Structures of photointermediates and their implications for the proton pump mechanism.

It is widely accepted that bacteriorhodopsin undergoes global conformational changes during its photocycle. In this review, the structural properties of the M and N intermediates are described in detail. Based on the clarified global conformational change, we propose a model for the molecular mechanism of the proton pump. The global structural change is suggested to be a key component in establishing vectorial proton transport.

Direct observation of three conformations of MutS protein regulated by adenine nucleotides.

Mismatched base-pairs, which are caused by either DNA replication errors, DNA damage or genetic recombination, are repaired by the mismatch-repair system. The MutS protein, a component of the mismatch-repair system, recognizes mismatched base-pairs in DNA, and its DNA-binding activity is affected by ATP and ADP. Here, we show that the MutS protein from Thermus thermophilus HB8 can have three different conformations in solution, based on direct observations made by small-angle X-ray scattering. The conformation of MutS in solution is drastically influenced by the presence of ADP and ATP; the ATP-bound form has the most compact conformation, the ADP-bound form the most stretched, and the nucleotide-free form has a conformation intermediate between the two. Based on these findings, we conclude that the DNA-binding activity of MutS may depend on conformational changes triggered by both the binding and hydrolysis of ATP.

Native-like beta-hairpin retained in the cold-denatured state of bovine beta-lactoglobulin.

Bovine beta-lactoglobulin is denatured by increased temperature (heat denaturation) and by decreased temperature (cold-denaturation) in the presence of 4 M urea at pH 2.5. We characterized the structure of the cold-denatured state of beta-lactoglobulin using circular dichroism (CD), small-angle X-ray scattering (SAXS) and heteronuclear nuclear magnetic resonance (NMR). CD and SAXS indicated that the cold-denatured state, in comparison with the highly denatured state induced by urea, is rather compact, retaining some secondary structure, but no tertiary structure. The location of the residual structures in the cold-denatured state and their stability were characterized by 1H/2H exchange combined with heteronuclear NMR. The results indicated that the residues adjacent to the disulfide bond (C106-C119) connecting beta-strands G and H had markedly high protection factors, suggesting the presence of a native-like beta-hairpin stabilized by the disulfide bond. Since this beta-hairpin is conserved between different conformational states, including the kinetic refolding intermediate, it should be of paramount importance for the folding and stability of beta-lactoglobulin. On the other hand, the non-native alpha-helix suggested for the folding intermediate was not detected in the cold-denatured state. The 1H/2H exchange experiments showed that the protection factors of a mixture of the native and cold-denatured states is strongly biased by that of the labile cold-denatured state, consistent with a two-process model of the exchange.

Energy coupling in an ion pump. The reprotonation switch of bacteriorhodopsin.

The active site of an ion pump must communicate alternately with the two opposite membrane surfaces. In the light-driven proton pump, bacteriorhodopsin, the retinal Schiff base is first the proton donor to D85 (with access to the extracellular side), and then it becomes the acceptor of the proton of D96 (with access to the cytoplasmic side). This "reprotonation switch" has been associated with a protein conformation change observed during the photocycle. When D85 is replaced with asparagine, the pKa value of the Schiff base is lowered from above 13 to about 9. We determined the direction of the loss or gain of the Schiff base proton in unphotolyzed and in photoexcited D85N, and the D85N/D96N and D85N/D96A double mutants, in order to understand the intrinsic and the induced connectivities of the Schiff base to the two membrane surfaces. The influence of D96 mutations on proton exchange and on acceleration of proton shuttling to the surface by azide indicated that in either case the access of the Schiff base on D85N mutants is to the cytoplasmic side. In the wild-type protein (but with the pKa of the Schiff base lowered by 13-trifluoromethyl retinal substitution) the results suggested that the Schiff base can communicate also with the extracellular side. Raising the pH without illumination of D85N so as to deprotonate the Schiff base caused the same, or nearly the same, change of X-ray scattering as observed when the Schiff base deprotonates during the wild-type photocycle. The results link the charge state of the active site to the global protein conformation and to the connectivity of the Schiff base proton to the membrane surfaces. Their relationship suggests that the conformation of the unphotolyzed wild-type protein is stabilized by coulombic interaction of the Schiff base with its counter-ion. A proton is translocated across the membrane after light-induced transfer of the Schiff base proton to D85, because the protein assumes an alternative conformation that separates the donor from the acceptor and opens new conduction pathways between the active site and the two membrane surfaces.

