Development of photo-cidnp methods for nmr sensitivity enhancement in solution & conformational changes of the drkn sh3 protein upon interaction with the hsp70 molecular chaperone

The first part of my Ph.D. thesis involves developing photochemically induced dynamic nuclear polarization (photo-CIDNP) methods to enhance NMR sensitivity and facilitate the analysis of small and large biomolecules in solution. First, I developed a 1H-detected 13C Photo-CIDNP pulse sequence (Chapter 2), which exploits 13C photo-CIDNP-inducing laser irradiation followed by polarization transfer to 1H for detection. Using this sequence, I was able to achieve large NMR sensitivity enhancements (up to 16-fold compared to SE-HSQC), for both side chain and backbone resonances of model polypeptides in solution. Second, I introduced a novel tri-enzyme system to simultaneously recycle the photosensitizer inactive form and prevent sample photodegradation during prolonged high-power laser pulse irradiation (Chapter 3). The addition of catalytic amounts of the tri-enzyme system to NMR samples enables long-term sensitivity-enhanced photo-CIDNP data collection, with up to 48-fold greater sensitivity relative to SE-HSQC, at low-micromolar biomolecule concentrations. The second part of this thesis discusses the interaction of the Hsp70 chaperone with its substrate. First, the fact that most proteins have Hsp70 binding sites raises the question of whether Hsp70 interacts with even nonobligatory substrate, i.e., proteins that do not require chaperones to fold. Kinetic studies showed that the interaction of DnaK, the E. coli Hsp70, and its co-chaperones with a nonobligatory substrate is short-lived, not interfering with the timely production of substrate and enabling the chaperone to assist other proteins that are prone to aggregation (Chapter 4). Second, although the conformational changes experienced by chaperone substrates provide key insights into DnaK's function, very few studies were carried out toward this end, especially with full-length protein substrates. By using multidimensional NMR and native gel analysis, we show here that the majority of the marginally stable full-length protein substrate drkN SH3 is associated with DnaK, when the chaperone is added to the medium (Chapter 5). A large shift in population towards the unfolded state, which is believed to be mostly bound, was observed. One or more NMR-invisible "dark states" are proposed to explain the decrease in the overall NMR-detectable populations upon addition of DnaK to the medium. Future directions are discussed in Chapter 6.