Modelling copper-containing proteins
Bosch, Marieke van den
Citation
Bosch, M. van den. (2006, January 18). Modelling copper-containing proteins. Retrieved
from https://hdl.handle.net/1887/4361
Version:
Corrected Publisher’s Version
Chapter 4
Summary
Azurin is a very stable protein thatonly unfolds atvery unusualconditions.Also the Trp residue in the hydrophobic core of the apo-protein has one of the longest lived phosphorescence signals ever measured,indicating a very solid environment.Two point mutations located near the Trp48 residue, Ile7ÆSer and Phe110ÆSer, replace a hydrophobic residue by a smaller, hydrophylic serine residue. The lifetime of the phosphoresence signal of the I7S variant is shorter whereas the signal of the F110S variantis even longer than thatof wt.This is unexpected because the smaller residues are thought to enhance the mobility of the Trp residue and its environment and thereby shorten the phosphorescence lifetime.
In this study,molecular dynamics simulations were performed to obtain information on the dynamics of the hydrophobic core of the three proteins in the apo form as wellas the holo form.The dynamic behaviour of the hydrophobic core was analysed using the root -mean-square fluctuation and the number of dihedral angle transitions. Furthermore the diffusion of solvent molecules to the protein core was examined and used for comparison with studies, in which small organic compounds like glycerol and acrylamide are shown to have a different influence on the unfolding and the spectroscopic features of the differentproteins.
4.1 Introduction
The study of protein dynamics has gained a prominent place in recent biophysical research for at least two reasons. M obility is essential for function, like enzyme catalysis, molecular recognition and signal transduction. Secondly, to understand protein folding it is necessary to know how protein dynamics is related to secondary and tertiary structure and to the physical properties of individual side chains. The fluorescence and phosphorescence of tryptophans provide excellent tools to study local mobility in proteins by photophysical means. Emission wavelength and bandwidth give information about local polarity and conformational variety of the protein. Lifetime distributions provide information about heterogeneity of the protein matrix. Fluorescence depolarisation studies provide information about local dynamics on the time scale of the fluorescence. Finally, quenching by small molecules may give insight into the flexibility of the protein matrix in terms of an effective viscosity.
The blue copper protein azurin has served as an excellent test case for studying protein mobility. Azurin from Pseudomonas aeruginosa carries a single tryptophan (Trp 48) with one of the most blue shifted absorptions and emissions described in the literature, indicative of a strongly hydrophobic and rigid interior. Extensive X-Ray Diffraction (XRD) and NM R studies have been reported for this protein as well as for variants produced by site-directed mutagenesis. Its dynamics have also been studied by steady state and time resolved fluorescence and phosphorescence spectroscopy. These techniques have also been applied to azurin in which Ile 7 or Phe 110 were replaced by a serine, see Figure 4.1. These residues are close to Trp 48 and their replacement by a small side chain was expected to enhance local mobility. Consequently several groups have studied the fluorescence and phosphorescence emission properties of the tryptophan in these azurin variants.
b
c
a
d
Figure 4.1: Ribbon representation of wt azurin (a), some residues with their residue numbers (italics) are depicted as well as the numbers of the strands (bold) according to (van de Kamp et al., 1992). Detailed view of the hydrophobic core of wt (b), I7S (c) and F110S azurin variants (d) (Hammann et al., 1996; Nar et al., 1991).
