Peter Nollert

High-resolution X-ray structure of an early intermediate in the bacteriorhodopsin photocycle

Bacteriorhodopsin is the simplest known photon-driven proton pump and as such provides a model for the study of a basic function in bioenergetics. Its seven transmembrane helices encompass a proton translocation pathway containing the chromophore, a retinal molecule covalently bound to lysine 216 through a protonated Schiff base, and a series of proton donors and acceptors. Photoisomerization of the all-trans retinal to the 13-cis configuration initiates the vectorial translocation of a proton from the Schiff base, the primary proton donor, to the extracellular side, followed by reprotonation of the Schiff base from the cytoplasm. Here we describe the high-resolution X-ray structure of an early intermediate in the photocycle of bacteriorhodopsin, which is formed directly after photoexcitation. A key water molecule is dislocated, allowing the primary proton acceptor, Asp 85, to move. Movement of the main-chain Lys 216 locally disrupts the hydrogen-bonding network of helix G, facilitating structural changes later in the photocycle.

X-ray structure of sensory rhodopsin II at 2.1-Å resolution

Sensory rhodopsins (SRs) belong to a subfamily of heptahelical transmembrane proteins containing a retinal chromophore. These photoreceptors mediate the cascade of vision in animal eyes and phototaxis in archaebacteria and unicellular flagellated algae. Signal transduction by these photoreceptors occurs by means of transducer proteins. The two archaebacterial sensory rhodopsins SRI and SRII are coupled to the membrane-bound HtrI and HtrII transducer proteins. Activation of these proteins initiates phosphorylation cascades that modulate the flagellar motors, resulting in either attractant (SRI) or repellent (SRII) phototaxis. In addition, transducer-free SRI and SRII were shown to operate as proton pumps, analogous to bacteriorhodopsin. Here, we present the x-ray structure of SRII from Natronobacterium pharaonis (pSRII) at 2.1-Å resolution, revealing a unique molecular architecture of the retinal-binding pocket. In particular, the structure of pSRII exhibits a largely unbent conformation of the retinal (as compared with bacteriorhodopsin and halorhodopsin), a hydroxyl group of Thr-204 in the vicinity of the Schiff base, and an outward orientation of the guanidinium group of Arg-72. Furthermore, the structure reveals a putative chloride ion that is coupled to the Schiff base by means of a hydrogen-bond network and a unique, positively charged surface patch for a probable interaction with HtrII. The high-resolution structure of pSRII provides a structural basis to elucidate the mechanisms of phototransduction and color tuning.

Molecular mechanism for the crystallization of bacteriorhodopsin in lipidic cubic phases.

Crystals of transmembrane proteins may be grown from detergent solutions or in a matrix of membranous lipid bilayers existing in a liquid crystalline state and forming a cubic phase (in cubo). While crystallization in micellar solutions appears analogous to that for soluble proteins, crystallization in lipidic matrices is poorly understood. As this method was shown to be applicable to several membrane proteins, understanding its mechanism will facilitate a rational design of crystallization, minimizing the laborious screening of a large number of parameters. Using polarization microscopy and low‐angle X‐ray diffraction, experimental evidence is provided to support a mechanistic model for the in cubo crystallization of bacteriorhodopsin in a lipid matrix. Membrane proteins are thought to reside in curved lipid bilayers, to diffuse into patches of lower curvature and to incorporate into lattices which associate to form highly ordered three‐dimensional crystals. Critical testing of this model is necessary to generalize it to other membrane proteins.

Protein Interactions and Membrane Geometry

The difficulty in growing crystals for x-ray diffraction analysis has hindered the determination of membrane protein structures. However, this is changing with the advent of a new method for growing high quality membrane protein crystals from the lipidic cubic phase. Although successful, the mechanism underlying this method has remained unclear. Here, we present a theoretical analysis of the process. We show that it is energetically favorable for proteins embedded in the highly curved cubic phase to cluster together in flattened regions of the membrane. This stabilizes the lamellar phase, permitting its outgrowth from the cubic phase. A kinetic barrier-crossing model is developed to determine the free energy barrier to crystallization from the time-dependent growth of protein clusters. Determining the values of key parameters provides both a rational basis for optimizing the experimental procedure for membrane proteins that have not yet been crystallized and insight into the analogous cubic to lamellar transitions in cells. We also discuss the implications of this mechanism for protein sorting at the exit sites of the Golgi and endoplasmic reticulum and the general stabilization of membrane structures.

Crystallization of membrane proteins in Cubo

Our understanding of lipidic cubic phases for the crystallization of membrane proteins has advanced greatly since the inception of the concept in 1996, and the method is becoming well accepted. Several protocols that allow the efficient screening of crystallization conditions and handling of crystals are presented. State-of-the art micro techniques allow a large number of crystallization conditions to be tested using very small amounts of protein, and diffraction quality crystals can be grown in larger volumes in glass vials. In cubo crystallization conditions differ from those employed for detergent-solubilized proteins. Variations comprise the type of lipid matrix, detergent, protein, salt, temperature, hydration, pH, and pressure. Commercially available screening kits may be applied in order to define lead conditions. Once obtained, crystals may be removed from the surrounding cubic phase mechanically, by enzymatic hydrolysis, or by detergent solubilization. We anticipate this set of protocols to be applied successfully to larger, less stable, and noncolored membrane proteins in order to obtain well-diffracting crystals of membrane proteins that have so far evaded crystallization in the detergent-solubilized state.

