Design and characterization of a de-novo adenine binding protein

This study explores the de novo design and characterization of a protein engineered to selectively bind adenine; a molecule critical to ATP function, nucleotide recognition, and a wide range of cellular processes [2]. Our objective is to determine whether targeted structural mutations can enhance adenine binding affinity beyond the levels achieved by the original computational model. The designed protein will be expressed via recombinant DNA techniques and purified using Ni-NTA affinity chromatography. Structural and functional characterization will be carried out using a variety of analytical techniques. Those include SDS-PAGE, circular dichroism (CD), fluorescence spectroscopy, surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC). These methods will evaluate protein purity, secondary structure, and protein-ligand binding behavior. Through this approach we aim to identify key structural determinants that improve adenine specificity, offering new insights into rational protein design and the molecular basis of protein-ligand recognition.

My name is Amber Middleton and my project is called the design and characterization of a Doo adding binding protein. Our research question is can specific structure mutations and Danovo designed protein enhanced binding affinity and specificity for adding compared to the original computational design so why adding all nucleotide bases are rigid and aromatic, but addinine has a hydrogen bon or donor arrangement that is unique and specific um for selective and specific binding to proteins so it’s better suited for binding when interacted with ATP. The mutations that we made are from Alline to Isosine Alline to veailinging to 3ine and glycine to searin all these mutations were done by a regent PhD student Robert Spain for our methods, we had to express the protein purify it check the purity check the secondary structure and do a series of binding assets. The first thing is the expression of the proteins and recombinent DNA techniques, TB Media, LB Media, and inoculation overnight then we move on to NINTA chomatography to purify your protein where you’ll put your protein down into the column. It’ll run through. You’ll rise it with binding buffer samples and then you’ll rinse it with mixtures of binding buffer and ausion buffer samples and then you’ll collect each of those for analysis separately you then check the purity via SDS page so you’ll take those samples that you collected from the column, put them in run them at 180 V and they will separate by mass ideally, the thicker or darker the bands they hire their protein concentration. You’ll take the thickest ones, darkest ones, and do dialysis to remove all small salts and then we’ll move on a circulularichroism to check the secondary structure for for alpha helix proteins. You’ll see two negative peaks one at 208 n and one at 222 n which we do see in both of the pictures to the right the top being the wild type and the bottom being the mutant we then moved on to our first binding assay, which is surface plasma residence. We were only able to do a negative control with the wild type in adding proving that adding does not bind to the wild type. We then moved to our second binding assay fluorescence, and isodropy. This measures molecular interacts by detecting changes in fluorescent molecules, rotations so the faster the tumbling, the less binding that’s happening and the slower the tumbling, the more binding that is happening. This is some of our results from the first few anisropy experiments as you can see in yellow these are a little bit weird values. They imply that they’ protein technal gives more of a signal than protein addinine does, which essentially means that addingine quitching the protein signal or other things such as G-factor issues are going wrong our values are specific are expected to be between zero and 0.4 for anisatropy, but that’s not what we see in the highlighted so because of this, we wanted to move on to isothermal titration calorimetry, which measures heat released or absorbed, and these are the results that followed that on the left we have a IC thermogram of Valerab into the wild type AT&D, which is our positive controls and we were able to see that there is decreasing exothermic peaks, which proves that there was um functional liggin interaction and binding with the wild type protein, but on the right we have a ITC thermogram of adding with AT&D mutant, which looks nothing like the one on the left showing that there were only small producible changes in heat changes and there was no binding happening so our conclusions and future directions again we were able to purify and express our proteins and get up for alpha helix bundle, but upon doing finding assays, we were able to determine that addine and our mutant do not bond to each other so we have to go back to insilical design to re-engineer the binding pocket for addinine recognition using structural modeling and computational design followed by the validation CDEFSPRNITC and we aim to create a new mutant capable of selectively binding addine and this will help our understanding of targeted mutations to shape Lan specificity and enobble protein scaffolds