We propose to use time-resolved fluorescence energy transfer to investigate protein-induced DNA bending in solution. We will measure multiple protein-to-DNA and DNA-to-DNA distances in each of two protein-DNA complexes that exhibit dramatic protein-induced DNA bending in the crystalline state: the CAP-DNA complex and the TBP-DNA complex. We will determine DNA bend angles and trajectories in solution, assess the salt-dependence of the protein-induced DNA bending, assess the effects of protein mutants on protein-induced DNA bending, assess the effects of DNA mutants on protein-induced DNA bending. We have chosen AEDANS (lex=340nm; lem=480nm) as the donor fluorochrome and fluorescein (lex=490 nm; lem=520 nm) as the acceptor fluorochrome. We have incorporated AEDANS at specific sites in protein by site-directed mutagenesis to introduce unique surface cysteines followed by cysteine-specific chemical modification, and we have incorporated fluorescein at specific sites in DNA by total synthesis. Pilot experiments support the feasibility of the proposed work but indicate the need for a laser system with a more stable baseline and with subnanosecond time resolution.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR001348-15
Application #
5223318
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
15
Fiscal Year
1996
Total Cost
Indirect Cost
Sheth, Rahul A; Arellano, Ronald S; Uppot, Raul N et al. (2015) Prospective trial with optical molecular imaging for percutaneous interventions in focal hepatic lesions. Radiology 274:917-26
Roussakis, Emmanuel; Spencer, Joel A; Lin, Charles P et al. (2014) Two-photon antenna-core oxygen probe with enhanced performance. Anal Chem 86:5937-45
Courter, Joel R; Abdo, Mohannad; Brown, Stephen P et al. (2014) The design and synthesis of alanine-rich ?-helical peptides constrained by an S,S-tetrazine photochemical trigger: a fragment union approach. J Org Chem 79:759-68
Kuroda, Daniel G; Bauman, Joseph D; Challa, J Reddy et al. (2013) Snapshot of the equilibrium dynamics of a drug bound to HIV-1 reverse transcriptase. Nat Chem 5:174-81
Lam, A R; Moran, S D; Preketes, N K et al. (2013) Study of the ?D-crystallin protein using two-dimensional infrared (2DIR) spectroscopy: experiment and simulation. J Phys Chem B 117:15436-43
Kuroda, Daniel G; Singh, Prabhat K; Hochstrasser, Robin M (2013) Differential hydration of tricyanomethanide observed by time resolved vibrational spectroscopy. J Phys Chem B 117:4354-64
Singh, Prabhat K; Kuroda, Daniel G; Hochstrasser, Robin M (2013) An ion's perspective on the molecular motions of nanoconfined water: a two-dimensional infrared spectroscopy study. J Phys Chem B 117:9775-84
Chuntonov, Lev; Ma, Jianqiang (2013) Quantum process tomography quantifies coherence transfer dynamics in vibrational exciton. J Phys Chem B 117:13631-8
Culik, Robert M; Annavarapu, Srinivas; Nanda, Vikas et al. (2013) Using D-Amino Acids to Delineate the Mechanism of Protein Folding: Application to Trp-cage. Chem Phys 422:
Goldberg, Jacob M; Speight, Lee C; Fegley, Mark W et al. (2012) Minimalist probes for studying protein dynamics: thioamide quenching of selectively excitable fluorescent amino acids. J Am Chem Soc 134:6088-91

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