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Ultrafast Vibrational Spectroscopy of Allosteric Proteins (Extension)

English title Ultrafast Vibrational Spectroscopy of Allosteric Proteins (Extension)
Applicant Hamm Peter
Number 188694
Funding scheme Project funding (Div. I-III)
Research institution Institut für Chemie Universität Zürich
Institution of higher education University of Zurich - ZH
Main discipline Physical Chemistry
Start/End 01.01.2020 - 31.12.2022
Approved amount 773'577.00
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Keywords (4)

Infrared Spectroscopy; Ultrafast Spectrosocpy; Protein Dynamics; Allosterie

Lay Summary (German)

Lead
Allosterie ist ein wichtiges Konzept in der Biochemie, das für die Regulation chemischer Prozesse in einer Zelle verantwortlich ist. Allosterische Proteine besitzen in der Regel zwei Bindungszentren; ein enzymatisches Zentrum, in dem ein bestimmtes Molekül chemisch modifiziert wird, sowie ein allosterisches Zentrum. In Abhängigkeit davon, ob ein bestimmtes anderes Molekül (Ligand) an dieses allosterische Zentrum bindet, wird die chemische Aktivität im enzymatischen Zentrum hoch- bzw. runtergefahren. Oft sind die beiden Bindungszentren relativ weit voneinander entfernt, und bis heute ist weitgehend unklar, wie sie miteinander „kommunizieren“. Man geht davon aus, dass ein Ligand im allosterischen Zentrum eine, wenn auch oft sehr kleine, strukturelle Veränderung des Proteins als Ganzes verursacht, die einen Einfluss auf die chemische Aktivität des enzymatischen Zentrums hat.
Lay summary

Wir entwickeln speziell designte Proteine und/oder Liganden, um mit Hilfe der zeitaufgelösten Infrarotspektroskopie die Ausbreitung eines Signals in einem Protein messen zu können. Dazu stören wir das Protein gezielt im Bereich des allosterischen Zentrums in dem wir z.B. einen durch einen kurzen Lichtblitz schaltbaren Liganden vom Protein dissoziieren, und messen die spektroskopische Antwort des Proteins im Bereich der sog. Amide I Bande, die zum gewissen Grad die Struktur des Proteins widerspiegelt. In Kombination mit Computersimulationen werden diese Experimente neue Einblicke in die relavanten Zeitskalen, und die Mechanismen vom allosterischer Kommunikation ermöglichen.

Direct link to Lay Summary Last update: 16.10.2019

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Number Title Start Funding scheme
183615 NCCR MUST: Molecular Ultrafast Science and Technology (phase III) 01.07.2018 National Centres of Competence in Research (NCCRs)
165789 Ultrafast Vibrational Spectroscopy of Allosteric Proteins 01.01.2017 Project funding (Div. I-III)

Abstract

With the term "allostery" one describes the coupling of two separated sites of a protein, where binding of a ligand at the so-called allosteric site changes the function of the protein at a remote active site. Allostery is one of the fundamental mechanisms of regulatory processes in life. The very question of how these two sites communicate with each other remains an intriguing and controversial problem, with the ultimate question of how an allosteric signal "propagates" through a protein. Transient IR spectroscopy provides the time resolution combined with the chemical selectivity necessary to study these non-equilibrium processes. In these experiments, an allosteric protein is light-trigger with the help of a photo-isomerizing azobenzene moiety, which is incorporated into the protein in a way that it mimics an allosteric process, and the response of the protein is recorded by transient IR spectroscopy. Measuring that response in a site-selective manner would reveal the propagation of an allosteric signal.In order to build on a wide basis of protein systems for these experiments, we focused on the design and characterization of three different protein systems during the first two years of the previous grant period. That is, we have characterized two protein/ligand complexes (i.e., RNase with a photo-switchable S-peptide and the PDZ2 domain with a photo-switchable peptide ligand), which are designed in a way that the binding affinity of the photo-switchable ligand is significantly different in the two states of the photo-switch. Hence, we can photo-trigger unbinding of the ligand. In addition, we considered the PDZ3 domain, photo-switching its terminal a3-helix, which has been shown to modulate the binding affinity at the remote binding groove significantly. That model system is probably the smallest protein that can be considered a full allosteric system with two clearly identifiable sites that communicate with each other.In the second grant period, we will continue and finalize time-resolved IR experiments on all molecular systems that are now established. In addition, we will introduce the MDM2/p53-TAD complex as a 4th protein system, whose construct is similar to the RNase/S-peptide complex, but whose biological relevance is more significant. In combination with accompanying MD simulations (performed in the group of our collaborator Gerhard Stock, University of Freiburg), these experiments will provide an unprecedented new, and hopefully unifying, view of the mechanism of allosteric communication, which will eventually pave the way towards a more rational design of new drugs that interact with allosteric regulation networks.
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