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Figure 1 Ribbon diagram of bacteriorhodopsin including seven trans membrane helices (A-G) and retinal chromophore in ball and stick form, click here for reference website.
 

ABSTRACT

In the science of structural biology, great leaps in knowledge in the past ten years have increased our knowledge of protein structure and function. One class of proteins which have been slow to yield information about both structure and functional dynamics has been the integral membrane protein (IMP). A good example of a well-studied membrane protein is bacteriorhodopsin, whose structure was identified using cryo-electron microscopy twenty-five years ago using naturally occurring 2D crystals by Henderson and Unwin at Cambridge UK[1]. The applications of understanding not only the structure of the seven trans-membrane helices, but the manner in which this protein functions as a light-driven proton pump, have contributed to the development of a large body of research. A complete understanding of the photoisomerization process in other organisms as well as the bioenergetics and biomechanics of membran proteins will be much closer using the model obtained from bacteriorhodopsin.Subsequently, structural models have been developed for both the unilluminated state of bacteriorhodopsin [2,3] and several intermediates of the photochemical cycle [4,5,6]. There are several theories as to the conformational relationships involved in the transport of protons across the membrane, x-ray diffraction maps to resolutions of 1.9 Å [2] and 1.55 Å [7] have revealed significant details concerning the active center of the protein and the protonation of the retinal Schiff base.
 

Figure 2 A view an x-ray structural representation of the proton pathway with a few of the key amino acids in red, the retinal cofactor in green, and some catalytically important waters in white.   The top of the figure is the cytoplasm and the bottom is the extracellular matrix.  Note the increased number of waters found in the vicinity of the extracellular matrix.
 

HISTORICAL BACKGROUND
 

The first information concerning bacteriorhodopsin dates back to the early 1960s when Mitchell first speculated on the nature of its structure and function [8]. Due to the natural formation of two-dimensional crystals called purple membrane, bacteriorhodopsin presented the rare opportunity to examine a membrane protiein without the difficulty of producing adequate quantities for crystallization.  Initial research focused on the elucidation of its orientation in the membrane, which revealed its transmembrane alpha-helical character. The number of analytical approaches taken with bacteriorhodopsin since its discovery include several techniques including high resolution electron microscopy, x-ray crystallography, Fourier Transform IR, neutron scattering, and, most recently, Solid-state nuclear magnetic resonance [9].  The two organisms of choice in structural research have been Halobacterium salinarum and H. halobium.  Bacteriorhodopsin became an ideal choice for study because of its naturally high levels in the plasma membrane of these halophilic archaeans under conditions of low oxygen tension and high light. A relatively small protein of about 26 kDa in molecular weight, bacteriorhodopsin is similar in structure and size to halorhodopsin and sensory rhodopsins [11].  The difficulty with obtaining a more detailed understanding of the molecular mechanisms of a membrane protein lies in the maintenance of biological activity in the process of purification. This is due to the fact that because it has both hydrophobic and hydrophilic regions that are not soluble in aqueous buffer solutions and denature in organic solvents.  Several approaches have been developed to crystallize the protein in its "native" conformation, including an entirely novel technique employed  by Landau and Rosenbusch called lipidic cubic phases, the first able to "form a structured, transparent, and complex thre-dimensioal lipidic array pervaded by an intercommunicating aqueous channel system" [10]. This resulted in the creation of a vital protocol for the creation of crystals for not only bacteriorhodopsin but other membrane proteins as well.

    The presence of a molecule of retinal, whose isomerization is key in the visual process, made it even more interesting as an object of study.  It forms a two-dimensional hexagonal crystal of trimers in the plasma membrane.  Spanning the lipid bilayer seven times, the keys to unlocking its mechanism lie in understanding the complex conformational changes it undergoes in its photochemical cycle.  Studies of the cycle have shown it can be divided into a series of intermediate states (termed K, L, M1, M2, N, and O).  Researchers are still attempting to determine the thermodynamics of each step of conformational changes which allow for the vectorial transport of protons across the membrane against a concentration gradient.

