Effect of Mutations on Structure and Function of Proteins
Focus:
I have research experience with DHFR and enzyme inhibitor assays.
These papers include high quality protein structure figures that are useful for a protein structure class.
One of the authors, Amy Anderson, is likely to respond/have her students respond to our interview.
Each paper highlights the effect of mutations on structure and function and discusses resistance.
Each paper deals with inhibition constants and assays and students will be able to review these important concepts.
The papers follow the story of drug discovery against a resistant S. aureus DHFR. This will be interesting to our student population, many of whom are in the M.S. program for pharmaceutical biochemistry. Three of the four articles are by the group of Amy Anderson and the fourth paper presents results the follow up on the work in papers 1-3.
The continuation of articles by one group (articles 1-3) allows students to see how the thoughts of Anderson group evolve over time.
There is much overlap in the methods and content in the articles allowing student to gain confidence.
Overview:
Applicable for Courses:
Educational Level:
Roadmap Objectives:
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- Article: Anderson, A. (2005) "Two Crystal Structures of Dihydrofolate Reductase-Thymidylate Synthase from Cryptosporidium hominis Reveal Protein:Ligand Interactions Including a Structural Basis for Observed Antifolate Resistance" Acta Cryst. F 61:258-262.
- Content area/major concepts: Protein structure
Protein domains
Protein representations
Ligand binding
Intramolecular interactions
Protein cofactors
Ligand induced conformational change
Drug selectivity
Sequence alignments
Enzyme inhibitors
Drug resistance
Structure function relationships - Methods or technology used to obtain data: Enzyme activity assays Crystallization and data collection Structure solution
- How the CREATE strategy was used:
- Biggest teaching challenge: Will not thoroughly study X-ray data collection and solution
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- Article: Frey, K., Liu, J., Lombardo, N., Bolstad, D., Wright, D. and Anderson, A. (2009) "Crystal Structures of Wild-type and Mutant Methicillin-resistant Staphylococcus aureus Dihydrofolate Reductase Reveal an Alternative Conformation of NADPH that may be Linked to Trimethoprim Resistance" J. Mol. Biol. 387: 1298-1308
- Content area/major concepts: Resistance mutations
Intermolecular interactions
IC50
Selectivity
Structure/function relationships
Protein conformation
Sequence alignmnet - Methods or technology used to obtain data: Cloning Protein expression Protein purification Enzyme inhibition assays Crystallization Structure determination Sequence alignment
- How the CREATE strategy was used:
- Biggest teaching challenge: A protein with two domains (two activities) – potentially confusing for students. Paper uses some organic chemistry terms to describe molecules that students may not know
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- Article: Frey, K., Lombardo, M., Wright, D. and Anderson, A. (2010) "Towards the understanding of resistance mechanisms in clinically isolated trimethoprim-resistant Staphylococcus aureus dihydrofolate reductase" J. Struc. Biol. 170: 93-97.
- Content area/major concepts: Enzyme purification
Cloning
Enzyme assays
Crystallization
X-ray crystallography - Methods or technology used to obtain data: Km IC50 Ki values Resistance mutations Intramolecular interactions
- How the CREATE strategy was used:
- Biggest teaching challenge: Paper has 8 different inhibitor molecules
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- Article: Oefner, C., Bandera, M., Haldimann, A., Laue, H., Schulz, H., Mukhija, S., ... & Dale, G. E. (2009). Increased hydrophobic interactions of iclaprim with Staphylococcus aureus dihydrofolate reductase are responsible for the increase in affinity and antibacterial activity. Journal of Antimicrobial Chemotherapy, 63(4), 687-698.
- Content area/major concepts: Enzyme activity assays
Isothermal titration calorimetry
X-ray crystallography - Methods or technology used to obtain data: Drug resistance Inhibitor binding Structure/function relationships IC50 values Dixon Plots
- How the CREATE strategy was used:
- Biggest teaching challenge: New author New technique: isothermal titration calorimetry New concepts: binding enthalpy and entropy