pymzML v2.0: introducing a highly compressed and seekable gzip format.

Motivation: In the new release of pymzML (v2.0), we have optimized the speed of this established tool for mass spectrometry data analysis to adapt to increasing amounts of data in mass spectrometry. Thus, we integrated faster libraries for numerical calculations, improved data retrieving algorithms and have optimized the source code. Importantly, to adapt to rapidly growing file sizes, we developed a generalizable compression scheme for very fast random access and applied this concept to mzML files to retrieve spectral data. Results: pymzML performs at par with established C programs when it comes to processing times. However, it offers the versatility of a scripting language, while adding unprecedented fast random access to compressed files. Additionally, we designed our compression scheme in such a general way that it can be applied to any field where fast random access to large data blocks in compressed files is desired. Availability and implementation: pymzML is freely available on https://github.com/pymzML/pymzML under GPL license. pymzML requires Python3.4+ and optionally numpy. Documentation available on http://pymzml.readthedocs.io.

Robust label-free biosensing using microdisk laser arrays with on-chip references.

Whispering-gallery mode (WGM) microdisk lasers show great potential for highly sensitive label-free detection in large-scale sensor arrays. However, when used in practical applications under normal ambient conditions, these devices suffer from temperature fluctuations and photobleaching. Here we demonstrate that these challenges can be overcome by a novel referencing scheme that allows for simultaneous compensation of temperature drift and photobleaching. The technique relies on reference structures protected by locally dispensed passivation materials, and can be scaled to extended arrays of hundreds of devices. We prove the viability of the concept in a series of experiments, demonstrating robust and sensitive label-free detection over a wide range of constant or continuously varying temperatures. To the best of our knowledge, these measurements represent the first demonstration of biosensing in active WGM devices with simultaneous compensation of both photobleaching and temperature drift.

Chloroplast site-directed mutagenesis of photosystem I in Chlamydomonas: electron transfer reactions and light sensitivity.

The photosystem I (PSI) complex is a multisubunit protein-pigment complex embedded in the thylakoid membrane which acts as a light-driven plastocyanin/cytochrome c(6)-ferredoxin oxido-reductase. The use of chloroplast transformation and site-directed mutagenesis coupled with the biochemical and biophysical analysis of mutants of the green alga Chlamydomonas reinhardtii with specific amino acid changes in several subunits of PSI has provided new insights into the structure-function relationship of this important photosynthetic complex. In particular, this molecular-genetic analysis has identified key residues of the reaction center polypeptides of PSI which are the ligands of some of the redox cofactors and it has also provided important insights into the orientation of the terminal electron acceptors of this complex. Finally this analysis has also shown that mutations affecting the donor side of PSI are limiting for overall electron transfer under high light and that electron trapping within the terminal electron acceptors of PSI is highly deleterious to the cells.

Fast electron transfer from cytochrome c6 and plastocyanin to photosystem I of Chlamydomonas reinhardtii requires PsaF.

To study the function of the PsaF subunit of photosystem I (PSI), the interactions between plastocyanin or cytochrome c6 and PSI isolated from wild-type and a PsaF-deficient mutant of Chlamydomonas reinhardtii have been examined using cross-linking techniques and flash absorption spectroscopy. We show that efficient electron transfer from both plastocyanin and cytochrome c6 to PSI depends on PsaF. A remarkable feature of the PSI complex of C. reinhardtii is that both plastocyanin and cytochrome c6 reduce P700+ with first-order kinetics and a half-time of 3 micros, which is unique among photosynthetic organisms examined.

Insertion of the N-terminal part of PsaF from Chlamydomonas reinhardtii into photosystem I from Synechococcus elongatus enables efficient binding of algal plastocyanin and cytochrome c6.

