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Scripps research study reveals structural dynamics of single prion molecules
February 13, 2007New techniques paint clearer picture of amyloid formation associated with protein-based inheritance and neurodegenerative diseases such as mad cow, Alzheimer's
The new findings, which are being published the week of February 12 in an online edition of the Proceedings of the National Academy of Sciences, offer significant insights into normal folding mechanisms as well as those that lead to abnormal amyloid fibril conversion. The new insights may lead to the discovery of novel therapeutic targets for neurodegenerative diseases.
Intriguingly, certain prions and amyloids can play beneficial roles. The subject of the new study, Sup35, enables protein-based inheritance in yeast. When this prion protein misfolds, it converts into self-perpetuating amyloid fibrils, thus altering its function in an inheritable manner. The research team used a combination of advanced biophysical methods to investigate these processes.
"By focusing on single unfolded prions, we were able to define the dynamics of two distinct regions or domains that determine conversion dynamics," said Ashok A. Deniz, a Scripps Research scientist who led the study. "Our research techniques can now be used to probe the structures of other amyloidogenic proteins. This could prove important in understanding the basic biology of amyloid formation, as well as in designing strategies against misfolding diseases."
Interestingly, the new study revealed that yeast prion protein Sup35 lacks a specific, static structure in its native collapsed state. Instead, the compact protein fluctuates among several different structures before forming intermediate shapes during the amyloid assembly process.
The intermediate stages of the process are critically important, Deniz noted: "No single native unfolded protein is capable of initiating the amyloid cascade because of this constant shape-shifting. To start the amyloid conversion process, it has to first convert to an intermediate species, consisting of multiple protein molecules. This insight may be important to finding potential new therapeutic targets for disease-causing amyloids."
To define the dynamic structural details of individual prions, Deniz and his colleagues employed several novel technologies including single-molecule fluorescence resonance energy transfer (SM-FRET) and fluorescence correlation spectroscopy (FCS).
Fluorescence resonance energy transfer is a highly sensitive tool used to measure molecular structure and dynamics such as in single proteins at the angstrom level, a measurement unit used to define molecular distances (a 10th of a millionth of a millimeter). Fluorescence correlation spectroscopy is a high resolution technique that measures time fluctuations in fluorescent emissions from tagged proteins, which provided information about changes in shape of Sup35 taking place on the nanosecond timescale (billionths of seconds).
A third technology, single molecule fluorescence coincidence, was used in an unusual way-to prove that the protein species under scrutiny were not oligomeric (consisting of multiple proteins in an aggregate). The technology, based on measuring fluorescence bursts from individual tagged proteins, enabled the scientists to determine that the proteins being studied were, in fact, single monomers and not aggregates.
Deniz said that future work with yeast prion mutants might resolve some of the questions that remain unanswered. "Our laboratory has spent a great deal of time in improving these techniques, and we have used them to uncover some very intriguing information about this particular monomer," he said. "This combination of techniques can now be used to study other amyloidogenic proteins, including prions, particularly small assemblies and intermediate stages of the aggregation process. These are currently considered the most toxic forms of amyloid-disease associated proteins."
While mammalian prion proteins are different from those of yeast in their amino acid sequence, they do share some basic features, including their ability to catalyze the conversion to amyloid fibers. Some studies suggest that prions may also play key roles in certain critical processes such as long-term memory. Other authors of the study, A Natively Unfolded Yeast Prion Monomer Adopts An Ensemble of Collapsed and Rapidly Fluctuating Structures, are Samrat Mukhopadhyay and Edward A. Lemke of The Scripps Research Institute; and Susan Lindquist and Rajaraman Krishnan of the Whitehead Institute for Biomedical Research.
Scripps Research Institute
"By focusing on single unfolded prions, we were able to define the dynamics of two distinct regions or domains that determine conversion dynamics," said Ashok A. Deniz, a Scripps Research scientist who led the study. "Our research techniques can now be used to probe the structures of other amyloidogenic proteins. This could prove important in understanding the basic biology of amyloid formation, as well as in designing strategies against misfolding diseases."
Interestingly, the new study revealed that yeast prion protein Sup35 lacks a specific, static structure in its native collapsed state. Instead, the compact protein fluctuates among several different structures before forming intermediate shapes during the amyloid assembly process.
The intermediate stages of the process are critically important, Deniz noted: "No single native unfolded protein is capable of initiating the amyloid cascade because of this constant shape-shifting. To start the amyloid conversion process, it has to first convert to an intermediate species, consisting of multiple protein molecules. This insight may be important to finding potential new therapeutic targets for disease-causing amyloids."
