Link to the University of Pittsburgh Homepage
Link to the University Library System Homepage Link to the Contact Us Form

Copper-Ion Based Electron Spin Resonance Sheds light on Protein-DNA Interaction

Ji, Ming (2014) Copper-Ion Based Electron Spin Resonance Sheds light on Protein-DNA Interaction. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

Primary Text

Download (2MB) | Preview


This thesis focuses on Cu2+-based electron spin resonance (ESR) spectroscopy and its application in biophysics. First, we review the use of paramagnetic metal ions like Cu2+, to measure distance constraints in protein by double electron electron resonance (DEER) spectroscopy. Although lower sensitivity and orientational selectivity complicate data collection and analysis, the simple sample preparation process and limited spatial flexibility makes Cu2+ an attractive probe.
We then use Cu2+ as spin probe to develop insights into the molecular determinants of catalysis in type II restriction endonuclease EcoRI. We show that Cu2+ does not catalyze the DNA cleavage by EcoRI. More strikingly, it inhibits the Mg2+-dependent DNA cleavage. We perform X-band (~9.5 GHz) ESR spectroscopy as well as biochemical studies to understand the functional difference between Cu2+ and Mg2+. These results reveal a hitherto unknown metal ion binding site. Molecular dynamics (MD) simulations reveal that the Cu2+-H114 coordination disrupts a critical protein-DNA interaction, which is essential for catalysis. This structural change may lead to inhibition of Mg2+-dependent cleavage. We also propose an electrostatic basis for the positive cooperativity of Mg2+ binding to the EcoRI-DNA complex. Such cooperativity helps EcoRI inactivate DNA by double-strand cuts instead of single-strand nicks.
Next, we use W-band (~95 GHz) ENDOR as well as X-band electron spin echo envelope modulation (ESEEM) spectroscopy to further refine the Cu2+ coordination environment in the EcoRI-DNA complex. The existence of equatorially coordinated water molecules is confirmed by the ENDOR results. Simulations of the X-band ESEEM spectra at different magnetic fields indicate that the Cu2+-H114 coordination is similar in the two Cu2+ sites. However, the relative orientation between the histidine imidazole ring and the Cu2+ center is possibly different.
Lastly, we apply Double Quantum Coherence (DQC) spectroscopy to measure the Cu2+-Cu2+ distance in the EcoRI-DNA complex. The DQC signal has contributions from nuclear hyperfine and quadrupole interactions. The effect of such interactions swamps the modulation due to the electron-electron dipolar interactions and makes it difficult to measure the interspin distance. We show a simple method to reduce these electron-nuclei interactions and resolve the dipolar interaction between two Cu2+ centers with high sensitivity.


Social Networking:
Share |


Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Ji, Mingmij13@pitt.eduMIJ13
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairSaxena, Sunilsksaxena@pitt.eduSKSAXENA
Committee MemberWaldeck, Daviddave@pitt.eduDAVE
Committee MemberWeber, Stevesweber@pitt.eduSWEBER
Committee MemberTang, Pei
Date: 29 May 2014
Date Type: Publication
Defense Date: 13 December 2013
Approval Date: 29 May 2014
Submission Date: 16 December 2013
Access Restriction: 1 year -- Restrict access to University of Pittsburgh for a period of 1 year.
Number of Pages: 164
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Chemistry
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Electron spin resonance, molecular dynamics simulation, Continuous Wave-ESR, Electron spin echo envelope modulation, Double electron-electron resonance, Electron nuclear double resonance, double quantum coherence, protein-DNA complex
Date Deposited: 29 May 2014 15:29
Last Modified: 15 Nov 2016 14:16


Monthly Views for the past 3 years

Plum Analytics

Actions (login required)

View Item View Item