Three-dimensional structure of yellow fluorescent protein zYFP538 from <Emphasis Type="Italic">Zoanthus</Emphasis> sp. at the resolution 1.8 Å

The three-dimensional structure of yellow fluorescent proteins zYFP538 (zFP538) from the button polyp Zoanthus sp. was determined at a resolution of 1.8 Å by X-ray analysis. The monomer of zYFP538 adopts a structure characteristic of the green fluorescent protein (GFP) family, a β-barrel formed from 11 antiparallel β segments and one internal α helix with a chromophore embedded into it. Like the TurboGFP, the β-barrel of zYFP538 contains a water-filled pore leading to the chromophore Tyr67 residue, which presumably provides access of molecular oxygen necessary for the maturation process. The post-translational modification of the chromophore-forming triad Lys66-Tyr67-Gly68 results in a tricyclic structure consisting of a five-membered imidazolinone ring, a phenol ring of the Tyr67 residue, and an additional six-membered tetrahydropyridine ring. The chromophore formation is completed by cleavage of the protein backbone at the C ^α -N bond of Lys66. It was suggested that the energy conflict between the buried positive charge of the intact Lys66 side chain in the hydrophobic pocket formed by the Ile44, Leu46, Phe65, Leu204 and Leu219 side chains is the most probable trigger that induces the transformation of the bicyclic green form to the tricyclic yellow form. A stereochemical analysis of the contacting surfaces at the intratetramer interfaces helped reveal a group of conserved key residues responsible for the oligomerization. Along with others, these residues should be taken into account in designing monomeric forms suitable for practical application as markers of proteins and cell organelles.     

Molecular dynamics simulations of a bacterial autotransporter: NalP from Neisseria meningitidis

NalP is an autotransporter secretory protein found in the outer membrane of Neisseria meningitidis. The crystal structure of the NalP translocator domain revealed a transmembrane β-barrel containing a central α-helix. The role of this α-helix, and of the conformational dynamics of the β-barrel pore have been studied via atomistic molecular dynamics simulations. Three simulations, each of 10 ns duration, of NalP embedded within a solvated DMPC bilayer were performed. The helix was removed from the barrel interior in one simulation. The conformational stability of the protein is similar to that of other outer membrane proteins, e.g., OmpA, in comparable simulations. The transmembrane β-barrel is stable even in the absence of the α-helix. Removal of the helix results in an influx of water into the pore region, suggesting the helix acts as a ‘plug’. Water molecules entering the resultant pore form hydrogen bonds with the barrel lining that compensate for the loss of helix-barrel hydrogen bonds. The dimensions of the pore fluctuate over the course of the simulation revealing it to be flexible, but only wide enough to allow transport of the passenger domain in an unfolded or extended conformation. The simulations help us to understand the role of the central helix in plugging the pore and in maintaining the width of the barrel, and show that the NalP monomer is sufficient for the transport of the passenger domain in an unfolded or extended conformation.     

Thermodynamic stability of water molecules in the bacteriorhodopsin proton channel: a molecular dynamics free energy perturbation study.

The proton transfer activity of the light-driven proton pump, bacteriorhodopsin (bR) in the photochemical cycle might imply internal water molecules. The free energy of inserting water molecules in specific sites along the bR transmembrane channel has been calculated using molecular dynamics simulations based on a microscopic model. The existence of internal hydration is related to the free energy change on transfer of a water molecule from bulk solvent into a specific binding site. Thermodynamic integration and perturbation methods were used to calculate free energies of hydration for each hydrated model from molecular dynamics simulations of the creation of water molecules into specific protein-binding sites. A rigorous statistical mechanical formulation allowing the calculation of the free energy of transfer of water molecules from the bulk to a protein cavity is used to estimate the probabilities of occupancy in the putative bR proton channel. The channel contains a region lined primarily by nonpolar side-chains. Nevertheless, the results indicate that the transfer of four water molecules from bulk water to this apparently hydrophobic region is thermodynamically permitted. The column forms a continuous hydrogen-bonded chain over 12 A between a proton donor, Asp 96, and the retinal Schiff base acceptor. The presence of two water molecules in direct hydrogen-bonding association with the Schiff base is found to be strongly favorable thermodynamically. The implications of these results for the mechanism of proton transfer in bR are discussed.     

