You are on page 1of 4

Accessible Conformations of Bacteriorhodopsin during 0.

05 ns
Alejandro G´mez Espinosa o
Escuela Polit´cnica Nacional e Quito - Ecuador

June 30, 2010

Abstract The study of structure differences characterizes protein conformation. The protein Bacteriorhodopsin is one of the best studied retinal proteins and despite of this study a clue of the folding protein problem could be found. In the present project, molecular dynamics simulation of bR during 0.05 ns equilibration time was perform and using cluster analysis, accessible conformation of this protein was found.

Retinylidene proteins (RP), also called Opsins, are a family of proteins found in photoreceptor cells that have an structure of seven transmembrane helices that enclose a chromophore called retinal. Bacteriorhodopsin is an example of such proteins. Nature uses this chromophore in processes such as vision, bioenergetics or phototaxis which is the movement of organisms in response to light stimuli (Hayashi et al 2003). Bacteriorhodopsin is a protein found in a purple membrane of the Halobacterium salinariumin, a Halobacteria archaea which are unicellular organisms who live where the salt concentration is very high, and weighs about 26 kDa according to Humphrey et al 1998. The study of this protein is because bR possesses the simplest known biological device of energy conversion, i.e. it carries out the simplest known form of biological photosynthetic energy storage (Ben-Nun et al 1998, Lanyi 1997 and Tajkhorshid et al. 2000); and is the best understood ion pump (Luecke et al. 1998). bR is built of seven transmembrane helix span its 45 ˚ width with the chromophore retinal linked A via a protonated Schiff base to Lysine 216 (Lys216) close to the middle of helix G as shown in Figure 1 and Figure 2. bR secondary structure, Figure 2, reveals that most of amino acids in alpha helices are nonpolar and hence this part of the protein is denominate as the hydrophobic part. Such caracteristic could be understood because these residues

are in contact with the hydrocarbon core of the membrane (Stryer 1995).

Figure 1: Graphical representation of bR. Alpha Helix are shown in purple in a New Cartoon representation and retinal in green in a WdV rep. [5]

Ion gradients are important forms of energy storage in nature and bR is a light-driven proton pump in the purple membrane. bR absorbs light and generates a proton gradient across the cellular membrane (Hayashi et al 2003). The extracellular and cytoplasmic sides are divided inside the interhelical cavity by the Schiff base, this channel describes the trajectory of the proton (Lanyi 1998). The proton gradient moves protons across


the cytoplasmic side to the extracellular side of the membrane in bacteria and other ATP-producing organelles (Hayashi et al 2003). Due to its unusual stability, the exhibition of a strong spectral shifts within the 500-650 nm range and the possibility of measure its vibrational spectra, bR is an ideal system resource for research (Luecke et al 1999; Humphrey et al 1998).

accessible conformations of bR in a lipid membrane at 300 K through a molecular dynamics simulation made by the NAMD (NAnoscalable Molecular Dynamics)software and the statistical analysis of the results.

Visual Molecular Dynamics (VMD) sotfware was utilised to get the protein in the right environment. The Protein Data Bank (PDB) file used here was 1C3W, but only the protein part of the file was taken out. Then, hydrogen were added to the protein. In addition, a lipid membrane of palmitoyl oleoyl phosphatidylcholine (POPC) of 60 ˚ was A formed on each side. The protein was located inside the membrane and overlapping atoms were removed. The final system had to be inside a solvate and for this reason membrane was solvated in a water located at each hidrophilic side of the membrane. Finally, some chlorine and sodium atoms were added to neutralize the final configuration, as shown in Figure 4.

Figure 2: A secondary structure model of bR showing the amino acids involved in proton translocation. Retinal is linked to Lys216 as a Schiff base. The arrow indicates the direction of proton transport. [3]

Photoisomerization processes change the retinal’s conformation from all-trans to 13-cis in the case of bR, as shown in Figure 3. It produces a large displacement of the methyl group C13 that makes a conformational change in E and F helices of bR and its cytoplasmic loops. When bR is activated by light, it opens the cytoplasmic channel that allows a proton to enter into the protein (Balu et al 2008).

Figure 4: bR (purple) inside a POPC membrane
(green) with water layers (red). [5]

Molecular Dynamics
After the system was completed, molecular dynamics simulations were performed with NAMD software. First, system was minimized in ten thousand steps of simulation. Once the system had the minimium energy configuration, it was warmed up to 300 K (room temperature) in twenty thousand steps. Finally, equilibrium process was realized in five hundred thousand steps i.e. 0.05 ns. Due to computational expense, only this time had been used but a greater time could give better results.