A strain of the cyanobacterium Synechococcus elongatus was generated that expresses a hybrid version of the photosystem I subunit PsaF consisting of the first 83 amino acids of PsaF from the green alga Chlamydomonas reinhardtii fused to the C-terminal portion of PsaF from S. elongatus. The corresponding modified gene was introduced into the genome of the psaF-deletion strain FK2 by cointegration with an antibiotic resistance gene. The transformants express a new PsaF subunit similar in size to PsaF from C. reinhardtii that is assembled into photosystem I (PSI). Hybrid PSI complexes isolated from these strains show an increase by 2 or 3 orders of magnitude in the rate of P700(+) reduction by C. reinhardtii cytochrome c6 or plastocyanin in 30% of the complexes as compared with wild type cyanobacterial PSI. The corresponding optimum second-order rate constants (k2 = 4.0 and 1.7 x 10(7) M1 s1 for cytochrome c6 and plastocyanin) are similar to those of PSI from C. reinhardtii. The remaining complexes are reduced at a slow rate similar to that observed with wild type PSI from S. elongatus and the algal donors. At high concentrations of C. reinhardtii cytochrome c6, a fast first-order kinetic component (t(1)/(2) = 4 microseconds) is revealed, indicative of intramolecular electron transfer within a complex between the hybrid PSI and cytochrome c6. This first-order phase is characteristic for P700(+) reduction by cytochrome c6 or plastocyanin in algae and higher plants. However, a similar fast phase is not detected for plastocyanin. Cross-linking studies show that, in contrast to PSI from wild type S. elongatus, the chimeric PsaF of PSI from the transformed strain cross-links to cytochrome c6 or plastocyanin with a similar efficiency as PsaF from C. reinhardtii PSI. Our data indicate that development of a eukaryotic type of reaction mechanism for binding and electron transfer between PSI and its electron donors required structural changes in both PSI and cytochrome c6 or plastocyanin.

The N-terminal domain of PsaF: precise recognition site for binding and fast electron transfer from cytochrome c6 and plastocyanin to photosystem I of Chlamydomonas reinhardtii.

The PsaF-deficient mutant 3bF of Chlamydomonas reinhardtii was used to modify PsaF by nuclear transformation and site-directed mutagenesis. Four lysine residues in the N-terminal domain of PsaF, which have been postulated to form the positively charged face of a putative amphipathic alpha-helical structure were altered to K12P, K16Q, K23Q, and K30Q. The interactions between plastocyanin (pc) or cytochrome c6 (cyt c6) and photosystem I (PSI) isolated from wild type and the different mutants were analyzed using crosslinking techniques and flash absorption spectroscopy. The K23Q change drastically affected crosslinking of pc to PSI and electron transfer from pc and cyt c6 to PSI. The corresponding second order rate constants for binding of pc and cyt c6 were reduced by a factor of 13 and 7, respectively. Smaller effects were observed for mutations K16Q and K30Q, whereas in K12P the binding was not changed relative to wild type. None of the mutations affected the half-life of the microsecond electron transfer performed within the intermolecular complex between the donors and PSI. The fact that these single amino acid changes within the N-terminal domain of PsaF have different effects on the electron transfer rate constants and dissociation constants for both electron donors suggests the existence of a rather precise recognition site for pc and cyt c6 that leads to the stabilization of the final electron transfer complex through electrostatic interactions.

Binding dynamics and electron transfer between plastocyanin and photosystem I.

The mechanism of the electron transfer from the soluble protein plastocyanin to the multiprotein complex of photosystem I from spinach has been studied in detail. The two kinetic components of P700+ reduction by plastocyanin after a laser flash, showing a constant half-life of 11 microseconds and a variable half-life of the second-order reaction, respectively, are used to monitor the electron transfer from bound and soluble plastocyanin. The effect of increasing concentration of reduced plastocyanin on both of these kinetic components and the competition by oxidized plastocyanin is used to estimate the individual dissociation constants of the complex between the proteins in each of its oxidized and reduced state. The dissociation constant of oxidized plastocyanin is about six times larger than that of 7 microM found for reduced plastocyanin and purified PSI. Consistent with this result the midpoint redox potential of plastocyanin bound to photosystem I either in equilibrium with soluble plastocyanin or after cross-linking to photosystem I is found to be 50-60 mV higher than that of soluble plastocyanin. It is concluded that the driving force of the intracomplex electron transfer is decreased in favor of an optimized turnover of photosystem I. Double-flash excitation shows that oxidized plastocyanin has to leave the complex after the electron transfer before a new reduced plastocyanin molecule can bind to photosystem I. This release of oxidized plastocyanin with a half-life of about 60 microseconds limits the turnover of photosystem I. All data are consistently described by a model including the formation of a complex at a single binding site of photosystem I. Differences in the rate and binding constants are discussed with respect to the structure and the electrostatic and hydrophobic interactions stabilizing the complex as well as their modification by the membrane environment in situ.

