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You are here: Home Events Prof. Tudor Luchian "Nanoscopic Interrogation of Molecular Interactions With Protein Nanopores"

Prof. Tudor Luchian "Nanoscopic Interrogation of Molecular Interactions With Protein Nanopores"

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Department of Physics, Laboratory of Molecular Biophysics and Medical Physics, Alexandru I. Cuza University, Iasi, Romania

What
  • Seminar
When Dec 10, 2014
from 02:15 PM to 03:00 PM
Where Seminarraum A, FMF, Stefan-Meier-Str. 21, Freiburg
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Currently, a large number of complementary techniques are employed to analyze membrane proteins, able to describe their behavior at various lengths and time scales. Of these, nanopore-based, single-molecule techniques have several unique attributes when it comes to single molecule sensing, e.g. high temporal (microsecond scale) and spatial (nanometer scale) resolution. The working principle is simple, whereby the single macromolecule capture, entry and subsequent translocations through voltage-biased nanopores, depend upon their physico-chemical and topological features of the analyte. While the specificity of the technique is essentially dictated by the nanopore’s confining volume molecular architecture, which rules the energetics of analyte-nanopore interactions, the volumetric and kinetic information that is embedded in the blockade measurements allows the direct characterization of the sensed analytes.  In general, nanopore-based, single-molecule techniques do not require labeled molecules, so the preparation of the samples is simpler and the molecules are not chemically modified. As a result, nanopore technologies have immense impact in ultra-sensitive detection of molecules, monitoring biochemical reactions at a single-molecule level, development of biocompatible semiconductors, and DNA sequencing with relevance in clinical diagnosis and anti-bioterorism.

Herein, by employing model peptides of physiological relevance, we will demonstrate the utility of the Staphylococcus aureus α-hemolysin (-HL) protein nanopore to exploring at uni-molecular level the morphological changes of peptides as a result of varying the pH, ionic strength, and exposure to a selection of metal ions relevant for the onset of misfolding diseases. By using pH-tuned single-molecule electrophysiology, we demonstrate how peptide movement through the α-HL protein can be slowed down, as to control residence time, direction and the sequence of spatio-temporal state-to-state dynamics of a single peptide, which allow the clear discrimination between various partially unfolded conformations of peptide inside the nanopore. We present evidence supporting the possibility of quantifying the dissociation constants of certain metals to peptide fragments differing by just an aminoacid. We will also describe the use of single -HL nanopores and engineered polypeptides to increase both the polypeptide capture rate and mean residence time of the molecules in the pore at increasing transmembrane potentials, irrespective of polarity. The results advance the possibility of studying microscopic features of peptides or other biological molecules with nanopores, over voltage-dependent controllable, wide range of times while a single peptide dwells inside the nanopore, with improved in the signal to noise ratio.
 

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