Forum for Science, Industry and Business

Sponsored by:     3M 
Search our Site:

 

Single-molecule technique captures calcium sensor calmodulin in action

12.08.2009
Unknown workings of a signaling protein unfold in the hands of biophysicists, through atomic-force spectroscopy

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:
Prof. Matthias Rief
Chair for Experimental Physics
Technische Universitaet Muenchen (TUM)
James-Franck-Str. 1
85748 Garching, Germany
Tel: +49 89 289 12471
Fax: +49 89 289 12523
E-mail: mrief@ph.tum.de

Patrick Regan | EurekAlert!
Further information:
http://www.tum.de

More articles from Life Sciences:

nachricht A novel socio-ecological approach helps identifying suitable wolf habitats
17.02.2017 | Universität Zürich

nachricht New, ultra-flexible probes form reliable, scar-free integration with the brain
16.02.2017 | University of Texas at Austin

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

Die letzten 5 Focus-News des innovations-reports im Überblick:

Im Focus: Breakthrough with a chain of gold atoms

In the field of nanoscience, an international team of physicists with participants from Konstanz has achieved a breakthrough in understanding heat transport

In the field of nanoscience, an international team of physicists with participants from Konstanz has achieved a breakthrough in understanding heat transport

Im Focus: DNA repair: a new letter in the cell alphabet

Results reveal how discoveries may be hidden in scientific “blind spots”

Cells need to repair damaged DNA in our genes to prevent the development of cancer and other diseases. Our cells therefore activate and send “repair-proteins”...

Im Focus: Dresdner scientists print tomorrow’s world

The Fraunhofer IWS Dresden and Technische Universität Dresden inaugurated their jointly operated Center for Additive Manufacturing Dresden (AMCD) with a festive ceremony on February 7, 2017. Scientists from various disciplines perform research on materials, additive manufacturing processes and innovative technologies, which build up components in a layer by layer process. This technology opens up new horizons for component design and combinations of functions. For example during fabrication, electrical conductors and sensors are already able to be additively manufactured into components. They provide information about stress conditions of a product during operation.

The 3D-printing technology, or additive manufacturing as it is often called, has long made the step out of scientific research laboratories into industrial...

Im Focus: Mimicking nature's cellular architectures via 3-D printing

Research offers new level of control over the structure of 3-D printed materials

Nature does amazing things with limited design materials. Grass, for example, can support its own weight, resist strong wind loads, and recover after being...

Im Focus: Three Magnetic States for Each Hole

Nanometer-scale magnetic perforated grids could create new possibilities for computing. Together with international colleagues, scientists from the Helmholtz Zentrum Dresden-Rossendorf (HZDR) have shown how a cobalt grid can be reliably programmed at room temperature. In addition they discovered that for every hole ("antidot") three magnetic states can be configured. The results have been published in the journal "Scientific Reports".

Physicist Dr. Rantej Bali from the HZDR, together with scientists from Singapore and Australia, designed a special grid structure in a thin layer of cobalt in...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

Event News

Booth and panel discussion – The Lindau Nobel Laureate Meetings at the AAAS 2017 Annual Meeting

13.02.2017 | Event News

Complex Loading versus Hidden Reserves

10.02.2017 | Event News

International Conference on Crystal Growth in Freiburg

09.02.2017 | Event News

 
Latest News

Switched-on DNA

20.02.2017 | Materials Sciences

Second cause of hidden hearing loss identified

20.02.2017 | Health and Medicine

Prospect for more effective treatment of nerve pain

20.02.2017 | Health and Medicine

VideoLinks
B2B-VideoLinks
More VideoLinks >>>