presence of water molecules was established in the core of the F110S mutant. In the latter case the number of water molecules differs for the different molecules in the asymmetric unit. The study does not show any water molecules in the I7S variant at the position of the mutation. Gilardi et al. have used the steady-state fluorescence spectra to look at the effect of the mutations on the mobility of the Trp 48 residue (Gilardi et al., 1994). They have shown that the introduction of the mutations causes a red-shift of the emission maxima and an increase in bandwidth, which points to an increased polarity and a greater inhomogeneity of the Trp surroundings, respectively. The increase in polarity was ascribed to the introduction of a polar side chain (Ser) close to Trp 48 and the presence of water molecules in the protein interior at the site of the mutation. They
a 0,01 0,1 1 0 1000 2000 3000 Time (ms) P h o s p h o re s c e n c e I n te n s it y wt I7S F110S apo azurin b 0,01 0,1 1 0 2000 4000 6000 8000 Time (ms) P h o p h o re s c e n c e I n te n s it y wt I7S F110S holo azurin
Figure 4.2: Phosphorescence properties decays of wt, I7S and F110S azurin in apo (a) and Cd (b) forms. Data from Cioni et al. (Cioni et al., 2004).
a 0 5 10 15 20 25 30 35 40 45 50 0 0,0005 0,001 0,0015 0,002 [Acryl] (M) 1 /Wa v -1 /Wa v 0 wt I7S F110S apo azurin b 0 5 10 15 20 25 30 35 40 45 50 0 0,002 0,004 0,006 0,008 0,01 [Acryl] (M) 1 /Wa v -1 /Wa v 0 wt I7S F110S holo azurin
Figure 4.3: Lifetime Stern-Volmer plots for the quencing of azurin phosphorescence by acrylamide of wt, I7S and F110S azurin in apo (a) and Cd (b) forms. Data from Cioni et al. (Cioni et al., 2004).
dielectric permittivity of the microenvironment of Trp 48 and by changes in mobility of the mutated residues (Hammann et al., 1996). Mei et al. performed unfolding studies showing that azurin and apo-azurin were practically unaffected by hydrostatic pressure up to 3000 bar (Mei et al., 1999). The mutants are less stable, especially in the apo form where the introduced cavity is relatively easy to compress. These results show that the tight packing of the hydrophobic residues that characterise the inner structure of azurin is fundamental for the protein stability. Also the addition of GndHCl has a much higher impact on the unfolding of the mutant proteins compared to wt azurin.
Phosphorescence experiments, as presented in Figure 4.2, show that the I7S mutant has a much higher flexibility than wt azurin: the lifetime has decreased a 3.6 fold for the apo form and 2.2 fold for the holo form (Cioni et al., 2004). This corresponds well with the earlier mentioned conclusions by Mei, Hammann and Gilardi et al. of higher heterogeneity, mobility and less stability. The lifetime of the phosphorescence signal of the F110S mutant, however, shows an opposite effect and increases a factor of 1.7 in the apo form and holo F110S has a lifetime 6 times higher than wt azurin, the longest ever measured for a protein. It implies a more stable environment, in contrast to the above-described experiments. Upon removing the metal in the F110S mutant, the lifetime is drastically reduced.
Furthermore, phosphorescence-quenching experiments, using acrylamide, showed a considerable reduction of internal 'viscosity' of the protein matrix in the case of both mutants compared to wt, see Figure 4.3. Upon adding glycerol to the solvent Kroes et al. also showed that the internal ‘viscosity’ of the mutants increases while the wt remains unaffected (Kroes et al., 1998).
Figure 4.4: RMS deviation of the protein with respect to the initial structure after an atom-positional least-squares fit on the backbone. A running average was taken over 100 configurations (50 ps). The different grey scales represent the different simulations.
4.2 Methods
Figure 4.5: Number of backbone N-H C-O hydrogen bonds in time using a running average over 100 points (50 ps).
was placed inside the structure and behaviour of the water molecule was monitored. Four MD runs of 9 ns for each of the six proteins were performed varying the initial velocities and a slightly different initial position of the explicitly inserted water molecules. The positions and orientations of the water molecules were energy minimised for 50 steps using the steepest descent algorithm followed by minimising the potential energy of the entire system by another 50 stepsbefore starting the MD-simulations.
4.3 Results
4.3.1 Stability
type-I Cu-site, consisting of the five co-ordinating residues, shows a large maximum deviation of 0.25 nm in the apo form compared to 0.17 nm in the holo form. In the case of the F110S protein these values are 0.28 nm and 0.15 nm respectively and for the I7S mutant 0.31 nm and 0.18 nm. As seen for the backbone, also the deviation of the type-I Cu-site is higher for the apo forms.