Limitation in electron transfer in photosystem I donor side mutants of Chlamydomonas reinhardtii. Lethal photo-oxidative damage in high light is overcome in a suppressor strain deficient in the assembly of the light harvesting complex.

Strains of Chlamydomonas reinhardtii lacking the PsaF gene or containing the mutation K23Q within the N-terminal part of PsaF are sensitive to high light (>400 microE m(-2) s(-1)) under aerobic conditions. In vitro experiments indicate that the sensitivity to high light of the isolated photosystem I (PSI) complex from wild type and from PsaF mutants is similar. In vivo measurements of photochemical quenching and oxygen evolution show that impairment of the donor side of PSI in the PsaF mutants leads to a diminished linear electron transfer and/or a decrease of photosystem II (PSII) activity in high light. Thermoluminescence measurements indicate that the PSII reaction center is directly affected under photo-oxidative stress when the rate of electron transfer becomes limiting in the PsaF-deficient strain and in the PsaF mutant K23Q. We have isolated a high light-resistant PsaF-deficient suppressor strain that has a high chlorophyll a/b ratio and is affected in the assembly of light-harvesting complex. These results indicate that under high light a functionally intact donor side of PSI is essential for protection of C. reinhardtii against photo-oxidative damage when the photosystems are properly connected to their light-harvesting antennae.

The PsaC subunit of photosystem I provides an essential lysine residue for fast electron transfer to ferredoxin.

PsaC is the stromal subunit of photosystem I (PSI) which binds the two terminal electron acceptors FA and FB. This subunit resembles 2[4Fe-4S] bacterial ferredoxins but contains two additional sequences: an internal loop and a C-terminal extension. To gain new insights into the function of the internal loop, we used an in vivo degenerate oligonucleotide-directed mutagenesis approach for analysing this region in the green alga Chlamydomonas reinhardtii. Analysis of several psaC mutants affected in PSI function or assembly revealed that K35 is a main interaction site between PsaC and ferredoxin (Fd) and that it plays a key role in the electrostatic interaction between Fd and PSI. This is based upon the observation that the mutations K35T, K35D and K35E drastically affect electron transfer from PSI to Fd, as measured by flash-absorption spectroscopy, whereas the K35R change has no effect on Fd reduction. Chemical cross-linking experiments show that Fd interacts not only with PsaD and PsaE, but also with the PsaC subunit of PSI. Replacement of K35 by T, D, E or R abolishes Fd cross-linking to PsaC, and cross-linking to PsaD and PsaE is reduced in the K35T, K35D and K35E mutants. In contrast, replacement of any other lysine of PsaC does not alter the cross-linking pattern, thus indicating that K35 is an interaction site between PsaC and its redox partner Fd.

A large fraction of PsaF is nonfunctional in photosystem I complexes lacking the PsaJ subunit.

PsaJ is a small hydrophobic subunit of the photosystem I complex (PSI) whose function is not yet fully understood. Here we describe mutants of the green alga Chlamydomonas reinhardtii, in which the psaJ chloroplast gene has been inactivated either in a wild-type or in a PsaF-deficient nuclear background. Cells lacking one or both subunits grow photoautotrophically and contain normal levels of PSI. Flash-absorption spectroscopy performed with isolated PSI particles isolated from the PsaJ-deficient strain indicates that only 30% of the PSI complexes oxidize plastocyanin (Pc) or cytochrome c6 (Cyt c6) with kinetics identical to wild type, whereas the remaining 70% follow slow kinetics similar to those observed with PsaF-deficient PSI complexes. This feature is not due to partial loss of PsaF, as the PsaJ-less PSI complex contains normal levels of the PsaF subunit. The N-terminal domain of PsaF can be cross-linked to Pc and Cyt c6 indicating that in the absence of PsaJ, this domain is exposed in the lumenal space. Therefore, the decreased amount of functional PsaF revealed by the electron-transfer measurements is best explained by a displacement of the N-terminal domain of PsaF which is known to provide the docking site for Pc and Cyt c6. We propose that one function of PsaJ is to maintain PsaF in a proper orientation which allows fast electron transfer from soluble donor proteins to P700(+).

