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Ruth Horn

Publications and source records attributed to Ruth Horn.

6 recordsLinked to original sources
PubMed2006

Consecutive binding of chlorophylls a and b during the assembly in vitro of light-harvesting chlorophyll-a/b protein (LHCIIb).

The apoprotein of the major light-harvesting chlorophyll a/b complex (LHCIIb) is post-translationally imported into the chloroplast, where membrane insertion, protein folding, and pigment binding take place. The sequence and molecular mechanism of the latter steps is largely unknown. The complex spontaneously self-organises in vitro to form structurally authentic LHCIIb upon reconstituting the unfolded recombinant protein with the pigments chlorophyll a, b, and carotenoids in detergent micelles. Former measurements of LHCIIb assembly had revealed two apparent kinetic phases, a faster one (tau1) in the range of 10 s to 1 min, and a slower one (tau2) in the range of several min. To unravel the sequence of events we analysed the binding of chlorophylls into the complex by using time-resolved fluorescence measurements of resonance energy transfer from chlorophylls to an acceptor dye attached to the apoprotein. Chlorophyll a, offered in the absence of chlorophyll b, bound with the faster kinetics (tau1) exclusively whereas chlorophyll b, in the absence of chlorophyll a, bound predominantly with the slower kinetics (tau2). In double-jump experiments, LHCIIb assembly could be dissected into a faster chlorophyll a and a subsequent, predominantly slower chlorophyll b-binding step. The assignment of the faster and the slower kinetic phase to predominantly chlorophyll a and exclusively chlorophyll b binding, respectively, was verified by analysing the assembly kinetics with a circular dichroism signal in the visible domain presumably reflecting the establishment of pigment-pigment interactions. We propose that slow chlorophyll binding is confined to the exclusively chlorophyll b binding sites whereas faster binding occurs to the chlorophyll a binding sites. The latter sites can bind both chlorophylls a and b but in a reversible fashion as long as the complex is not stabilised by proper occupation of the chlorophyll b sites. The resulting two-step model of LHCIIb assembly is able to reconcile the highly specific binding sites containing either chlorophyll a or b, as seen in the recent crystal structures of LHCIIb, with the observation of promiscuous binding sites able to bind both chlorophyll a and b in numerous reconstitution analyses of LHCIIb assembly.

Animals↗
PubMed2005

A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality.

BACKGROUND: In March 2003, several hospitals in Quebec, Canada, noted a marked increase in the incidence of Clostridium difficile-associated diarrhea. METHODS: In 2004 we conducted a prospective study at 12 Quebec hospitals to determine the incidence of nosocomial C. difficile-associated diarrhea and its complications and a case-control study to identify risk factors for the disease. Isolates of C. difficile were typed by pulsed-field gel electrophoresis and analyzed for binary toxin genes and partial deletions in the toxin A and B repressor gene tcdC. Antimicrobial susceptibility was evaluated in a subgroup of isolates. RESULTS: A total of 1703 patients with 1719 episodes of nosocomial C. difficile-associated diarrhea were identified. The incidence was 22.5 per 1000 admissions. The 30-day attributable mortality rate was 6.9 percent. Case patients were more likely than matched controls to have received fluoroquinolones (odds ratio, 3.9; 95 percent confidence interval, 2.3 to 6.6) or cephalosporins (odds ratio, 3.8; 95 percent confidence interval, 2.2 to 6.6). A predominant strain, resistant to fluoroquinolones, was found in 129 of 157 isolates (82.2 percent), and the binary toxin genes and partial deletions in the tcdC gene were present in 132 isolates (84.1 percent). CONCLUSIONS: A strain of C. difficile that was resistant to fluoroquinolones and had binary toxin and a partial deletion of the tcdC gene was responsible for this outbreak of C. difficile-associated diarrhea. Exposure to fluoroquinolones or cephalosporins was a risk factor.

Aged↗
PubMed2004

Early steps in the assembly of light-harvesting chlorophyll a/b complex: time-resolved fluorescence measurements.