Early Structural Rearrangements in the Photocycle of an Integral Membrane Sensory Receptor

Sensory rhodopsins are the primary receptors of vision in animals and phototaxis in microorganisms. Light triggers the rapid isomerization of a buried retinal chromophore, which the protein both accommodates and amplifies into the larger structural rearrangements required for signaling. We trapped an early intermediate of the photocycle of sensory rhodopsin II from Natronobacterium pharaonis (pSRII) in 3D crystals and determined its X-ray structure to 2.3 A resolution. The observed structural rearrangements were localized near the retinal chromophore, with a key water molecule becoming disordered and the retinals beta-ionone ring undergoing a prominent movement. Comparison with the early structural rearrangements of bacteriorhodopsin illustrates how modifications in the retinal binding pocket of pSRII allow subtle differences in the early relaxation of photoisomerized retinal.

Atomic structure of a glycerol channel and implications for substrate permeation in aqua(glycero)porins

The structure of a glycerol channel from Escherichia coli at 2.2 Å resolution serves as a basis for the understanding of selective transmembrane substrate permeation. In the course of permeation, glycerol molecules diffuse through a tripathic channel with their alkyl backbone wedged against a hydrophobic corner, such that OH groups become acceptors and donors of hydrogen bonds at the same time. The structure of the channel explains the preferential permeability for linear carbohydrates and absolute exclusion of ions and charged solutes. Its gene‐duplicated sequence has a structural counterpart in a pseudo two‐fold symmetry within the monomeric channel protein.

From test tube to plate: a simple procedure for the rapid preparation of microcrystallization experiments using the cubic phase method

Crystals of transmembrane proteins for X-ray diffraction experiments may be grown either by employing mixed protein–detergent complexes, or in a matrix of liquid-crystalline membraneous material forming a lipidic cubic phase (in cubo). Widespread use of the in cubo method has been severely hampered by its tediousness and the large amounts of protein required. Here a simple procedure is presented that by virtue of its simplicity and small setup size substantially reduces the preparation time as well as the amount of protein. Crystallization trials are set up in conventional multi-well plates using a semi-automatic dispenser-driven microsyringe. The microprocedure is amenable to full automation and further miniaturization. Its feasibility is demonstrated by screening for new crystallization conditions for bacteriorhodopsin using volumes of ca 200 nl of lipidic cubic phase. New crystallization conditions were identified that avoid the necessity of weighing solid precipitation agents.

The glycerol facilitator GlpF its aquaporin family of channels, and their selectivity.

Publisher Summary The “glycerol facilitator” GlpF, a highly selective transmembrane channel that conducts glycerol and certain other small uncharged organic molecules, was the first member of the large aquaporin family (AQP family) to be identified at the genetic level and functionally characterized. Once glycerol is passaged inside the cell, it is rapidly phosphorylated by glycerol kinase to produce glycerol 3-phosphate that proceeds by dehydrogenation to dihydroxyacetone phosphate (DHAP) or to phospholipid synthesis where glycerol provides the basis for attachment of fatty acid chains and phophatidyl head groups in ∼2/3 of cellular phospholipids. The glp regulon is inducible by glycerol 3-phosphate which provides an advantage for growth of Escherichia coli on glycerol. The glpF gene is the first gene on the glp operon that also contains the gene for glycerol kinase.

Encoding Selectivity of a Transmembrane Channel

Membrane channel proteins of the aquaporin family are integral plasma membrane proteins that are highly selective for permeation of specific small molecules, with absolute exclusion of ions including protons or OHanions and charged solutes, and without dissipating the electrochemical potential across the cell membrane. There are 11 human family members. The family includes channels that conduct water, called aquaporins, and channels that conduct glycerol or other linear polyalcohols (aquaglyceroporins), and ion conducting channels. The E. coli glycerol facilitator (GlpF) was cloned, expressed, purified, and crystallized with its primary permeant substrate glycerol. The crystal structure of GlpF, determined by multiple isomorphous replacement at 2.2 A resolution illustrate the basis for its selectivity. Glycerol molecules G1, G2, G3 line up in an amphipathic channel in single file. In the narrow selectivity filter of the channel, the glycerol alkyl backbone is wedged against a hydrophobic corner and successive OH groups form hydrogen bonds with a pair of acceptor, and donor atoms. These hydrogen bonds serve to orient the CHXOH groups within the narrower regions of the channel, and explain the stereo, and enantio selectivity measured for this channel. Two conserved -Asn Pro Alamotifs meet in the center of the membrane and form a conserved interface between two gene-duplicated segments that each encode three and one half membrane spanning helices around the channel. This structure elucidates the mechanism of selective permeability for linear carbohydrates and suggests how ions and water are excluded. A functional assay was developed, and shows that the channel is both stereoand enantio-selective. The conservation of sequence shows how the water channels of the same family conduct water and exclude glycerol.