Molecular mechanisms of the anomalous thermal aggregation of green fluorescent protein

The peculiarities of thermal denaturation and interaction with water of the cycle-3 mutant of green fluorescent protein (GFP) were analyzed by NMR techniques and compared with those of bovine carbonic anhydrase II (BCA-II). Irreversible thermal denaturation was accompanied by massive GFP aggregation with no detectable accumulation of soluble denatured protein. Analysis of the spin diffusion data suggested that the internal part of the GFP β-can is involved in intensive interactions with water molecules. As a result, at high temperatures, the GFP structure does not unfold but rather breaks, consequently leading to enhanced protein aggregation. This is very different from typical BCA-II behavior.     

Function and structure of GFP-like proteins in the protein data bank

The RCSB protein databank contains 266 crystal structures of green fluorescent proteins (GFP) and GFP-like proteins. This is the first systematic analysis of all the GFP-like structures in the pdb. We have used the pdb to examine the function of fluorescent proteins (FP) in nature, aspects of excited state proton transfer (ESPT) in FPs, deformation from planarity of the chromophore and chromophore maturation. The conclusions reached in this review are that (1) The lid residues are highly conserved, particularly those on the "top" of the β-barrel. They are important to the function of GFP-like proteins, perhaps in protecting the chromophore or in β-barrel formation. (2) The primary/ancestral function of GFP-like proteins may well be to aid in light induced electron transfer. (3) The structural prerequisites for light activated proton pumps exist in many structures and it's possible that like bioluminescence, proton pumps are secondary functions of GFP-like proteins. (4) In most GFP-like proteins the protein matrix exerts a significant strain on planar chromophores forcing most GFP-like proteins to adopt non-planar chromophores. These chromophoric deviations from planarity play an important role in determining the fluorescence quantum yield. (5) The chemospatial characteristics of the chromophore cavity determine the isomerization state of the chromophore. The cavities of highlighter proteins that can undergo cis/trans isomerization have chemospatial properties that are common to both cis and trans GFP-like proteins.     

Structural and dynamic roles of permanent water molecules in ligand molecular recognition by chicken liver bile acid binding protein

Chicken liver bile acid binding protein (cL-BABP) crystallizes with water molecules in its binding site. To obtain insights on the role of internal water, we performed two 100 ns molecular dynamics (MD) simulations in explicit solvent for cL-BABP, as apo form and as a complex with two molecules of cholic acid, and analyzed in detail the dynamics properties of all water molecules. The diffusion coefficients of the more persistent internal water molecules are significantly different from the bulk, but similar between the two protein forms. A different number of molecules and a different organization are observed for apo- and holo-cL-BABP. Most water molecules identified in the binding site of the apo-crystal diffuse to the bulk during the simulation. In contrast, almost all the internal waters of the holo-crystal maintain the same interactions with internal sidechains and ligands, which suggests they have a relevant role in protein-ligand molecular recognition. Only in the presence of these water molecules we were able to reproduce, by a classical molecular docking approach, the structure of the complex cL-BABP::cholic acid with a low ligand root mean square deviation (RMSD) with respect to its reference positioning. Literature data reported a conserved pattern of hydrogen bonds between a single water molecule and three amino acid residues of the binding site in a series of crystallized FABP. In cL-BABP, the interactions between this conserved water molecule and the three residues are present in the crystal of both apo- and holo-cL-BABP but are lost immediately after the start of molecular dynamics. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Hydration of enzyme in nonaqueous media is consistent with solvent dependence of its activity