The light-harvesting chlorophyll a/b complex (LHCIIb) spontaneously assembles from its pigment and protein components in detergent solution. The formation of functional LHCIIb can be detected in time-resolved experiments by monitoring the establishment of excitation energy transfer from protein-bound chlorophyll b to chlorophyll a. To detect the possible initial steps of chlorophyll binding that may not yet give rise to chlorophyll b-to-a energy transfer, we have monitored LHCIIb assembly by measuring excitation energy transfer from a fluorescent dye, covalently bound to the protein, to the chlorophylls. In order to exclude interference of the dye with protein folding or pigment binding, the experiments were repeated with the dye bound to four different positions in the protein. Initial chlorophyll binding occurs at roughly the same rate as the establishment of chlorophyll b-to-a energy transfer, in the range of 10 s. However, under limiting chlorophyll concentrations, the binding of chlorophyll a clearly precedes that of chlorophyll b. The complex containing the apoprotein, carotenoids, and chlorophyll a but no chlorophyll b is biochemically unstable and therefore cannot be isolated. However, chlorophyll a binding into this weak complex is specific, as it does not occur with a C-terminal deletion mutant of Lhcb1 which still contains most chlorophyll-ligating amino acids but is unable to fold and assemble into functional LHCIIb. As a scenario for LHCIIb assembly in the thylakoid, we propose the initial formation of a labile Lhcb1-chlorophyll a-carotenoid complex that then becomes stabilized by the binding (or formation in situ) of chlorophyll b.

Animals↗
PubMed2003

The light-harvesting chlorophyll a/b complex can be reconstituted in vitro from its completely unfolded apoprotein.

The major light-harvesting chlorophyll a/b protein (LHCIIb) of higher plants is one of the few membrane proteins that can be refolded in vitro. During folding, the apoprotein is assembled with pigments to form a structurally authentic and functional pigment--protein complex. All reconstitution procedures used so far include solubilization of the apoprotein in sodium dodecyl sulfate (SDS) where the protein adopts approximately half of its alpha-helical folding present in the native structure. This paper shows that this preformed alpha-helix is not a prerequisite for LHCIIb folding in vitro. The apoprotein can also be reconstituted starting from a solution in guanidinium hydrochloride (Gnd) where the protein contains no detectable helical structure. Reconstitution yields are somewhat lower in the Gnd than in the SDS procedure, but the reconstitution products exhibit very similar biochemical and spectroscopic properties. The kinetics of LHCIIb assembly, as assessed by time-resolved fluorescence measurements, are virtually the same in both reconstitution procedures. This demonstrates that the initiation of alpha-helix formation is not a rate-limiting step in LHCIIb apoprotein folding.

Apoproteins↗
PubMed2002

Folding in vitro of light-harvesting chlorophyll a/b protein is coupled with pigment binding.

The major light-harvesting chlorophyll a/b protein (LHCIIb) of the plant photosynthetic apparatus is able to self-organise in vitro. When the recombinant apoprotein, Lhcb1, is solubilised in the denaturing detergent sodium (or lithium) dodecylsulfate (SDS or LDS) and then mixed with chlorophylls and carotenoids under renaturing conditions, structurally authentic LHCIIb forms. Assembly of functional LHCIIb, as indicated by the establishment of energy transfer between complex-bound chlorophyll molecules, occurs in two apparent kinetic steps with time constants of 10 to 30 seconds and 50 to 300 seconds, depending on the reaction conditions. Here, we use circular dichroism (CD) in the far-UV range to monitor the folding of the LHCIIb apoprotein as it is complexed with pigments. The alpha-helix content in the protein's secondary structure increases in two apparent kinetic steps with time constants similar to those observed for the establishment of chlorophyll energy transfer. When the carotenoid concentration in the reaction mixture is reduced, the time constants of alpha-helix formation increase, as do those for the appearance of chlorophyll energy transfer. This indicates that both processes, pigment assembly and secondary structure formation, are tightly coupled. A substantial amount of alpha-helix is present in dodecylsulfate-solubilised LHCIIb apoprotein and appears to be distributed among various protein domains.

Circular Dichroism↗