As basis for the structural models of the bR, O and O* state served a recently released crystal structure of the bR state which was obtained by time-resolved serial femtosecond crystallography25. The heavy atom coordinates (see Figure 6.1A) of this structure were obtained from the PDB file with the ID 5B6V. Beside the amino acid sequence, this file also contained 17 entries for crystal water which were not deleted. This is mainly because most of these water molecules were located in the active site cavity close to D85, D212,
aFor example the observation of a water wire between D85 and the PRG68,69,160.
48 CHAPTER 6. STRUCTURAL MODELS FOR THE BR/O/O* STATE
Figure 6.1: Workflow of the bR model setup. The crystal structure with the PDB-ID 5B6V (A) was embedded in a POPC bilayer (during step I). The protein/membrane-complex was subsequently solvated and a chloride ion was added in order to gain charge neutrality of the system (during step II). The whole system comprised the truncated 230 amino acid long, ground state bR, 284 POPC lipids, 16381 TIP3P waters and one chloride counterion (C).
R82, E204, E194 and it is well known that this region contains structurally important water molecules which build up a HBN. This decision is furthermore underpinned by the relatively high crystallographic resolution of 2.0 Å and the very low, thus reliable, B-factors (38.9 - 72.7 Å2) of the water oxygens.
For MD simulations, Gromacs v.5.0161in combination with the Plumed v.2.1.1 plugin162
was used. The CHARMM36 force field was used for the description of the whole system comprising protein, lipid phase, water phase and ions. Parameters for the retinal molecule were taken from refs. 163–167. During topology creation with pdb2gmx all titratable amino acids were kept in their physiological protonation state, identical to the bR state. As lipid environment, a 500 ns preequilibrated POPC bilayer was taken which was provided by Violetta Schneider.
For the embedding of the bR protein into the POPC bilayer the InflateGRO168method- ology was used. During this workflow the protein was centered in the lipid bilayer. In the next step, overlapping lipids within a cutoff radius of 14Å were removed, and the bilayer was artificially inflated in it’s plane. At the end, several shrinking steps followed which compressed the bilayer and packed the lipids around the protein. The shrinking procedure was seen as “converged”, when the area per lipid value of the POPC bilayer was close to the experimental value169 of 65.8 Å2 (see Figure A.1).
The protein/membrane-complex (see Figure 6.1B) was solvated by using the gmx solvate routine with the CHARMM TIP3P as water model. During solvation, small gaps between the lipid acyl chains also got filled with water. These misplaced water molecules were removed from the structure with the help of a Python script which was written by Dr.
6.1. SIMULATION SETUP 49
Sabine ReiSSer. In order to gain charge neutrality of the system one chloride counterion was added with the gmx genion routine. The whole workflow of the system setup is summarized in Figure 6.1. The whole system comprised the truncated 230 amino acid long, ground state bR, 284 POPC lipids, 16381 TIP3P waters and one chloride counterion (see Figure 6.1C). Energy minimization was conducted using the steepest descent minimization algorithm with a maximum force threshold of 1000 kJ/(mol· nm). The system was equilibrated in the NVT ensemble for 200 ps at 300 K using the V-rescale170 thermostat. Furthermore, xyz- position restraints were placed on the heavy atoms of protein and lipids. An unrestrained NPT simulation over 100 ns followed with a temperature and pressure coupling to 300 K and 1.0 bar. The temperature was controlled via the Nosé-Hoover thermostat and the pressure via the Parrinello-Rahman barostat. Since it is known that MD simulations are insufficient for the preservation of the pentagonal HBN67, an additional bias potential was applied which should ensure the intactness of the HBN. Therefore, distance-dependent, harmonic restraints were applied between the HBN water molecules and D85, D212, K216RET. The biasing force of κ = 8000 kJ/(mol·nm2) was activated, when the distance between the water oxygen atom and the respective amino acid atom was larger than 2.8 Å.
6.1.1 Protonation States and Hamiltonian Replica Exchange Simulations
On basis of the 100 ns equilibrated bR state structure the two further models for the O and O* state were created. Therefore, protonation states of the aspartates/glutamates were changed. More precisely, for the O state model, E204 was deprotonated and D85 protonated. The same was done to mimic the O* state but instead of D85, D212 was protonated. The O and O* state model were minimized and NVT equilibrated like before (cf. section 6.1, p 47).
The three protonation models were then prepared for enhanced sampling simulations employing HREX151. Since it is known that the NPT ensemble disrupts the HREX exchange rates, these simulations were conducted in the NVT ensemble with the V-rescale thermostat. All three protonation models were equilibrated over 200 ps. The HREX simulations had for each protonation model the same setup: 16 replicas were used which spanned a λ scaling range from 1 – 0.16. This corresponds to a simulation temperature range of 300 – 1900 K. The frequency for an exchange-attempt was set to 4 ps. The region, where the scaling was applied to, consisted of the active site amino acids D85, D212, K216RET, R82, E194, E204 and all interacting amino acids in and around this region. A complete list with the amino acids of the “hot region” and the justification for their selection is shown in Table 6.1. The finished simulations were then analyzed for a proper exchange of the individual replicas. All three simulations exhibited a frequent exchange of their replicas (see Figure A.2) which is a prerequisite for efficient sampling. As basis for the analysis of each state, served the corresponding unscaled (λ = 1) replica.
50 CHAPTER 6. STRUCTURAL MODELS FOR THE BR/O/O* STATE
Table 6.1: Selected amino acids of the HREX “hot region” are listed together with their justification of selection.
Amino acid Function
D85 proton donor + member of the pentagonal HBN
D212 proton acceptor/donor + member of the pentagonal HBN K216RET Member of the pentagonal HBN
R82 Up-/Downswing movement during photocycle
E194 Part of the PRG region
E204 Part of the PRG region
Y185 Hydrogen-bonded to D212
W86 Hydrogen-bonded to D212
E9 Polar amino acid near to the PRG
Y83 Polar amino acid near to the PRG
S193 Hydrogen-bonded to the E204
P77 Polar amino acid near to the PRG + hydrogen-bonded to S193
Y57 Hydrogen-bonded to D212
F208 Serves together with R82 as hydrophobic plug
Y79 Polar amino acid near to the PRG + hydrogen-bonded to E9
P200 Hydrogen-bonded to E204
W189 Polar amino acid near to the PRG + hydrogen-bonded to Y83