the sequence of the diblock ELPs (VPGVG)x1 -(VPGXG)x2with X = A,G (1:1)∪˙ X = S, b) structural formula of 16-DOXYL stearic acid (16-DSA).
a)
N C
N C
SKGPG
SKGPG
WP
WP
(VAPVG)n
(TPVAVG)n
b)
O−N• O O
Figure 5.2: a) Schematic depiction of the sequence of the hysteretic polymers (VAPVG)n
and (TPVAVG)n, b) structural formula of 4-Hydroxy-TEMPO benzoate (TB).
behaviour with the use of turbidimetry measurements (unpublished data). Synthesis, ex-pression and purication of these peptide polymers has been reported in detail by Quiroz and Chilkoti.24
Two dierent polymer sequences were investigated (see Figure 5.2 a): i) composed of the amino acid sequence (VAPVG)n with n ∈ {30,40,80} and ii) composed of the amino acid sequence (TPVAVG)n withn∈ {40,80}. The so-called leader sequence SKGPG is encoded at the N-terminus and the respective trailer sequence WP at the C-terminus.
5.1.2 Methods
For the measurement with CW EPR the diblock ELPs were dissolved in PBS (pH 7.4) cooled by ice to avoid an early phase transition. The amphiphilic spin probe 16-DSA (16-DOXYL stearic acid, purchased from Sigma-Aldrich Co. LLC., St. Louis, USA, Fig-ure 5.1 b), dissolved in ethanol, was added. Dierent concentrations of polymer and spin probe were tested to nd the most suitable approach. The nal samples contained 300µM polymer and 1 mM 16-DSA. Micropipettes (BLAUBRAND intraMARK, Wertheim, Ger-many) were lled with about 15µl of the solution and sealed with CRITOSEAL (Le-ica Microsystems, Wetzlar, Germany) for subsequent EPR measurements. Temperatures were adjusted with the Temperature Controller H03 (Magnettech, Berlin, Germany) with an accuracy of±0.2◦C and a heating rate of 1◦C/min. To distinguish the eects of the temperature on the probe from the eects of the polymer aggregates on the probe, refer-ence spectra of 1 mM 16-DSA in pure buer were measured.
For the measurement with CW EPR the hysteretic polymers were dissolved in PBS
84
5.2 Myelin basic protein