To define the dynamic structural details of individual prions, Deniz and his colleagues employed several novel technologies including single-molecule fluorescence resonance energy transfer (SM-FRET) and fluorescence correlation spectroscopy (FCS).
Fluorescence resonance energy transfer is a highly sensitive tool used to measure molecular structure and dynamics such as in single proteins at the angstrom level, a measurement unit used to define molecular distances (a 10th of a millionth of a millimeter). Fluorescence correlation spectroscopy is a high resolution technique that measures time fluctuations in fluorescent emissions from tagged proteins, which provided information about changes in shape of Sup35 taking place on the nanosecond timescale (billionths of seconds).
A third technology, single molecule fluorescence coincidence, was used in an unusual way-to prove that the protein species under scrutiny were not oligomeric (consisting of multiple proteins in an aggregate). The technology, based on measuring fluorescence bursts from individual tagged proteins, enabled the scientists to determine that the proteins being studied were, in fact, single monomers and not aggregates.
Deniz said that future work with yeast prion mutants might resolve some of the questions that remain unanswered. "Our laboratory has spent a great deal of time in improving these techniques, and we have used them to uncover some very intriguing information about this particular monomer," he said. "This combination of techniques can now be used to study other amyloidogenic proteins, including prions, particularly small assemblies and intermediate stages of the aggregation process. These are currently considered the most toxic forms of amyloid-disease associated proteins."
While mammalian prion proteins are different from those of yeast in their amino acid sequence, they do share some basic features, including their ability to catalyze the conversion to amyloid fibers. Some studies suggest that prions may also play key roles in certain critical processes such as long-term memory. Other authors of the study, A Natively Unfolded Yeast Prion Monomer Adopts An Ensemble of Collapsed and Rapidly Fluctuating Structures, are Samrat Mukhopadhyay and Edward A. Lemke of The Scripps Research Institute; and Susan Lindquist and Rajaraman Krishnan of the Whitehead Institute for Biomedical Research.
Scripps Research Institute
Prion Current Events and Prion News RSSYale-developed test can help predict and diagnose preeclampsia
Researchers at Yale School of Medicine have developed a simple urine test to rapidly predict and diagnose preeclampsia, a common, but serious hypertensive complication of pregnancy.
Scripps Florida scientists show 'lifeless' prions capable of evolutionary change and adaptation
Scientists from The Scripps Research Institute have determined for the first time that prions, bits of infectious protein devoid of DNA or RNA that can cause fatal neurodegenerative disease, are capable of Darwinian evolution.
Proline repeats help protein grow tooth enamel, study shows
A simple amino acid that is repeated in the center of proteins found in tooth enamel makes teeth stronger and more resilient, according to new research at the University of Illinois at Chicago.
Metastasis formation revealed in detail and real time
Up to 25% of cancer patients develop metastases in the brain - often long after successful treatment of the primary tumor. In almost all such cases, the prognosis is poor.
The Protein Srebp2 Drives Cholesterol Formation in Prion-Infected Neuronal Cells Which May Promote Prion-Dependent Diseases
The regulating protein Srebp2 drives cholesterol formation, which prions need for their propagation, in prion-infected neuronal cells.
Prion study reveals first direct information about the protein's molecular structure
A collaboration between scientists at Vanderbilt University and the University of California, San Francisco has led to the first direct information about the molecular structure of prions.
U of T led research team uncovers evolutionary origins of prion disease gene
A University of Toronto-led team has uncovered the evolutionary ancestry of the prion gene, which may reveal new understandings of how the prion protein causes diseases such as bovine spongiform encephalopathy (BSE), also known as "mad cow disease."
Species barrier may protect macaques from chronic wasting disease
Data from an ongoing multi-year study suggest that people who consume deer and elk with chronic wasting disease (CWD) may be protected from infection by an inability of the CWD infectious agent to spread to people.
A Penny for Your Prions
North Carolina State University researchers have discovered a link between copper and the normal functioning of prion proteins, which are associated with transmissible spongiform encephalopathy diseases such as Cruetzfeldt-Jakob in humans or "mad cow" disease in cattle.
Scripps Florida scientists devise accelerated method to determine infectious prion strainsScripps Florida scientists devise accelerated method to determine infectious prion strains
Current tests to identify specific strains of infectious prions, which cause a range of transmissible diseases (such as mad cow) in animals and humans, can take anywhere from six months to a year to yield results - a time-lag that may put human populations at risk.
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