Trimeric mutant bacteriorhodopsin, D85N, shows a monophasic CD spectrum.

The structure of mutant bacteriorhodopsin (bR), D85N, was examined by CD and X-ray diffraction at pH 7. The absorption maximum of D85N at pH 7 is located at 605 nm, which is similar to the acid-blue form of wild-type bR. D85N shows a monophasic CD band, the maximum of which is at 575 nm, although the crystalline arrangement and the trimeric structure is maintained. The acid-blue form of wild-type bR shows a biphasic CD despite the similarity in absorption spectra.

Observation of RecA protein monomer by small angle X-ray scattering with synchrotron radiation.

RecA protein is capable of forming homo-oligomers in solution. The oligomeric and monomeric states of Thermus thermophilus RecA protein were studied by small angle X-ray scattering, a direct method used to measure the overall dimensions of a macromolecule. In the presence of 3 M urea or 0.2 M lithium perchlorate, RecA dissociates from higher oligomeric states to form a hexamer with a radius of gyration (R(g)) of 52 A. The value of R(g) decreased to 36 A at a higher lithium perchlorate concentration (1.0 M). The zero angle intensity, I(0), was consistent with the identification of the former state as a hexamer and the latter as a monomer.

X-ray diffraction studies of bacteriorhodopsin. Determination of the positions of mercury label at several engineered cysteine residues.

The single cysteine-containing bacteriorhodopsin mutants F27C, L100C, T170C, F171C and I222C were labeled with p-chloromercuribenzoic acid, which specifically reacts with sulfhydryl groups. These cysteines should be located at the cytoplasmic ends of the transmembrane helices A, C, F or G. We determined the positions of the bound mercury atoms by X-ray diffraction of purple membrane films, with better than 1 A accuracy. The determined mercury positions were compared with the structural model from cryoelectron microscopy (N. Grigorieff, T. A. Ceska, K. H. Downing, J. M. Baldwin and R. Henderson, J. Mol. Biol. 259, 393-421, 1996). Given that the distance between the mercury and the C alpha atom of the cysteine in the xy plane must be shorter than 4.5 A and that the mercury atom is located at the delta position, the positions obtained for the mercury labels agree with their expected positions from the structural model. The present results give a rationale for detecting structural changes upon illumination as shifts occur in the mercury label position.

A partially unfolded structure of the alkaline-denatured state of pepsin and its implication for stability of the zymogen-derived protein.

Pepsin, a gastric aspartic proteinase, is a zymogen-derived protein that undergoes irreversible alkaline denaturation at pH 6-7. Detailed knowledge of the structure of the alkaline-denatured state is an important step in understanding the mechanism of the formation of the active enzyme. An extensive analysis of the denatured state at pH 8.0 was performed using a variety of techniques including (1)H nuclear magnetic resonance spectroscopy and solution X-ray scattering. This analysis indicates that the denatured state under these conditions has a compact and globular conformation with a substantial amount of secondary and tertiary structures. The data suggest that this partially structured species has a highly folded region and a flexible region. The NMR measurements suggest that the folded region contains His53 and is located at least partly in the N-terminal lobe of the protein. The alkaline-denatured state experiences a further reversible denaturation step at higher pH or on heating; the midpoints of the unfolding transition are pH 11.5 (at 25 degrees C) and 53.1 degrees C (at pH 8.0), respectively. The present findings suggest that the proteolytic processing of pepsinogen has substantially modified the ability of the protein to fold, such that its folding process cannot progress beyond the partially folded intermediate of pepsin.

The last phase of the reprotonation switch in bacteriorhodopsin: the transition between the M-type and the N-type protein conformation depends on hydration.

In order to elucidate the mechanism of the reprotonation switch of bacteriorhodopsin, the protein conformation of the M intermediate of the D96N mutant was examined at various hydration conditions by X-ray diffraction and FTIR spectroscopy. We observed two distinct protein conformations at different levels of hydration. One is like in the N photointermediate, although in this case with an unprotonated Schiff base. It is stabilized in highly hydrated samples. The other is a protein conformation identical to that in the normal M intermediate of wild-type bacteriorhodopsin, which is stabilized in partially dehydrated samples. The hydration dependence of the structural transition between the M-type and the N-type conformations suggests that there is a change in the binding of water at the cytoplasmic surface. Thus, more water molecules bind in the N-type structure than in the M-type. This is consistent with the idea that the conformational change from the M-type to the N-type corresponds to the opening of the proton channel to the cytoplasmic surface by tilt of the cytoplasmic end of helix F, and that this is required for proton transfer from Asp-96 to the retinal Schiff base.