 Beginning in about 1990, however, pioneering work by Henderson and coworkers produced the first atomic level models of bacteriorhodopsin (PDB codes 1BRD and 2BRD) [12].  A recent review by Luecke notes that there are twenty atomic coordinate entries deposited in the Protein Data Bank for bacteriorhodopsin [13].  The various structures include both electron diffraction and x-ray crystallographic models as well as wild-type and mutant forms.  Point mutations have allowed researchers to determine the role of certain amino acid residues on proton transport.  The twenty entries in PDB can also be divided in terms of ground state structure vs. various cryo-trapped photocycle intermediates.  The most recent structure of ground state, wild-type bacteriorhodopsin is at 1.5 Å resolution by Luecke et al (PDB code 1C3W) [7].  His work allows structural feactures such as all intramembrane residue and backbone interactions to be seen with more clarity than previous work [7] The ultimate goal for researches is to resolve beyond the current level of resolution to determine the exact location of key water molecules involved in the catalytic pathway and pinpoint the sequence of events leading to transport of protons across the plasma membrane.

MOLECULAR MECHANISMS OF PHOTOCYCLE
 

The main focus of modern research on bacteriorhodopsin has been the exact mechanism behind the light-driven proton pump which it forms.  A clear sequence of events was determined from early x-ray diffraction models [14,15,16] which showed the involvement of a retinal photoisomerization reaction.  As time went by, higher resolution models have shown a clearer picture of the interaction between the retinal and the side chains of the nearby alpha helical substructures [17].  As mentioned above, a large body of research has concerned itself with the effect of changing key amino acids on the catalytic activity of bacteriorhodopsin by synthesizing point mutations.

The photocycle of bacteriorhodopsin can be broken down into a series of discrete steps.  The first step is the ground state conformation of the protein when retinal is in an all-trans conformation.  Click here for a closer view of a the retinal cofactor.  The position of the covalent attachment between the retinal and the Lys216 residue of the bacteriorhodopsin is shown here with the lysine residue in red and the retinal in white.  This is termed the Schiff base of the retinal cofactor.  The first step in the catalytic cycle is the photoisomerization of retinal from all-trans to 13-cis which shifts the Schiff base in the direction of the proton acceptor on the extracellular side of the protein.  The isomerization of retinal is believed to change the geometry and the electrostatics at the Schiff base. The putative initial proton acceptor has been proven to be Asp85 and lies in the extracellular channel [18].

Once the proton has shifted from Schiff base to Asp85, the orientation of the retinal is shifted from in the extracellular direction to toward the cytoplasm as part of a complex shift of hydrogen bonds between Schiff base and surrounding residues and water molecules [3].  This prevents the reprotonation of the Schiff base by Asp85 and facilitates the transfer of a proton from the cytoplasmic channel.  Consequently, the retinal is reisomerized back to all-trans and the Schiff base is reprotonated by Asp96 (which is between the retinal and the cytoplasm) and resettles in its original position toward the extracellular channel.  In the process a proton is vectorially transported from the cytoplasm to the extracellular matrix against concentration gradient and provides energy in the process to fuel adjacent the catalytic activity of adjacent ATP synthase.

Photocycle Breakdown

I. Ground State

The ground state, or light-adapted state, is commonly referred to as BR in the literature.  It is the best-defined molecular state in the photocycle.  The active site on BR contains the retinal cofactor in an all-trans conformation and in close contact with surrounding protein residues.  The protonated Schiff base is in direct hydrogen-bonding contact to water 402.  On the extracellular side, a network of hydrogen bonds leads from the Schiff base to a terminal proton release group consisting of two glutamate residues (Glu194 and Glu204) via a series of amino acids including Asp85.  Initially Asp85 is deprotonated.  The beginning of the photocycle is marked the absorption of a photon and deposition of approximately 50 kcal/mol of energy into the retinal.

II. K State

After the absorption of a photon, the K state forms within a few picoseconds.  This intermediate has been determined to have a highly strained retinal configuration in the 13-cis,15-anti form.  Edman et al. has done work to trap the low-temperature K intermediate at temperatures between 110 K and 125 K for the purpose of showing changes around the retinal chromophore early in the photocyle [16].  The successful trapping and characterizing of the K intermediate has shown the key role of a pi bulge near Lys216 and several water molecules located in the hydrogen-bond network around the Schiff base in the helical shifts surrounding proton transport.  The pi bulge in helix G has been implicated in chain of covalent bonds that connects the region of the retinal in the cytoplasmic direction toward Asp96.  The event of photoexcitation and isomerization of retinal is linked to the beginning of a series of reorienting of hydrogen bonds which creates an energy pathway favoring proton movement across the membrane [16].  As an early intermediate, it is key to understand the sequence of events between photoexcitation and initial deprotonation of the Schiff base (early M state).