Water plays an important role in enzyme structure and function in aqueous media. That role becomes even more important when one focuses on enzymes in low water media. Here we present results from molecular dynamics simulations of surfactant-solubilized subtilisin BPN' in three organic solvents (octane, tetrahydrofuran, and acetonitrile) and in pure water. Trajectories from simulations are analyzed with a focus on enzyme structure, flexibility, and the details of enzyme hydration. The overall enzyme and backbone structures, as well as individual residue flexibility, do not show significant differences between water and the three organic solvents over a timescale of several nanoseconds currently accessible to large-scale molecular dynamics simulations. The key factor that distinguishes molecular-level details in different media is the partitioning of hydration water between the enzyme and the bulk solvent. The enzyme surface and the active site region are well hydrated in aqueous medium, whereas with increasing polarity of the organic solvent (octane --> tetrahydrofuran --> acetonitrile) the hydration water is stripped from the enzyme surface. Water stripping is accompanied by the penetration of tetrahydrofuran and acetonitrile molecules into crevices on the enzyme surface and especially into the active site. More polar organic solvents (tetrahydrofuran and acetonitrile) replace mobile and weakly bound water molecules in the active site and leave primarily the tightly bound water in that region. In contrast, the lack of water stripping in octane allows efficient hydration of the active site uniformly by mobile and weakly bound water and some structural water similar to that in aqueous solution. These differences in the active site hydration are consistent with the inverse dependence of enzymatic activity on organic solvent polarity and indicate that the behavior of hydration water on the enzyme surface and in the active site is an important determinant of biological function especially in low water media.     

Simulations of apo and holo-fatty acid binding protein: structure and dynamics of protein, ligand and internal water.

Two molecular dynamics simulations of 5 ns each have been carried out for rat intestinal fatty acid binding protein, in apo-form and with bound palmitate. The fatty acid and a number of water molecules are encapsulated in a large interior cavity of the barrel-shaped protein. The simulations are compared to experimental data and analyzed in terms of root mean square deviations, atomic B-factors, secondary structure elements, hydrogen bond patterns, and distance constraints derived from nuclear Overhauser experiments. Excellent agreement is found between simulated and experimental solution structures of holo-FABP, but a number of differences are observed for the apo-form. The ligand in holo-FABP shows considerable displacement after about 1.5 ns and displays significant configurational entropy. A novel computational approach has been employed to identify internal water and to capture exchange pathways. Orifices in the portal and gap regions of the protein, discussed in the experimental literature, have been confirmed as major openings for solvent exchange between the internal cavity and bulk water. A third opening on the opposite side of the barrel experiences significant exchange but it does not provide a pathway for further passage to the central cavity. Internal water is characterized in terms of density distributions, interaction energies, mobility, protein contact times, and water molecule coordination. A number of differences are observed between the apo and holo-forms and related to differences in the protein structure. Solvent inside apo-FABP, for example, shows characteristics of a water droplet, while solvent in holo-FABP benefits from interactions with the ligand headgroup and slightly stronger interactions with protein residues.     

The protein import pore Tom40 in the microsporidian Nosema bombycis

Microsporidia, an unusual group of unicellular parasites related to fungi, possess a highly reduced mitochondrion known as the mitosome. Since mitosomes lack an organellar genome, their proteins must be translated in the cytosol before being imported into the mitosome via translocases. We have identified a Tom40 gene (NbTom40), the main component of the translocase of the outer mitochondrial membrane, in the genome of the microsporidian Nosema bombycis. NbTom40 is reduced in size, but it is predicted to form a β-barrel structure composed of 19 β-strands. Phylogenetic analysis confirms that NbTom40 forms a clade with Tom40 sequences from other species, distinct from a related clade of voltage-dependent anion channels (VDACs). The NbTom40 contains a β-signal motif that the polar residue is substituted by glycine. Furthermore, we show that expression of NbTom40, as a GFP fusion protein within yeast cells, directs GFP to mitochondria of yeast. These findings suggest that NbTom40 may serve as an import channel of the microsporidian mitosome and facilitate protein translocation into this organelle.     

