Protein and DNA Biophysics
Site-Directed Cu(II) Spin Labeling of Proteins
We have developed methodology to bind the paramagnetic Cu(II) ion to specific sites on a protein in order to measure structural constraints that better reflect backbone structure and flexibility compared to commonly used spin labels. A Cu(II) ion chelated to nitrilotriacetic acid is site specifically attached to proteins using a double histidine (dHis) mutation of the protein. The Cu(II) position is significantly restricted by coordination to protein side chains, and therefore, the resultant distances are remarkably precise, with a distance distribution width that is as much as five times narrower when compared to the nitroxide spin label. In addition, the room temperature EPR lineshape is sensitive to site specific backbone dynamics. This labeling scheme elucidates biophysical information that is costly or simply inaccessible by traditional EPR labels. Examples include the direct measurement of protein backbone dynamics at β-sheet sites and rigid Cu(II)−Cu(II) distance measurements that enable high precision in the analysis of protein conformations, conformational changes, interactions with other biomolecules, and the relative orientations of two labeled protein subunits. As described in the Molecular Dynamics section, we have developed force fields for this label, and MD simulations find remarkable agreement with experiment which has been a major challenge for traditional labels. Therefore we anticipate new combinations of MD and EPR to soon emerge that are likely to improve our ability to build an atomic level picture of protein structure, dynamics and function.
Site-Directed Cu(II) Spin Labeling of DNA
We introduce Cu(II) into DNA via a 2,2’-dipicolylamine phosphoramidite, which is incorporated into any nucleotide during initial DNA synthesis. Our approach places the ESR probe at the interior of the DNA helix, as opposed to ~1-1.5 nm outside in the helical perimeter as seen in current nitroxide-based labeling techniques. In addition, the small length of the linker ensures that the distance measurements based on our approach directly report on backbone distances and are maximally sensitive to DNA structure and flexibility. Much of this work has been furthered by systematic experimentation, development of force fields, and MD simulations.
Protein-DNA Interactions in Bacterial Metalloregulation
The interactions between proteins and specific DNA sequences are at the heart of many cellular processes. In the Saxena Group, we use robust EPR methodology to elucidate the structural relationship between protein and DNA that leads to repair, transcription, and cellular defense. This work is in collaboration with the Ruthstein group at Bar Ilan University. The copper efflux regulator (CueR) is a transcription metalloregulator that maintains E. coli and P. aeruginosa cellular Cu(I) homeostasis. We use protein and DNA Cu(II) spin labeling understand how the structural transitions and changes in dynamics of CueR and DNA explicitly facilitate initiation and termination of transcription. In return, we aim to create a fundamental understanding of this cellular process to aid in the development of new antibiotics that deploy copper to terminate the CueR defense response to combat rising antibiotic resistance.
Collaborator: Sharon Ruthstein
Protein-DNA Interactions in Restriction Enzymes
Restriction enzymes, such as EcoRI, bind to and cleave foreign viral DNA at a specific sequence to ensure native cell DNA is not destroyed. We have revealed the structural and dynamical details that underlie the binding and cleavage specificity measurements on the protein bound to specific and nonspecific DNA sequences. In addition, we have discovered a new metal-binding site in EcoRI and delineated the mechanistic role of metal binding at this site in the modulation of DNA cleavage. More recently, we have focused on the DNA-cleaving enzyme EcoRV, which has a 12,000-fold preference for binding to a specific DNA sequence than a sequence with one base pair mismatch. Remarkably, this specificity is quadrupled when bound to a metal cofactor. Such high protein-DNA binding specificity offers a model system for our group to methodically probe with EPR how flexibility, dynamics, and metal binding cumulatively affect a protein’s ability to bind to DNA.
Collaborator: Linda Jen-Jacobson