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Probing Individual Complexes of Self-Associating Autotransporters by Single Molecule Force Spectroscopy

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Probing Individual Complexes of Self-Associating Autotransporters by Single Molecule Force Spectroscopy

Kochuparampil, Jumey (2013) Probing Individual Complexes of Self-Associating Autotransporters by Single Molecule Force Spectroscopy. Masters thesis, Concordia University.

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Abstract

Self-associating autotransporters (SAAT) are proteins that play a major role in bacterial auto-aggregation. Members of this family include the adhesin involved in diffuse adherence (AIDA-I) and the enterotoxigenic invasion locus b (TibA) proteins, which represent powerful model systems to study how bacteria promote tight association leading to auto-aggregation. Herein, the homologous interactions of SAATs were systematically determined by using single molecule force spectroscopy (SMFS), which measures the force required to pull apart two interacting molecular entities at the piconewton scale. In this study, a detailed force curve evaluation and in depth histogram analyses were developed. The unbinding force of the AIDA-I/AIDA-I interaction was measured at different loading rates (ranging from 780 to 14000 pN/s). The rate of complex dissociation, koff (0.3 s-1), and the activation energy barrier distance, xb (0.4 nm) were calculated based on the rupture forces. This first single molecule study of the AIDA-I/AIDA-I interaction confirms a very strong self-association that is in line with the important role that SAATs play in the pathogenicity of bacteria. The TibA protein self-association showed a highly selective interaction similar to that of AIDA-I. These single molecule experiments are consistent with previously performed bulk measurements and indicate that self-association between the different SAAT proteins are more specific among the homo-protein pairs (AIDA-I/AIDA-I and TibA/TibA having an unbinding force of 52 ± 13 pN and 64 ± 14 pN, respectively, at a loading rate of 5020 pN/s) than in hetero-protein pairs (AIDA-I/TibA).

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (Masters)
Authors:Kochuparampil, Jumey
Institution:Concordia University
Degree Name:M. Sc.
Program:Chemistry
Date:30 April 2013
Thesis Supervisor(s):Cuccia, Louis A.
Keywords:Self-Associating Autotransporters, adhesin involved in diffuse adherence (AIDA-I), enterotoxigenic invasion locus b (TibA) proteinsSingle Molecule Force Spectroscopy
ID Code:977272
Deposited By: JUMMEY KOCHUPARAMPIL
Deposited On:13 Jun 2013 19:52
Last Modified:18 Jan 2018 17:44

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