The number of N-H C-O backbone hydrogen bonds is stable during the simulations, see Figure 4.5. Only in one case, for the holo wt protein, the number of hydrogen bonds decreases significantly after 2 ns, indicating unfolding of a part of the protein (upper right panel, black line). This coincides with the higher RMS deviation shown in Figure 4.4 and with the increased RMS fluctuation as shown further in Figure 4.8 (2nd panel from the top, black line). After 5 ns the protein has regained the proper number of hydrogen bonds, which means that the unfolding was temporary. Remarkable is the fact that the mutants have more backbone hydrogen bonds. Only in the case of one simulation in the holo form of the F110S protein is the number reduced to the same value as in wt azurin.
4.3.2 Structure
From the crystal structure two conformations are suggested for Ile/Ser 7 and Ser 110. From the simulations it becomes clear that both conformations are sampled, see Figure 4.6. Figure 4.7a and 4.7b show the distributions of the I, \, F1 and F2 dihedral angles of Trp 48
during the simulations. All distributions show well defined peaks. In the holo form of the three
proteins the tryptophan residue exhibits mostly one conformation: every dihedral angle is distributed along one central value. Only in the case of the holo F110S mutant does the backbone \ angle occasionally flip from 90 to 130 degrees. The I angle also has a broader distribution in this simulation. In the apo form the backbone I angle populates two conformations in all three proteins. The \ angle also shows more variability and in the case of one simulation of the F110S mutant it populates two conformations. The
Figure 4.6: Snapshot showing the double conformation of residue 7.
Phe 110
a b
Figure 4.7: Distribution of the backbone I and \ (a) and the sidechain F1 and F2 (b)
dihedral angles of Trp 48.
side-chain is not affected by the backbone flexibility and only one orientation is sampled, see Figure 4.7b.
4.3.3 Flexibility
Figure 4.8: RMS atom-positional fluctuation of the backbone atoms.
proteins in presence and absence of the Cu ion. The Cu-co-ordinating residues Gly 45 and His 46 show no significant increase in backbone flexibility upon Cu removal. However, the other Cu-co-ordinating residues Cys 112, His 117 and Met 121, all located in a turn between strand seven and eight, obtain a much higher backbone mobility in the apo -form. To obtain more information on the flexibility around the hydrophobic core the variations of the dihedral angles around the Trp 48 residue have been analysed. Dihedral-angle torsional profiles exhibit more than one optimal value and when the barrier between these values is not much larger than kT (~2.5 kJ.mol-1) a dihedral angle can occupy several conformations during an MD-simulation. The number of these dihedral transitions during a simulation is a measure for the flexibility of the protein. The
Table 4.1: Number of dihedral transitions per ns for 76 selected dihedral angles; in bold the number of the strand is designated in which the dihedral angle is situated according to Figure 4.1(a). In the summation below the F2
-dihedral angles of residues 7 and 110 are excluded.
Strand Angle wt I7S F110S
apo holo apo holo apo holo
Continuation of Table 4.1
Strand Angle wt I7S F110S
apo holo apo holo apo holo
Continuation of Table 4.1
Strand Angle wt I7S F110S
apo holo apo holo apo holo
8 FLeu125 1 0 0 0 3 2 \ Leu125 6 5 7 6 11 15 FLeu125 4 2 2 3 7 2 \ Thr126 9 14 11 5 7 13 Total number of \transitions per ns 186 158 252 197 237 206 Total number of Itransitions per ns 165 158 169 136 155 124 Total number of Ftransitions per ns 10 10 19 17 21 20 Total number of Ftransitions per ns 120 73 84 73 88 63 Total number of transitions per ns 481 399 524 423 501 413
dihedrals in close proximity of the Trp residue were selected excluding the stable omega backbone angles and the low-energy-barrier dihedrals involving a hydrogen atom. The potential energy profile of a dihedral angle consists of a complex combination of an explicit dihedral-angle term and contributions from (1,4) and other non-bonded interactions. W hen the potential energy profile is mostly governed by the non-bonded interactions and the energy barriers are low, an unusually high number of transitions may occur. This resulted in 74 dihedrals all belonging to the above mentioned residues. Table 4.1 shows the number of dihedral transitions per ns. The F2-dihedral angles of Ser
Table 4.2: Number of dihedral angle distribution peaks (N) of four runs for 76 selected dihedral angles during the simulation. In bold the number of the strand is designated in which the dihedral angle is situated according to Figure 4.1(a). The F2-dihedral angles of residue 7 and 110 were not included in the
heterogeneity analysis presented below, a value of N0=74 was used.
