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Development of a technique for the investigation of folding dynamics of single proteins for extended time periods.

ABSTRACT: A technique was developed for the detection of fluorescence signals from free single molecules for extended time periods and was applied to the characterization of the unfolded states of iso-1-cytochrome c (cyt c). Protein molecules labeled with fluorescent dye were slowly injected into a capillary at concentrations that allow for the observation of one molecule at a time. A laser was introduced into the capillary coaxially, and the fluorescence was imaged as traces by using a lens with a large focal depth and wide field of view. Thus, the traces reflect the time-dependent changes in the fluorescence signals from single proteins. Cyt c was labeled with Alexa Fluor 532 at the C-terminal cysteine (cyt c-Alexa). In bulk experiments, cyt c-Alexa was shown to possess different fluorescence intensity for the native state, the unfolded state (U), and the intermediate state. Single-molecule traces of cyt c-Alexa were recorded by using the device. Intensity histograms of the traces revealed two distributions with broad and narrow widths, which were interpreted to correspond to the U and intermediate state, respectively, observed in the bulk measurements. The broad width of the U suggested the existence of a relatively slow conformational dynamics, which might be consistent with the correlation time ( approximately 15 ms) estimated from the traces assignable to the U. The technique was expected to reveal dynamics of proteins along the folding processes without artifacts caused by immobilization.

SUBMITTER: Kinoshita M

PROVIDER: S-EPMC1965534 | biostudies-other | 2007 Jun

REPOSITORIES: biostudies-other

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Development of a technique for the investigation of folding dynamics of single proteins for extended time periods.

Kinoshita Masahito M Kamagata Kiyoto K Maeda Akio A Goto Yuji Y Komatsuzaki Tamiki T Takahashi Satoshi S

Proceedings of the National Academy of Sciences of the United States of America 20070611 25

A technique was developed for the detection of fluorescence signals from free single molecules for extended time periods and was applied to the characterization of the unfolded states of iso-1-cytochrome c (cyt c). Protein molecules labeled with fluorescent dye were slowly injected into a capillary at concentrations that allow for the observation of one molecule at a time. A laser was introduced into the capillary coaxially, and the fluorescence was imaged as traces by using a lens with a large ...[more]

PMID: 17563378

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Project description:Microbes evolve within complex ecological communities where biotic interactions impact both individual cells and the environment as a whole. Here we examine how cellular regulation in the marine cyanobacterium Prochlorococcus is influenced by a heterotrophic bacterium, Alteromonas macleodii, under different light conditions. We monitored the transcriptome of Prochlorococcus, grown either alone or in coculture, across a diel light:dark cycle and under the stress of extended darkness-a condition that cells would experience when mixed below the ocean's euphotic zone. More Prochlorococcus transcripts exhibited 24-h periodic oscillations in coculture than in pure culture, both over the normal diel cycle and after the shift to extended darkness. This demonstrates that biotic interactions, and not just light, can affect timing mechanisms in Prochlorococcus, which lacks a self-sustaining circadian oscillator. The transcriptomes of replicate pure cultures of Prochlorococcus lost their synchrony within 5 h of extended darkness and reflected changes in stress responses and metabolic functions consistent with growth cessation. In contrast, when grown with Alteromonas, replicate Prochlorococcus transcriptomes tracked each other for at least 13 h in the dark and showed signs of continued biosynthetic and metabolic activity. The transcriptome patterns suggest that the heterotroph may be providing energy or essential biosynthetic substrates to Prochlorococcus in the form of organic compounds, sustaining this autotroph when it is deprived of solar energy. Our findings reveal conditions where mixotrophic metabolism may benefit marine cyanobacteria and highlight new impacts of community interactions on basic Prochlorococcus cellular processes. IMPORTANCEProchlorococcus is the most abundant photosynthetic organism on the planet. These cells play a central role in the physiology of surrounding heterotrophs by supplying them with fixed organic carbon. It is becoming increasingly clear, however, that interactions with heterotrophs can affect autotrophs as well. Here we show that such interactions have a marked impact on the response of Prochlorococcus to the stress of extended periods of darkness, as reflected in transcriptional dynamics. These data suggest that diel transcriptional rhythms within Prochlorococcus, which are generally considered to be strictly under the control of light quantity, quality, and timing, can also be influenced by biotic interactions. Together, these findings provide new insights into the importance of microbial interactions on Prochlorococcus physiology and reveal conditions where heterotroph-derived compounds may support autotrophs-contrary to the canonical autotroph-to-heterotroph trophic paradigm.

Dataset Information

Development of a technique for the investigation of folding dynamics of single proteins for extended time periods.

Publications

Development of a technique for the investigation of folding dynamics of single proteins for extended time periods.

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