| Popis: |
Nonribosomal peptide synthetases (NRPSs) are modular, multi-domain, enzymatic assembly lines that combine simple substrates to synthesize complex natural products (e.g. penicillin, bacitracin, yersiniabactin). Substrates are each covalently attached to so-called carrier proteins (CP) via a post-translationally introduced phosphopantetheine arm. CPs present the substrates to catalytic domains in a series of transient, sequential domain/domain and domain/substrate interactions during biosynthesis. Thus, understanding the mechanism of NRPS assembly line synthesis necessitates both characterizing fleeting molecular interactions and unraveling the molecular determinants for the directionality of these interactions. Unfortunately, the large molecular weight of NRPS domains, the presence of dynamics between domains and within domains, and the lability of tethered substrates have precluded studying these processes at the molecular level. Here, we present methods that permitted us to overcome these limitations and we discuss our findings. First, by exploiting the non-invasive nature of NMR we found that chemical substrates interact with their carrier proteins and these interactions may modulate domain communication to provide directionality. Second, we present a method that facilitates resonance assignments in large proteins. By applying covariance to conventional spectra, simplified correlation maps permit to identify sequential residues or to assign side-chain resonances with a simple visual inspection. Hence, resonances of 37 kDa and 53 kDa NRPS domains could be assigned unambiguously, enabling us to describe molecular signatures of transient interactions involving these domains. |
