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20.109(F20): Laboratory Fundamentals of Biological Engineering

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Fall 2020 schedule        FYI        Assignments        Homework        Communication |        Accessibility

       M1: Genomic instability        M2: Drug discovery        M3: Metabolic engineering       


Proteins and small molecules interact in a process referred to as molecular recognition. In molecular recognition, the non-covalent complexes that form are defined by two characteristics: specificity and affinity.

  • Specificity distinguishes a specific binding partner from the milieu of potential binding partners in complex environments.
  • Affinity dictates the likelihood of binding based on the concentration of a specific binding partner in a milieu of potential binding partners such that high affinity partners at low concentrations are not outcompeted by low affinity partners and high concentrations.

In cells, proteins are critical macromolecules that perform numerous roles related to structure, mechanics, metabolism, and signaling. In these roles, proteins do not work in a vacuum; rather the biological roles of proteins are dependent on direct physical interactions with other molecules. Though our focus is on small molecule binders, proteins also interact with other proteins, nucleic acids, oxygen, and metals. In all cases, the interactions are characterized by protein-ligand-solvent binding kinetics.

Though beyond the scope of this module, protein-ligand-solvent binding kinetics are defined as a thermodynamic system composed of solute (proteins and binders) and solvent (liquid that contains the proteins and binders). In that protein-small molecule complexes result in heat transfer, the driving forces that promote these interactions are due to energy exchanges that are characterized by Gibbs free energy (ΔG). ΔG measures the capacity of a thermodynamic system to do maximum or reversible work at constant temperature and pressure. When at equilibrium with constant temperature and pressure, protein-small molecule binding occurs when the change in ΔG is negative. The magnitude of ΔG provides insight into the stability of a protein-small molecule complex.

Another method for assessing protein-small molecule binding is to visually inspect known small molecule binders for common features / structures. Though you may not realize it, you did this on M2D2! Remember the information regarding lactose and IPTG? In both of these examples, the molecules share a feature that enables binding to the same target (LacI for lactose and IPTG). Your goal for today is to carefully examine the hits identified by the class and identify any common features / structures. As in the image below, it is possible that multiple features will be present within the same small molecule.

Sp17 20.109 M1D7 chemical structure features.png


Part 1: Visually evaluate chemical structures of positive hits

With your partner, review the hits identified in the SMM. It may be easier to copy / paste the small molecule images into a powerpoint file so you can readily see all of the structures. Also, it may be helpful to use a color-coding system (like the one in the image provided in the Introduction) to highlight features / structures that are common to the small molecules that putatively bind PF3D7_1351100.

In your laboratory notebook, complete the following:

  • How many features did you identify that are present in two or more of the small molecules that putatively bind PF3D7_1351100? Are there more or less than you expected?
  • Is there a feature present in all of the identified small molecules? What might this suggest about the binding site(s) and / or binding ability of PF3D7_1351100?
  • Can you assign the identified small molecules to sub-groups based on the common features that are present?
  • What might the different features represent? More specifically, consider whether each subgroup has a unique binding site on the target protein or if each subgroup represents different solutions for interacting with the same binding site. They can propose studies (along with underlying rationale) that would help them distinguish these possibilities.
  • How might you make modifications to the small molecules / features to probe binding? As a hint, consider how different functional groups could be positioned at a given site without altering qualitative binding in the SMM assay to translate that into some testable ideas (e.g. quantitative binding properties may be occurring that are functionally relevant, but not discernible by SMM assays; or such a site is not critical for binding and may allow for modifications that confer beneficial properties of the compound).

These online resources may be helpful to learning more about the hits identified in the SMM:

  • Cloud version of ChemDraw here.
    • Copy and paste the small molecule smiles into the work space to get a chemical structure
  • Platform to transform the smiles information into a PubChem ID here.
    • Copy and paste the smiles into the input ID search to determine the ID number.
  • PubChem database of chemical information here.
    • Includes small molecule molecular weight and other useful information.

Part 2: Design experiments to test putative small molecule binders

Using the ideas discussed with your partner for Part 1, design the experiments that will follow your research. Remember, that the next steps to your project should build upon the work you completed.

In your laboratory notebook, complete the following:

  • Consider aspects of the experimental approach that could be improved.
    • What steps might you do differently to improve the results?
  • Consider two potential next step experiments that you might perform to test putative small molecule binders identified from the SMM screen. For each experiment:
    • What specifically will the experiment test?
    • How will this experiment bolster the results from the current research project?
    • Is the experiment a next step? Does the experimental approach make assumptions beyond the data acquired from the SMM screen?
    • Include some information regarding a method that could be used to complete the experiment. What controls should be included?
    • Hint: the last two bullets in Part 1 are next step ideas from Prof. Niles!!
  • Consider the implications of your research.
    • How does your research address the knowledge gap / research question? This should tie back to the information provided in the Introduction of your Research article.
    • How does your research advance the field of anti-malarial drug discovery? Don't overreach, but do state what is now known because of your work.

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