Figure 3: Retinal chromophores and their lightinduced isomerizations in Rh (left) and bR (right). [3]
Previous theoretical or experimental studies of bacteriorhodopsin made by Humphrey et al. 1998, Tajkorshid et al. 2000 or Balu et al. 2008 and others; have found some fundamental propierties of this protein. The purpose of this study is to get the


Figure 5: Dendogram found in CA of hundred sixty three different structures of a equilibrate bR in 0.05 ns. [11] Cluster Analysis
Cluster Analysis (CA) is a multivariate analysis method that classifizes a bunch of data in hierarchical levels depending on their similar characteristics, i.e. divides a group in subgroups of similar propierties. In the present study, Agglomerative Cluster Analysis was employed. Here, similar groups are formed from the bottom, i.e. similar objects are joined to form clusters that continued joined more general objects until all the objects are joined in a final cluster [9]. This arragenment could be shown graphically due to a dendogram. Once the system was equilibrated, hundred sixty three conformations was found during the equilibrate time. Then, dissimilarity matrix of bR conformations was calculated using the root mean square distance (RMSD) of each structure, with help of VMD. R software was utilized to perform CA and the dendogram shown in Figure 5 [11].

Figure 6: Graphical differences of seven accessible
conformations of bR. [5]

Results and Discussion
Seven accesible conformations of bR was found using Mojena Criteria (MC), such criteria divides dendogram in 2.5 distance. Once these seven similar groups were indentified, average structure of each group was calculated using VMD. Figure 6 shows the differences between the seven bR structures and Figure 7 shows such retinal differences. Because of the short time that DM simulation was perform, Figures 6 and 7 does not show great differences between this structures. Perhaps a greater simulation time could show better results but this study is a good start. In the future, a 1 ns simulation will be perform for bR and for a related retinale protein called Rhodopsin. The comparative study of these two proteins could figure out how different environment regulates conformational changes in retinal.

Figure 7: Graphical differences of seven accessible
conformations of retinal. [5]


[1] Balu, R., et al 2008. Terahertz Spectroscopy of Bacteriorhodopsin and Rhodopsin: Similarities and Differences. Biophysical Journal. 94(8). pp: 3217-3226. [2] Ben-Nun, M., et al., 1998. Quantum dynamics of the femtosecond photoisomerization of retinal in bacteriorhodopsin. Faraday Discuss. 110. pp:447-462. [3] Khorana, G. H., 1993. Two lighttransducing membrane proteins: Bacteriorhodopsin and the mammalian rhodopsin. Proc. Natl. Acad. Sci. USA. 90. pp:1166-1171. [4] Hayashi, S., Tajkhorshid, E., Schulten, K., 2003. Molecular Dynamics Simulation of Bacteriorhodopsin’s Photoisomerization Using Ab Initio Forces for the Excited Chromophore. Biophysical Journal. 85(3). pp:1440-1449. [5] Humphrey, W., Dalke, A., Schulten, K. 1996. VMD - Visual Molecular Dynamics, J. Molec. Graphics 14. pp:33-38. [6] Humphrey, W., et al, 1998. Three Electronic State Model of the Primary Phototransformation of Bacteriorhodopsin. Biophysical Journal. 75. pp:1689-1699. [7] Hoffmann, M., et al, 2006. Color Tuning in Rhodopsins: The Mechanism for the Spectral Shift between Bacteriorhodopsin and Sensory Rhodopsin II. J. Am. Chem. Soc. 128(33). pp:10808-10818.

[8] Lanyi, J., 1997. Mechanims of ion transport across membranes. Bacteriorhodopsin as a prototype for proton pumps. J. Biol. Chem. 272. pp:31209-31212. [9] Leps, J. Smilauer, P., 1999. Multivariate Analysis of Ecological Data. [Online] Czech Republic: Faculty of Biological Sciences. University of South Bohemia Ceske Budejovide. Available at pp: 61-64. [10] Luecke, H., et al, 1999. Structure of bacteriorhodopsin at 1.55 A resolution. J. Mol. Biol. 291. pp:899-911. [11] R Development Core Team, 2005. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-90005107-0, [12] Stryer, L., 1995. Biochemistry. 4th Ed. W.H. Freeman & Company. pp: 1319-1355. [13] Tajkhorshid, E., Baudry, J., Schulten, K., Suhai, S., 2000. Molecular dynamics study of the nature and origin of retinal’s twisted structure in bacteriorhodopsin. Biophysical Journal. 78(2). pp: 683-693. [14] Terakita, A., 2005. The Opsins. Genome Biol. [Online] 6(3). pp:213. Available at: http:www.ncbi.nlm.nih. govpmcarticlesPMC1088937?tool= pmcentrez [Accessed 12 June 2010]