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Published on-line at 11:01:12 AM on Tuesday, September 4th, 2012

Ultrafast force-clamp spectroscopy of single molecules reveals load dependence of myosin working stroke

Marco Capitanio, Carina Monico, Francesco Saverio Pavone, Francesco Vanzi on NATURE METHODS (NAT METHODS)

New ultrafast force-clamp spectroscopy to understand the molecular mechanism underlying muscle contraction, interactions between proteins and DNA, and more.

In this paper a technique able to capture for the first time the movements of a single biological molecule with a detail of a millionth of a millimeter and within a few millionths of second is presented and described. This technique has enabled a major step forward in our understanding of the molecular mechanism underlying the regulation of the speed of contraction of the muscles subjected to an applied load.

The muscle is shortened due to the interaction between two cyclic proteins present in all muscles: myosin, which is the molecular motor of contraction, and actin. At each cycle of interaction, myosin, consuming energy, changes its conformation and moves the actin a few millionths of a millimeter in times of the order of a few thousandths of a second. The sum of the activity of thousands of myosin molecules that repeat their cycle many times during contraction, produces the phenomenon that is valued at a macroscopic level as muscle shortening and movement.

Schematic of a dual-trap or dumbbell configuration. B can be actin, a microtubule, DNA, RNA or any polymer and A, any protein interacting with B (myosin, kinesin, dynein, DNA or RNA-binding proteins).

In this so called ultrafast force-clamp spectroscopy, the actin functions as a sort of leash and the optical trap pulls it to apply constant forces to myosin. The optical trap is able to pull on the leash about 10 microseconds after the myosin is bound to actin and displays the dynamic interaction actin-myosin with a temporal resolution of a few tens of microseconds. "The temporal resolution of this system, - declares Roberto Bottinelli -, about two orders of magnitude higher than that of previous systems, has allowed, for the first time, to directly measure the dynamics of conformational changes of myosin and their dependence on a external load, allowing to understand the molecular mechanism underlying the muscle contraction and its regulation in the presence of external forces".

"In addition, - explains Marco Capitanio - our technique is applicable to a wide variety of biological systems and in this work we have exploited its unique characteristics to study the interaction between proteins and DNA. In the cell, a variety of proteins and molecular motors interact with DNA and are involved in fundamental processes, such as the regulation of gene expression. Our technique has allowed us to detect interactions between proteins and DNA and so to try to clarify the molecular mechanisms underlying these fundamental phenomena of cellular life".

Source: Nature Methods

  • Title:
  • Ultrafast force-clamp spectroscopy of single molecules reveals load dependence of myosin working stroke
  • Authors:
  • Capitanio, M Canepari, M Maffei, M Beneventi, D Monico, C Vanzi, F Bottinelli, R Pavone, FS
  • Source:
  • NATURE METHODS
  • Vol. na, Issue: na, pages: na-na, Article Number: na, SEP 02 2012, DOI: 10.1038/nmeth.2152