The structure of bacterial outer membrane proteins

Integral membrane proteins come in two types, α-helical and β-barrel proteins. In both types, all hydrogen bonding donors and acceptors of the polypeptide backbone are completely compensated and buried while nonpolar side chains point to the membrane. The α-helical type is more abundant and occurs in cytoplasmic (or inner) membranes, whereas the β-barrels are known from outer membranes of bacteria. The β-barrel construction is described by the number of strands and the shear number, which is a measure for the inclination angle of the β-strands against the barrel axis. The common right-handed β-twist requires shear numbers slightly larger than the number of strands. Membrane protein β-barrels contain between 8 and 22 β-strands and have a simple topology that is probably enforced by the folding process. The smallest barrels form inverse micelles and work as enzymes or they bind to other macromolecules. The medium-range barrels form more or less specific pores for nutrient uptake, whereas the largest barrels occur in active Fe²⁺ transporters. The β-barrels are suitable objects for channel engineering, because the structures are simple and because many of these proteins can be produced into inclusion bodies and recovered therefrom in the exact native conformation.     

Molecular modeling of green fluorescent protein: structural effects of chromophore deprotonation

Molecular dynamics (MD) simulations were carried out to study the conformational rearrangement induced by deprotonation of the fluorescent chromophore in GFP, as well as the associated changes in the hydrogen-bonding network. For both the structures with either a neutral or an anionic chromophore, it was found that the beta-barrel was stable and rigid, and the conformation of the chromophore was consistent with the available x-ray structure. The conformational change in Thr203 due to deprotonation was also found to be consistent with the three-state isomerization model. Although GFP is highly fluorescent, denatured-GFP is nonfluorescent, indicating that the environment of the protein plays an important role in its fluorescence behavior. Our MD simulations, which explore the effect of the protein shell on the conformation of the chromophore, find the flexibility of the central chromophore to be significantly restricted due to the rigid nature of the protein shell. The hydrogen-bonding between the chromophore and neighboring residues was also shown to contribute to the chromophore rigidity. In addition to the MD studies, quantum mechanics/molecular mechanics (QM/MM) ONIOM calculations were carried out to investigate the effect of the beta-barrel on the internal rotation in the chromophore. Along with providing quantitative values for torsional rotation barriers about the bridging bond in the chromophore, the ONIOM calculations also validate our MD force field parameters.     

Distinct Effects of Guanidine Thiocyanate on the Structure of Superfolder GFP

Having a high folding efficiency and a low tendency to aggregate, the superfolder GFP (sfGFP) offers a unique opportunity to study the folding of proteins that have a β-barrel topology. Here, we studied the unfolding–refolding of sfGFP that was induced by guanidine thiocyanate (GTC), which is a stronger denaturing agent than GdnHCl or urea. Structural perturbations of sfGFP were studied by spectroscopic methods (absorbance, fluorescence, and circular dichroism), by acrylamide quenching of tryptophan and green chromophore fluorescence, and by size-exclusion chromatography. Low concentrations of GTC (up to 0.1 M) induce subtle changes in the sfGFP structure. The pronounced changes in the visible absorption spectrum of sfGFP which are accompanied by a dramatic decrease in tryptophan and green chromophore fluorescence was recorded in the range 0–0.7 M GNC. These alterations of sfGFP characteristics that erroneously can be mixed up with appearance of intermediate state in fact have pure spectroscopic but not structural nature. Higher concentrations of GTC (from 0.7 to 1.7 M), induce a disruption of the sfGFP structure, that is manifested in simultaneous changes of all of the detected parameters. Full recovery of native properties of denaturated sfGFP was observed after denaturant removal. The refolding of sfGFP passes through the accumulation of the off-pathway intermediate state, in which inner alpha-helix and hence green chromophore and Trp57 are still not tuned up to (properly integrated into) the already formed β-barrel scaffold of protein. Incorporation of the chromophore in the β-barrel in the pathway of refolding and restoration of its ability to fluoresce occur in a narrow range of GTC concentrations from 1.0 to 0.7 M, and a correct insertion of Trp 57 occurs at concentrations ranging from 0.7 to 0 M GTC. These two processes determine the hysteresis of protein unfolding and refolding.     

