Small shifts offer big insights

Last week we said our goodbye to Rebecca Courtemanche, who finished up her MSc degree. Rebecca is now in Vancouver and we miss her already because she has been instrumental in our efforts to measure small molecule/protein interactions by NMR. I asked Rebecca to write a blog entry dedicated to some of the research she performed while at the University of Toronto. Here is her post:

“As a chemist, I have an innate curiosity to study molecular interactions in biological settings. Chemical probes are small molecules that interact with protein targets. Understanding these interactions give insight into a protein’s role in a disease and can guide future drug discovery. Interest in chemical probes has significantly grown over the past decade and their importance is addressed in Stephen Frye’s article entitled “The art of the chemical probe”:

http://www.nature.com/nchembio/journal/v6/n3/full/nchembio.296.html

When I was given the opportunity to research chemical probes for biologically relevant proteins last Fall I was very excited to ‘nerd-out’ (according to my non-chemist friends). Dr. Peter Brown’s group at the Structural Genomics Consortium, SGC, has been actively pursuing probes for epigenetic proteins. Along the way, we found that 1H-15N HSQC chemical shift mapping has been particularly insightful in distinguishing the strength of interaction between a molecule and a protein as well as the molecule’s binding location. Proteins were expressed and purified such that they were 15N enriched (we used 15N-labelled ammonium formate while growing cells). This way, for a 1H-15N-HSQC experiment, all H-N correlations from the backbone amide groups as well as some side chain H-N correlations can be observed. Together, they provide a map of chemical shifts unique for every protein. When a small molecule is added to the protein, the region of the protein directly involved in binding will undergo a structural rearrangement, thus inducing a change in the region’s magnetic environment. On an HSQC map you see below (the nature of the protein is still confidential), the unbound protein will have a different chemical shift map as compared to the bound protein. The amount of chemical shift movement relative to the concentration of the binding molecule provides a sense of the strength of binding and it is possible to calculate a dissociation constant (Kd).

Image

Figure 1. 1H-15N HSQC spectrum overlay: blue contours are the unbound protein and the red and green contours represent 1:1 and 1:3 molar ratios of protein to compound, respectively. 

Understanding the binding location on a protein can be achieved if it is known which NH correlations correspond to which amino acid. We are currently assigning our proteins by 3D NMR techniques using doubly labeled protein (C-13 and N-15). For more information pertaining to protein NMR theory see http://www.protein-nmr.org.uk/.”

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