It's well known that the protein calmodulin specifically targets and steers the activities of hundreds of other proteins – mostly kinases – in our cells, thus playing a role in physiologically important processes ranging from gene transcription to nerve growth and muscle contraction But just how it distinguishes between target proteins is not well understood.
Methods developed by biophysicists at the Technische Universitaet Muenchen (TUM) have enabled them to manipulate and observe calmodulin in action, on the single-molecule scale. In recent experiments, as they report in the early edition of PNAS, the Proceedings of the National Academy of Sciences, they compared the sequences of structural and kinetic changes involved in binding two different kinases. The results reveal new details of how calmodulin binds and regulates its target proteins.
A so-called signaling protein and "calcium sensor," calmodulin gives start and stop signals for a great number of intracellular activities by binding and releasing other proteins. Calmodulin can bind up to four calcium ions, and the three-dimensional spatial structure of calmodulin varies with the number of calcium ions bound to it. This structure in turn helps to determine which amino acid chains – peptides and proteins – the calmodulin will bind.
Techniques such as X-ray structural analysis offer snapshots, at best, of steps in this intracellular work flow. But single-molecule atomic-force spectroscopy has opened a new window on such dynamic processes.
Professor Matthias Rief and colleagues at the Technische Universitaet Muenchen had previously shown that they could fix a single calmodulin molecule between a surface and the cantilever tip of a specially built atomic-force microscope, expose it to calcium ions in solution, induce peptide binding and unbinding, and measure changes in the molecule's mechanical properties as it did its work.
"What is special about our technique," Rief says, "is that we can work directly in aqueous solution. We can make our measurements in exactly the conditions under which the protein works in its natural environment. So we can directly observe how the calmodulin snatches the amino acid chain and folds itself, to hold its target fast." Measuring the force needed to bend the calmodulin molecule out of its stable condition at any given moment enables the researchers to compute the energies associated with binding both the calcium ions and the amino acid chains. And by following changes in the molecule's mechanical properties over time, they also can determine how long a protein fragment remains bound.
The results Rief and biophysicist Jan Philipp Junker report in the early edition of PNAS show that their approach also enables detailed comparative studies of binding sequences for different target proteins. The target sequences observed in these experiments are called skMLCK and CaMKK. Rief and Junker used mechanical force – actually pulling on complexes of calmodulin and the target peptides at rates of 1 nanometer per second or less – to slow down the processes to observable time scales and to clearly separate the individual unbinding steps.
"By applying mechanical force," Junker says, "we are able to dismantle the calmodulin-target peptide complex with surgical precision. Using conventional methods, this would be very difficult to do."
Among the detailed insights this approach made accessible are the hierarchy of folding and target binding, the sequence of unbinding events, and target-specific differences in terms of what is called cooperative binding.
Original paper: "Single-molecule force spectroscopy distinguishes target binding modes of calmodulin," by Jan Philipp Junker and Matthias Rief, published in the online Early Edition of PNAS, Proceedings of the National Academy of Sciences for the week of August 10, 2009.Contact:
Patrick Regan | EurekAlert!
Desert ants cannot be fooled
23.11.2017 | Max-Planck-Institut für chemische Ökologie
Bacteria as pacemaker for the intestine
22.11.2017 | Christian-Albrechts-Universität zu Kiel
Heat from the friction of rocks caused by tidal forces could be the “engine” for the hydrothermal activity on Saturn's moon Enceladus. This presupposes that...
The WHO reports an estimated 429,000 malaria deaths each year. The disease mostly affects tropical and subtropical regions and in particular the African continent. The Fraunhofer Institute for Silicate Research ISC teamed up with the Fraunhofer Institute for Molecular Biology and Applied Ecology IME and the Institute of Tropical Medicine at the University of Tübingen for a new test method to detect malaria parasites in blood. The idea of the research project “NanoFRET” is to develop a highly sensitive and reliable rapid diagnostic test so that patient treatment can begin as early as possible.
Malaria is caused by parasites transmitted by mosquito bite. The most dangerous form of malaria is malaria tropica. Left untreated, it is fatal in most cases....
The formation of stars in distant galaxies is still largely unexplored. For the first time, astron-omers at the University of Geneva have now been able to closely observe a star system six billion light-years away. In doing so, they are confirming earlier simulations made by the University of Zurich. One special effect is made possible by the multiple reflections of images that run through the cosmos like a snake.
Today, astronomers have a pretty accurate idea of how stars were formed in the recent cosmic past. But do these laws also apply to older galaxies? For around a...
Just because someone is smart and well-motivated doesn't mean he or she can learn the visual skills needed to excel at tasks like matching fingerprints, interpreting medical X-rays, keeping track of aircraft on radar displays or forensic face matching.
That is the implication of a new study which shows for the first time that there is a broad range of differences in people's visual ability and that these...
Computer Tomography (CT) is a standard procedure in hospitals, but so far, the technology has not been suitable for imaging extremely small objects. In PNAS, a team from the Technical University of Munich (TUM) describes a Nano-CT device that creates three-dimensional x-ray images at resolutions up to 100 nanometers. The first test application: Together with colleagues from the University of Kassel and Helmholtz-Zentrum Geesthacht the researchers analyzed the locomotory system of a velvet worm.
During a CT analysis, the object under investigation is x-rayed and a detector measures the respective amount of radiation absorbed from various angles....
15.11.2017 | Event News
15.11.2017 | Event News
30.10.2017 | Event News
23.11.2017 | Earth Sciences
23.11.2017 | Physics and Astronomy
23.11.2017 | Power and Electrical Engineering