Active-site hydration and water diffusion in cytochrome P450cam: a highly dynamic process

Long-timescale molecular dynamics simulations (300 ns) are performed on both the apo- (i.e., camphor-free) and camphor-bound cytochrome P450cam (CYP101). Water diffusion into and out of the protein active site is observed without biased sampling methods. During the course of the molecular dynamics simulation, an average of 6.4 water molecules is observed in the camphor-binding site of the apo form, compared to zero water molecules in the binding site of the substrate-bound form, in agreement with the number of water molecules observed in crystal structures of the same species. However, as many as 12 water molecules can be present at a given time in the camphor-binding region of the active site in the case of apo-P450cam, revealing a highly dynamic process for hydration of the protein active site, with water molecules exchanging rapidly with the bulk solvent. Water molecules are also found to exchange locations frequently inside the active site, preferentially clustering in regions surrounding the water molecules observed in the crystal structure. Potential-of-mean-force calculations identify thermodynamically favored trans-protein pathways for the diffusion of water molecules between the protein active site and the bulk solvent. Binding of camphor in the active site modifies the free-energy landscape of P450cam channels toward favoring the diffusion of water molecules out of the protein active site.     

A multidomain outer membrane protein from Pasteurella multocida: Modelling and simulation studies of PmOmpA

PmOmpA is a two-domain outer membrane protein from Pasteurella multocida. The N-terminal domain of PmOmpA is a homologue of the transmembrane β-barrel domain of OmpA from Escherichia coli, whilst the C-terminal domain of PmOmpA is a homologue of the extra-membrane Neisseria meningitidis RmpM C-terminal domain. This enables a model of a complete two domain PmOmpA to be constructed and its conformational dynamics explored via MD simulations of the protein embedded within two different phospholipid bilayers (DMPC and DMPE). The conformational stability of the transmembrane β-barrel is similar to that of a homology model of OprF from Pseudomonas aeruginosa in bilayer simulations. There is a degree of water penetration into the interior of the β-barrel, suggestive of a possible transmembrane pore. Although the PmOmpA model is stable over 20 ns simulations, retaining its secondary structure and fold integrity throughout, substantial flexibility is observed in a short linker region between the N- and the C-terminal domains. At low ionic strength, the C-terminal domain moves to interact electrostatically with the lipid bilayer headgroups. This study demonstrates that computational approaches may be applied to more complex, multi-domain outer membrane proteins, rather than just to transmembrane β-barrels, opening the possibility of in silico proteomics approaches to such proteins.     

β-Barrel topology of Alzheimer's β-amyloid ion channels

Emerging evidence supports the ion channel mechanism for Alzheimer's disease pathophysiology wherein small β-amyloid (Aβ) oligomers insert into the cell membrane, forming toxic ion channels and destabilizing the cellular ionic homeostasis. Solid-state NMR-based data of amyloid oligomers in solution indicate that they consist of a double-layered β-sheets where each monomer folds into β-strand-turn-β-strand and the monomers are stacked atop each other. In the membrane, Aβ peptides are proposed to be β-type structures. Experimental structural data available from atomic force microscopy (AFM) imaging of Aβ oligomers in membranes reveal heterogeneous channel morphologies. Previously, we modeled the channels in a non-tilted organization, parallel with the cross-membrane normal. Here, we modeled a β-barrel-like organization. β-Barrels are common in transmembrane toxin pores, typically consisting of a monomeric chain forming a pore, organized in a single-layered β-sheet with antiparallel β-strands and a right-handed twist. Our explicit solvent molecular dynamics simulations of a range of channel sizes and polymorphic turns and comparisons of these with AFM image dimensions support a β-barrel channel organization. Different from the transmembrane β-barrels where the monomers are folded into a circular β-sheet with antiparallel β-strands stabilized by the connecting loops, these Aβ barrels consist of multimeric chains forming double β-sheets with parallel β-strands, where the strands of each monomer are connected by a turn. Although the Aβ barrels adopt the right-handed β-sheet twist, the barrels still break into heterogeneous, loosely attached subunits, in good agreement with AFM images and previous modeling. The subunits appear mobile, allowing unregulated, hence toxic, ion flux.     