Strand Angle wt I7S F110S
apo holo apo holo apo holo
Continuation of Table 4.2
Strand Angle wt I7S F110S
apo holo apo holo apo holo
Continuation of Table 4.2
Strand Angle wt I7S F110S
apo holo apo holo apo holo
8 \ Thr124 1 1 1 1 2 2 I Leu125 1 1 1 2 2 2 FLeu125 2 2 2 1 3 3 \ Leu125 2 2 2 2 2 2 FLeu125 3 3 3 3 4 4 \ Thr126 2 2 2 2 1 2
Total number of \ peaks 34 37 37 39 42 41
Total number of Ipeaks 28 30 31 30 36 29
Total number of Fpeaks 34 29 29 30 37 31
Total number of Fpeaks 22 16 19 18 25 18
Total number of peaks 118 112 116 117 140 119
0 0 N N N 0,59 0,51 0,57 0,58 0,89 0,61
apo form. The F110S mutant shows only a small increase of flexibility around the tryptophan residue compared to wt in both the apo- and holo form. An evident increase of flexibility is observed at the residues 124-126. These residues are situated nearby residue 110 in strand 8, see Figure 4.1a.
4.3.4 Heterogeneity
The heterogeneity of the protein was monitored by analysing the number of maxima in the dihedral angle distributions. Table 4.2 shows the number of conformations that the selected dihedrals adopt. A measure for the heterogeneity could be (N-N0)/N0 where N is
the total number of observed maxima in the conformational distribution of all dihedral angles and N0 the number of dihedral angles.
The heterogeneity index, (N-N0)/N0, is very similar for all proteins except for the apo
Figure 4.9: RMS fluctuation of the backbone atoms after a least squares fit of six concatenated MD-runs of 400 ps. Above the Figure the secondary structure of the protein is indicated. The blocks in the graphic indicate the numbers of the residues situated close to the Trp 48: 6-8, 14-16, 20, 30-33, 46-50, 81-85, 94-98, 109-112, 124-126.
fluctuations. The main reson for the high fluctuation was the increase of transitions of backbone dihedral angles of residues 94-95 while in the heterogeneity analysis these angles do not significantly influence the statistics. The holo form, however, a large increase in heterogeneity is observed for residues 95-98, in accordance with an increase of flexibility as discussed above. From the simulations, dihedral transitions with a lower frequency than 0.1 ns-1 will not be sampled sufficiently. To increase the sampling, the temperature was increased for six structures of the holo form for each of the three proteins. The six structures were obtained from the first six ns of the simulations at 300 K at 1 ns intervals (excluding t = 0 ns) and were simulated for 0.5 ns at 400 K under
Figure 4.10: Number of water molecules present within 0.6 nm of the NHatom of the Trp 48. A running average of 250 configurations (125 ps) was taken.
constant volume. After relaxing the protein for 0.1 ns at 300 K under constant volume, another 0.4 ns of simulation was performed under constant pressure. The six trajectories, each 0.4 ns, were concatenated and the differences in the trajectories were examined using the RMS fluctuation, see Figure 4.9. In the region around the Trp 48 (residues 6-8,14-16, 20, 30-33, 46-50, 81-85, 94-98, 109-112, 124-126) the fluctuation along thedifferent trajectories for wt is generally lower compared to both mutants. Especially at E-strand S2, residues 14-33, the mutants exhibit a much larger heterogeneity than wt azurin. The RMS fluctuations of the individual runs of 0.4 ns do not show this significant increase which implies that upon increasing the sampling, the mutants occupy many more conformations than wt.