Time-resolved x-ray diffraction reveals multiple conformations in the M-N transition of the bacteriorhodopsin photocycle.

We measured the M-N transition of wild-type bacteriorhodopsin (pH 9, 10 degrees C) by time-resolved x-ray diffraction study at SPring8 BL45XU-A. We confirmed the accumulation of M and N intermediates by absorbance measurements, and we found that the time resolution of x-ray diffraction experiments (244 ms) was sufficient to resolve the M-N transition. From the x-ray diffraction data, three components were decomposed by singular value decomposition analysis. The existence of three components in the M-->N-->BR reaction revealed that BR changes its structure during the M-N transition. Moreover, the difference Fourier maps of reconstituted fast and slow decay components clearly showed that the electron density distributions of the F helix changes in the M-N transition. The observed structural change at the F helix will increase access of the Schiff base and D96 to the cytoplasmic surface and facilitate the proton transfer steps that begin with the decay of the M state.

Conformational change of helix G in the bacteriorhodopsin photocycle: investigation with heavy atom labeling and x-ray diffraction.

According to the current structural model of bacteriorhodopsin, Ile222 is located at the cytoplasmic end of helix G. We labeled the single cysteine of the site-directed mutant Ile222 --> Cys with p-chloromercuribenzoic acid and determined the position of the labeled mercury by x-ray diffraction in the unphotolyzed state, and in the MN photointermediate accumulated in the presence of guanidine hydrochloride at pH 9.5. According to the difference Fourier maps between the MN intermediate and the unphotolyzed state, the structural change in the MN intermediate was not affected by mercury labeling. The difference Fourier map between the labeled and the unlabeled I222C gave the position of the mercury label. This information was obtained for both the unphotolyzed state and the MN intermediate. We found that the position of the mercury at residue 222 is shifted by 2.1 +/- 0.8 A in the MN intermediate. This agrees with earlier results that suggested a structural change in the G helix. The movement of the mercury label is so large that it must originate from a cooperative conformational change in the helix G at its cytoplasmic end, rather than from displacement of residue 222. Because Ile222 is located at the same level on the z coordinate as Asp96, the structural change in the G helix could have the functional role of perturbing the environment and therefore the pKa of this functionally important aspartate.

A local electrostatic change is the cause of the large-scale protein conformation shift in bacteriorhodopsin.

During light-driven proton transport bacteriorhodopsin shuttles between two protein conformations. A large-scale structural change similar to that in the photochemical cycle is produced in the D85N mutant upon raising the pH, even without illumination. We report here that (i) the pKa values for the change in crystallographic parameters and for deprotonation of the retinal Schiff base are the same, (ii) the retinal isomeric configuration is nearly unaffected by the protein conformation, and (iii) preventing rotation of the C13-C14 double bond by replacing the retinal with an all-trans locked analogue makes little difference to the Schiff base pKa. We conclude that the direct cause of the conformational shift is destabilization of the structure upon loss of interaction of the positively charged Schiff base with anionic residues that form its counter-ion.

Structure of the N intermediate of bacteriorhodopsin revealed by x-ray diffraction.

X-ray diffraction experiments revealed the structure of the N photointermediate of bacteriorhodopsin. Since the retinal Schiff base is reprotonated from Asp-96 during the M to N transition in the photocycle, and Asp-96 is reprotonated during the lifetime of the N intermediate, or immediately after, N is a key intermediate for understanding the light-driven proton pump. The N intermediate accumulates in large amounts during continuous illumination of the F171C mutant at pH 7 and 5 degrees Celsius. Small but significant changes of the structure were detected in the x-ray diffraction profile under these conditions. The changes were reversible and reproducible. The difference Fourier map indicates that the major change occurs near helix F. The observed diffraction changes between N and the original state were essentially identical to the diffraction changes reported for the M intermediate of the D96N mutant of bacteriorhodopsin. Thus, we find that the protein conformations of the M and N intermediates of the photocycle are essentially the same, in spite of the fact that in M the Schiff base is unprotonated and in N it is protonated. The observed structural change near helix F will increase access of the Schiff base and Asp-96 to the cytoplasmic surface and facilitate the proton transfer events that begin with the decay of the M state.