Low temperature FTIR spectroscopy provides new insights in the pH-dependent proton pathway of proteorhodopsin.

In the presented study the low pH photocycle of proteorhodopsin is extensively investigated by means of low temperature FTIR spectroscopy. Besides the already well-known characteristics of the all-trans and 13-cis retinal vibrations the 77K difference spectrum at pH 5.1 shows an additional negative signal at 1744 cm(-1) which is interpreted as indicator for the L state. The subsequent photocycle steps are investigated at temperatures higher than 200K. The combination of visible and FTIR spectroscopy enabled us to observe that the deprotonation of the Schiff base is linked to the protonation of an Asp or Glu side chain - the new proton acceptor under acidic conditions. The difference spectra of the late intermediates are characterized by large amide I changes and two further bands ((-)1751 cm(-1)/(+)1725 cm(-1)) in the spectral region of the Asp/Glu ν(C=O) vibrations. The band position of the negative signature points to a transient deprotonation of Asp-97. In addition, the pH dependence of the acidic photocycle was investigated. The difference spectra at pH 5.5 show distinct differences connected to changes in the protonation state of key residues. Based on our data we propose a three-state model that explains the complex pH dependence of PR.

Characterizing the structure and photocycle of PR 2D crystals with CD and FTIR spectroscopy.

We present here a study on proteorhodopsin (PR) 2D crystals with analytical ultracentrifugation, circular dichroism and Fourier transform infrared (FTIR) spectroscopy. The aim of our experiments was to test the activity of 2D crystal sample preparations and to gain further insight in PR structure, stability and function with these techniques. Our results demonstrate higher stability compared to detergent-solubilized or reconstituted samples. For different pH values, low pH 2D crystals tend to form bigger aggregates and are less stable than at basic pH. The pH 9 sample shows a sharp phase transition during heat denaturation and there is also evidence for protein-protein interaction due to the close proximity of the proteins in the 2D crystals. In the FTIR measurements at cryogenic temperatures (77 K), we characterized the first step in the PR photocycle. At pH 9, the K intermediate could be observed and the samples showed no orientation effects. At pH 5, we could trap the K/L intermediate, characterized by its negative IR signal at 1741 cm(-1). In rapid-scan FTIR experiments, we could also identify the M intermediate of the photocycle at basic pH. We conclude that the PR 2D crystals exhibit a fully functional photocycle and are therefore well suited for further studies on the proton transport mechanism of PR.

Solid-state NMR and functional studies on proteorhodopsin.

Proteins of the proteorhodopsin (PR) family are found abundantly in many marine bacteria in the photic zone of the oceans. They are colour-tuned to their environment. The green absorbing species has been shown to act as a light-driven proton pump and thus could form a potential source of energy. The pK(a) of the primary proton acceptor is close to the pH of seawater which could also indicate a regulatory role. Here, we review and summarize our own recent findings in the context of known data and present some new results. Proton transfer in vitro by PR is shown by a fluorescence assay which confirms a pH dependent vectoriality. Previously reported low diffracting 2D crystal preparations of PR are assessed for their use for solid-state NMR by two dimensional (13)C-(13)C DARR spectra. (15)N-(1)H HETCOR MAS NMR experiments show bound water in the vicinity of the protonated Schiff base which could play a role in proton transfer. The effect of highly conserved H75 onto the properties of the chromophore has been investigated by single site mutations. They do show a pronounced effect onto the optical absorption maximum and the pK(a) of the proton acceptor but have only a small effect onto the (15)N chemical shifts of the protonated Schiff base.

Prospective multicenter study evaluating fecal calprotectin in adult acute bacterial diarrhea.

BACKGROUND: Every year, about 2.2 million deaths occur worldwide due to diarrhea. Reliable diagnosis of patients with acute infectious diarrhea remains a formidable challenge to the clinicians. This is the first study reporting use of fecal calprotectin in diagnosing acute diarrhea. The aim was to compare the diagnostic accuracy of fecal calprotectin, fecal lactoferrin, and guaiac-based fecal occult blood test in a diverse group of consecutive patients with acute diarrhea in which routine bacterial stool cultures and cytotoxins for Clostridium difficile were performed. METHODS: This was a prospective case-control multicenter study from January 2004 until October 2007 in 2383 consecutive patients with acute diarrhea. They provided stool samples for performing cultures. Patients with positive cultures and an equal number of matched controls with negative cultures underwent fecal occult blood test and calprotectin and lactoferrin assays. RESULTS: Calprotectin, lactoferrin, and fecal occult blood tests demonstrated sensitivity and specificity of 83% and 87%, 78% and 54%, and 38% and 85%, respectively, for diagnosing acute bacterial diarrhea. CONCLUSIONS: Calprotectin showed high correlation with bacteriologically positive infectious diarrhea compared with lactoferrin and fecal occult blood test. It may potentially revolutionize management algorithm for patients with acute diarrhea. As a screening test, calprotectin can generate results within hours to support presumptive diagnosis of infectious diarrhea, which can decide suitability of stool samples for culture.