4.3.5 Diffusion of water
a b
Figure 4.11: Ribbon representation of two snapshots of the I7S molecule where in red the trajectories of water entering the protein core as in pathway A (a) and pathway B (b) are shown
in the cavity. In the crystal structure of holo I7S a water molecule was expected in the created cavity but not observed. Using the simulations, where water molecules were placed in the hydrophobic core, their diffusion was studied.
Figure 4.12: Value of the F2 dihedral angle of Phe 15 in
time during the simulations.
observed and all water molecules enter the core via the first pathway. In the F110S simulations water enters only via pathway B.
4.4 Discussion
4.4.1 Structure & Heterogeneity
4.4.2 Flexibility
The number of dihedral angle transitions in close vicinity of the Trp 48 residue provides a detailed picture of the flexibility of hydrophobic core. It is convincingly shown that the residues in the mutants exhibit a larger flexibility in both the apo- and holo form and that these can be ascribed to the mutated residues and residues nearby the position of the mutations, see Table 4.1. Phosphorescence emission of Trp is a sensitive probe that indicates the fluidity of the protein environment (Vanderkooi, 1991). The lifetime as measured by Cioni et al. of the I7S protein has decreased with a factor 2 and 3 with respect to wt for holo- and apo form respectively corresponding with the observed increase of flexibility (Cioni et al., 2004). The sizeable increase in flexibility in the apo-simulations emphasises the results even more.
The large increase of the lifetime of the holo form of the F110S protein (six-fold compared to wt holo-azurin) would suspect the formation a specific bonding arrangement resulting and complete immobilisation of the indole ring. In the simulations, however, only more flexibility is found. In the apo form (two-fold increase with respect to wt apo-azurin) even exchange of the water molecules with the bulk solvent is observed. Every property used to analyse the simulation confirms an increase of mobility and heterogeneity of the F110S mutant compared to wt.
4.4.3 Diffusion of water
of protein flexibility, by the elimination of an inner barrier to the diffusion process (Cioni et al., 2004).
4.5 Conclusion
The dynamics of the hydrophobic core of wt azurin and two variants of both the apo as well as the holo form was studied. The above presented analysis of the MD-simulations provide insight in their different behaviour.
The simulations agree with the crystallographic data: the presence of ordered water is established in the F110S holo-variant while this is not the case for the I7S holo form and both wt forms. The simulations also show that the Trp 48 is oriented in the same way with an equal spread of dihedral angle values for the different proteins complying with the equal B-factor along the different proteins.
The calculations stress that both mutants have an increased mobility and heterogeneity. It explains the shorter phosphorescence lifetime of the I7S variant with respect to wt azurin but no indication is found why the lifetime of the phosphorescence signal of F110S is larger than for wt azurin. The increased heterogeneity of the mutants resemble the conclusions based on steady-state fluorescence data.
Furthermore, the diffusion rate of water was examined. In both the apo- as well as the holo form of wt azurin no solvent molecules were found to penetrate into the protein core. In the case of the I7S variant, the core of the protein is relatively easily accessible for water molecules in both the apo as well as the holo form. In the case of the F110S variant the apo form shows a higher diffusion pattern than the holo form which is in agreement with the different dependence of the concentration of quencher on the phosphorescence lifetime. Since the mutants show an enormous increase of its dependence on the concentration of quencher compared to wt azurin, Cioni concluded that an opening was created by replacement the residues Ile 7 and Phe 110 (Cioni et al., 2004). This was confirmed by addressing the pathways of the solvent molecules entering the protein core.
