Probing Allosteric Responses in Large Peptide Synthetases by Solution NMR

Dr. Dominique Frueh, Johns Hopkins School of Medicine

Host: Dr. David Fushman

Abstract: In this presentation, we will illustrate how the non-invasive nature of NMR can be harnessed to probe molecular responses in macromolecules during biochemical reactions. We will first explain how NMR helps us understand domain communication in nonribosomal peptide synthetases, and later describe NMR methods we developed to overcome challenges in NMR studies of larger proteins. Nonribosomal peptide synthetases (NRPSs) use repeats of domains to tether substrates onto carrier protein domains (CP) and assemble them into complex products through intervening condensation or cyclization domains. A dynamic domain architecture has hampered NRPS engineering because it is unclear whether and how catalytic steps are required for synthesis to drive transient domain interactions. Here, we demonstrate experimentally that large-scale structural dynamics within a 52 kDa cyclization domain sense the attachment of substrates to its partner CP and promote binding while opening an allosteric path to a remote binding site. The demonstration relies on a nuclear magnetic resonance experimental design, in which we monitor the molecular response of a protein towards covalent modifications of its binding partner in situ. Assigning the NMR resonances of a 453 residue protein is challenging, and we will show how mathematical operations applied to NMR spectra can provide new correlation maps that facilitate their analysis. We will also briefly describe how we employ NMR as a tool to guarantee the integrity of our samples during our studies, irrespectively of the techniques providing readouts. Our results exemplify how structural dynamics within proteins can couple substrate recognition with active site remodeling and remote protein communication, and they illustrate the versatility of NMR as an atomic readout of biomolecular function. 

Biochemistry Seminar