III. L State

In this state, the strained retinal begins to ease and hydrogen bonds around the active state begin to shift toward the transition to the M state.  Royant et al. have been able to establish conditions under which a high population of low-temperature L intermediate build up within crystals of wild-type bacteriorhodopsin in order to determine its structure [5].  Similar to Edman et al., Royant's group used microspectrophotometric techniques to develop a trapping protocol for the purple membrane at extremely low temperatures.  They were able to show conclusively the beginnings of helical conformational changes prior to deprotonation of the Schiff base [5].  They theorize that the "early dislocation of a single key water molecule facilitates a cascade of structural rearrangements propagating from the active site towards the extracellular surface, inducing the transient deformation of an alpha-helix" [5].

IV. M State

It is in this state the deprotonation of the Schiff base to Asp85 occurs.  It is divided by researchers into two substate, termed M1 and M2.  Initially, a pKa difference between the proton donor or Schiff base and the proton accepton or Asp85 exists of over 11 units.  This must be reduced for the protonation reaction to proceed and this accomplished by transition between the L, M1, and M2 states.  After the proton has been transferred to Asp85, the Schiff base is reoriented away from the extracellular channel to prevent reprotonation and insure vectorial ion transport.  The use of a D96N mutant allowed for the full conversion to the M mutant and a slower decay.  This resulted in the first X-ray structure of an intermediate [19] which has allowed comparison to the light-adapted structure previously known.  Sass et al. determined the structure (PDB code 1CWQ) of a late M intermediate of wild-type bacteriorhodopsin at 2.25 Å resolution [6] and observed a net of water molecules involved in moving a proton from the donor group Asp96 towards the Schiff base.  They were able to observe a cavity around Asp96 which they believe to facilitate the de- and reprotonation of the donor group by shifting the water net around it [6].

V. N State

During this intermediate state, the Schiff base is reprotonated from a proton transported from Asp96. This can occur in either a single conformational change or a series of steps between late M state and early O state. The binding site of retinal is now in a relaxed state and still 13-cis,15-anti though it now prefers this conformation energetically.

VI. O State

During the O state, Asp96 has been reprotonated with a proton transported from the cytoplasm and retinal has been reisomerized to an all-trans conformation.  The low pKa of Asp85 is reestablished and the terminal proton release group is reprotonated from Asp85 as a result of this reestablishment.  The molecule now returns to a ground state.  Work continues to clarify the exact bioenergetics involved in the reisomerization of retinal and returning to the ground state.

The increasing resolution of molecular models of bacteriorhodopsin, both wild-type and mutant, has allowed researchers to view the more minute details of the photocycle.  There is evidence to tie water molecules integrated into the complex intrahelical hydrogen-bond network with the catatlytic activity.  Several water molecules (see figure 2 or click here) such as water 400, water 401, and water 402 have been shown to be actively involved in the orientation of the Schiff base toward either the cytoplasm or the extracellular matrix. Recently, solid-state NMR spectroscopy has revealed the geometrical arrangement of the Schiff base in the all-trans, 15-anti,13-cis, and the 15-syn conformations which allows for  closer examination of the active site around the time of proton exchange [20].

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ORAL PRESENTATION

SOME FINAL THOUGHTS
 

The transient nature of the photocycle intermediates compounds the difficulty of crystallizing these for visualization.  Molecular conformational changes on the level of femtoseconds make it difficult to capture the directional movement of a proton between the cytoplasm and the extracellular matrix via intermediate positions.  Improved techniques have allowed for advancing the research of bacteriorhodopsin and other membrane proteins.  Beginning with its effects on cryo-electron microscopy over twenty-five years ago, bacteriorhodopsin has served as a tool for molecular biologists and biophysicists to increase their knowledge of membrane dynamics and bioenergetics.  It continues to hold secrets which researchers today strive to unlock.