Role of bulk water in hydrolysis of the rhodopsin chromophore

Rhodopsin (Rho) is a prototypical G protein-coupled receptor that changes from an inactive conformational state to a G protein-activating state as a consequence of its retinal chromophore isomerization, 11-cis-retinal → all-trans-retinal. The photoisomerized chromophore covalently linked to Lys(296) by a Schiff base is subsequently hydrolyzed, but little is known about this reaction. Recent research indicates a significant role for tightly bound transmembrane water molecules in the Rho activation process. Atomic structures of Rho and hydroxyl radical footprinting reveal ordered waters within Rho transmembrane helices that are located close to highly conserved and functionally important receptor residues, forming a hydrogen bond network. Using (18)O-labeled H(2)O, we now report that water from bulk solvent, but not tightly bound water, is involved in the hydrolytic release of chromophore upon Rho activation by light. Moreover, small molecules (and presumably, water) enter the Rho structure from the cytoplasmic side of the membrane. Thus, this work indicates two distinct origins of water vital for Rho function.     

Can the fluorescence of green fluorescent protein chromophore be related directly to the nativity of protein structure?

In studies of green fluorescence protein (GFP) or other proteins with the use of GFP as a marker, the fluorescence of GFP is for the most part related directly to the nativity of its structure. Naturally, such a relation does exist since the chromophore of this protein is formed autocatalytically only just after GFP acquires its native structure. However, the fluorescence method may not yield reliable information on protein structure when studying renaturation and denaturation of this protein (with the formed chromophore). Using proteolysis, denaturant gradient gel electrophoresis and circular dichroism, we demonstrate herein that at major disturbances of the native structure of protein GFP-cycle3 the intensity of fluorescence of its chromophore can change insignificantly. In other words, the chromophore fluorescence does not reliably mirror alterations in protein structure. Since the main conclusions of this study are especially qualitative, it can be suggested that during renaturation/denaturation of wild-type GFP and its “multicolored” mutants their fluorescence is also not always associated with the changes in the structure of these proteins.     

Water penetration in the low and high pressure native states of ubiquitin

Theoretical studies on the solvation of methane molecules in water have shown that the effect of increased pressure is to stabilize solvent separated contacts relative to direct contacts. This suggests that high pressure stabilizes waters that have penetrated into a protein's core, indicating a mechanism for the high pressure denaturation of proteins. We test this theory on a folded protein by studying the penetration of water into the native state of ubiquitin at low and high pressures, using molecular dynamics. An ensemble of conformations sampled in the folded state of ubiquitin has been determined by NMR at two pressures below the protein's denaturation pressure, 30 atm and 3000 atm. We find that 1-5 more waters penetrate the high pressure conformations than the low pressure conformations. Low volume configurations of the system are favored at high pressures, but different components of the system may experience increases or decreases in their specific volumes. We find that penetrating waters have a higher volume per water than bulk waters, but that the volume per protein residue may be lowered by solvation. Furthermore, we find that penetration of the protein by water at high pressures is driven by the difference in the pressure dependence of the probability of cavity opening in the protein and pressure dependence of the probability of cavity opening in the bulk solvent. The volume changes associated with cavity opening and closing indicate that each penetrating water reduces the volume of the system by about 12 mL/mol. The experimental volume change going from the low pressure to the high pressure native state of ubiquitin is 24 mL/mol. Our results indicate that this volume change can be explained by penetration of the protein by two water molecules.