Light-induced charge separation between plastocyanin and the iron-sulfur clusters FA and FB in the complex of plastocyanin and photosystem I.

The light-induced electron transfer in a crosslinked complex between plastocyanin and photosystem I from spinach was studied by EPR at low temperature. Electron donation from reduced plastocyanin to P700+ was observed under illumination above a temperature of about 160 K, resulting in a second charge separation and an electron transfer from rereduced P700 to the terminal electron acceptors FA/FB. The charge-separated state Pc oxP700+ [FA/FB](2-) was found to be stable at 15 K. Implications of these results for the kinetic constants of the donation reaction and the backtransfer of electrons from reduced acceptors as well as for the structural models of the terminal acceptors are discussed.

Towards functional proteomics of membrane protein complexes: analysis of thylakoid membranes from Chlamydomonas reinhardtii.

Functional proteomics of membrane proteins is an important tool for the understanding of protein networks in biological membranes but structural studies on this part of the proteome are limited. In this study we undertook such an approach to analyse photosynthetic thylakoid membranes isolated from wild-type and mutant strains of Chlamydomonas reinhardtii. Thylakoid membrane proteins were separated by high-resolution two-dimensional gel electrophoresis (2-DE) and analysed by immuno-blotting and mass spectrometry for the presence of membrane-spanning proteins. Our data show that light-harvesting complex proteins (LHCP), that cross the membrane with three transmembrane domains, can be separated using this method. We have identified more than 30 different LHCP spots on our gels. Mass spectrometric analysis of 2-DE separated Lhcb1 indicates that this major LHCII protein can associate with the thylakoid membrane with part of its putative transit sequence. Separation of isolated photosystem I (PSI) complexes by 2-DE revealed the presence of 18 LHCI protein spots. The use of two peptide-specific antibodies directed against LHCI subunits supports the interpretation that some of these spots represent products arising from differential processing and post-translational modifications. In addition our data indicate that the reaction centre subunit of PSI, PsaA, that possesses 11 transmembrane domains, can be separated by 2-DE. Comparison between 2-DE maps from thylakoid membrane proteins isolated from a PSI-deficient (Deltaycf4) and a crd1 mutant, which is conditionally reduced in PSI and LHCI under copper-deficiency, showed the presence of most of the LHCI spots in the former but their absence in the latter. Our data demonstrate that (i) hydrophobic membrane proteins like the LHCPs can be faithfully separated by 2-DE, and (ii) that high-resolution 2-DE facilitates the comparative analysis of membrane protein complexes in wild-type and mutants cells.

The plastocyanin binding domain of photosystem I.

The molecular recognition between plastocyanin and photosystem I was studied. Photosystem I and plastocyanin can be cross-linked to an active electron transfer complex. Immunoblots and mass spectrometric analysis of proteolytic peptides indicate that the two negative patches conserved in plant plastocyanins are cross-linked with lysine residues of a domain near the N-terminus of the PsaF subunit of photosystem I. Conversion of these negative to uncharged patches of plastocyanin by site-directed mutation D42N/E43Q/D44N/E45Q and E59Q/E60Q/D61N respectively, reveals the first patch to be essential for the electrostatic interaction in the electron transfer complex with photosystem I and the second one to lower the redox potential. The domain in PsaF, not found in cyanobacteria, is predicted to fold into two amphipathic alpha-helices. The interacting N-terminal helix lines up six lysines on one side which may guide a fast one-dimensional diffusion of plastocyanin and provide the electrostatic attraction at the attachment site, in addition to the hydrophobic interaction in the area where the electron is transferred to P700 in the reaction center of photosystem I. This two-step interaction is likely to increase the electron transfer rate by more than two orders of magnitude in plants as compared with cyanobacteria. Our data resolve the controversy about the function of PsaF.