Download 1 - From protein structure to biological function through interactomics

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PCISBIO Day – 11 July 2016
http://www.itqb.unl.pt/pcisbio2016/
1st - From protein structure to biological function through
interactomics – an integrated view
Bruno Manadas1, Isaura Simões1, Pedro Castanheira2, Rui Brito3, Sandra Anjo1,3
1
CNC, Coimbra; 2Biocant, Cantanhede, 3University of Coimbra
The course was organized by the Center for Neurosciences and Cell Biology – University of
Coimbra, and took place at the UC-Biotech in Cantanhede at the Proteomics and Molecular
Biotechnology Units.
Protein-protein interactions (PPIs) are key elements for the normal function of a living cell.
The identification and quantitative and structural characterization of PPI networks allow for
an integrated view and a better understanding of the functioning of a living cell or an
organism. The course aimed to cover advanced experimental strategies to identify and
characterize PPI networks commonly known as interactomes. The course extensively
focused on the workflow of a protein interactomics project, consisting of: 1) the capture of a
network of interactions and the identification of interacting partners; 2) novel approaches for
parallel recombinant protein expression and purification of the interacting partners; 3)
physical-chemical characterization of the interactions and 4) identification and structural
characterization of binding sites. The integrative nature of the approach allowed a better
understanding of the works of living cells but also as an exercise in integration of data from
multiple disciplines and methodologies such as biochemistry, molecular biology, protein
chemistry, biophysics, thermodynamics, mass spectrometry and structural biology.
The course dedicated special emphasis to novel technical approaches, such as AP-SWATH
(affinity purification followed by quantitative mass spectrometry), new cloning methods such
as CPEC and SLiCE, multi-tiered protein expression approaches, in cell NMR, protein
binding-sites through cross-linking approaches, ITC, and several other auxiliary biophysical
techniques.
The course started with Msc Sandra Anjo and Dr Bruno Manadas providing a biological
context which was used as a backbone for the course, based on an FCT interactomics’
project. Then, Dr Antonio Serna (Sciex) – application specialist for proteomics and
quantitative proteomics provided detailed information on data acquisition and processing.
Dr. Germán Rivas Caballero (CSIC, Madrid) presented the study of molecular interactions
using analytical ultracentrifugation and light scattering in highly crowded environments.
Dr. Renaud Vincentelli (CNRS Aix Marseille) presented several aspects of his work, in
particular the high-throughput expression of small Cys-rich proteins and peptides from
venomous animals. He also presented a new high-throughput assay to quantify domainligand affinities and specificities.
Dr Attila Aranyos – FortéBio – Application Specialist of FortéBio – Presented the Biolayer
Interferomety technology.
Dr. Raul Pacheco-Gomez – Malvern – Application Specialist of Microcal – Presented
microcalorimetry techniques for the characterization of proteins and protein interactions, in
particular ITC and DSC.
Dr Rui Brito (CNC) introduced different protein structural characterization techniques,
including circular dichroism and Nuclear Magnetic Ressonance, both for quality control and
structural elucidation of binding partners.
Dr. Alexander Shekhtman (Associate Professor at University at Albany, USA) presented a
talk and a seminar on "Protein Interactions using live cell NMR".
Dr. Anthonius Heemskerk, (Sciex) Application Support Specialist introduced and presented
CESI-MS applications for the analysis of intact protein complexes.
The course ended with group presentations from the students, selection of the best project,
and an online anonymous evaluation of the course.