Double quantum filtering homonuclear MAS NMR correlation spectra: a tool for membrane protein studies.

13C homonuclear correlation spectra based on proton driven spin diffusion (PDSD) are becoming increasingly important for obtaining distance constraints from multiply labeled biomolecules by MAS NMR. One particular challenging situation arises when such constraints are to be obtained from spectra with a large natural abundance signal background which causes detrimental diagonal peak intensities. They obscure cross peaks, and furthermore impede the calculation of a buildup rates matrix which may be used to derive distance constraints, as carried out in "NMR crystallography". Here, we combine double quantum (DQ) filtering with 13C-13C dipolar assisted rotational resonance (DARR) experiments to yield correlation spectra free of natural abundance contributions. Two experimental schemes, using DQ filtering prior to evolution (DOPE), and after mixing (DOAM), have been evaluated. Diagonal peak intensities along the spectrum diagonal are removed completely, and crosspeaks close to the diagonal are easily identifiable. For DOAM spectra with negligible mixing times, it is possible to carry out 'assignment walks' which simplify peak identification substantially. The method is demonstrated on 13C-cys labeled proteorhodopsin, a 27 kDa membrane protein. The magnetization transfer characteristics were studied using buildup curves obtained on uniformly 13C labelled crystalline tripeptide MLF. Our data show that DQ filtered DARR experiments pave the way for obtaining through space constraints for structural studies on ligands, bound to membrane receptors, or on small fragments within large proteins.

Folding and assembly of proteorhodopsin.

Proteorhodopsins (PRs), the recently discovered light-driven proton pumps, play a major role in supplying energy for microbial organisms of oceans. In contrast to PR, rhodopsins found in Archaea and Eukarya are structurally well characterized. Using single-molecule microscopy and spectroscopy, we observed the oligomeric assembly of native PR molecules and detected their folding in the membrane. PR showed unfolding patterns identical with those of bacteriorhodopsin and halorhodopsin, indicating that PR folds similarly to archaeal rhodopsins. Surprisingly, PR predominantly assembles into hexameric oligomers, with a smaller fraction assembling into pentamers. Within these oligomers, PR arranged into radial assemblies. We suggest that this structural assembly of PR may have functional implications.

Characterisation of Schiff base and chromophore in green proteorhodopsin by solid-state NMR.

The proteorhodopsin family consists of hundreds of homologous retinal containing membrane proteins found in bacteria in the photic zone of the oceans. They are colour tuned to their environment and act as light-driven proton pumps with a potential energetic and regulatory function. Precise structural details are still unknown. Here, the green proteorhodopsin variant has been selected for a chemical shift analysis of retinal and Schiff base by solid-state NMR. Our data show that the chromophore exists in mainly all-trans configuration in the proteorhodopsin ground state. The optical absorption maximum together with retinal and Schiff base chemical shifts indicate a strong interaction network between chromophore and opsin.

Proteorhodopsin: characterisation of 2D crystals by electron microscopy and solid state NMR.

Proteorhodopsin (PR) a recent addition to retinal type 1 protein family, is a bacterial homologue of archaeal bacteriorhodopsin. It was found to high abundance in gamma-proteobacteria in the photic zone of the oceans and has been shown to act as a photoactive proton pump. It is therefore involved in the utilisation of light energy for energy production within the cell. Based on data from biodiversity screens, hundreds of variants were discovered worldwide, which are spectrally tuned to the available light at different locations in the sea. Here, we present a characterisation of 2D crystals of the green variant of proteorhodopsin by electron microscopy and solid state NMR. 2D crystal formation with hexagonal protein packing was observed under a very wide range of conditions indicating that PR might be also closely packed under native conditions. A low-resolution 2D projection map reveals a ring-shaped oligomeric assembly of PR. The protein state was analysed by 15N MAS NMR on lysine, tryptophan and methionine labelled samples. The chemical shift of the protonated Schiff base was almost identical to non-crystalline preparations. All residues could be cross-polarised in non-frozen samples. Lee-Goldberg cross-polarisation has been used to probe protein backbone mobility.