BASIL Biochemistry Curriculum

Key Points

Structural Alignment with PyMOL and ProMOL
  • Your students will use a computer alignment algorithm that includes quantitative measures (Levenshtein distance and RMSD values) and visual inspection to measure quality of alignments.

  • These results can be compared and contrasted with sequence alignment (BLAST, Pfam) and and full backbone alignment (Dali) results in other BASIL modules.

  • This module requires that you to install PyMOL and ProMOL on student-accessible computers and requires internet access.

Protein BLAST Search
  • Protein BLAST searches identify sequence similarity between a query protein and a database of other proteins.

  • Sequence similarity does not guarantee similarity in function.

Using Pfam to Predict Protein Function
  • Highly conserved amino acid residues does not guarantee similarity in function.

Structural Alignment with Dali
  • Dali searches identify backbone structure similarity between a query protein and 3D structures of proteins from the PDB.

  • Backbone structure similarity may not guarantee similarity in function since side chains are important for function.

Molecular Docking with AutoDock VINA and PyRx
  • Noncovalent interactions stabilize the binding of ligands to macromolecules like proteins.

  • Ligand binding is complex; chemical modifications to a ligand may not result in changes to the interaction energy due to competing stabilizing and destabilizing interactions.

Expression of Proteins from Lactose-Inducible Vectors in E. coli
  • Introduction of a foreign gene into E. coli allows researchers to express proteins of interest.

  • Protein expression can be controlled using knowledge of operators and repressors in E. coli.

Protein Purification
  • Purification of proteins is usually accomplished by lysis, removal of nucleic acids, and affinity chromatography utilizing a 6-histidine tag or fusion protein tag.

Measurement of Protein Concentration
  • Measuring protein concentration is important for determining protein purification yield and amounts to use in further analyses.

Using SDS-PAGE to assess the purification of protein
  • To carry out this lab students need vertical electrophoresis equipment.

  • The students can use pre-purchased gels or pour their own.

Protein Activity Assay
  • Activity assays with a broad substrate can be used to identify the class of enzyme while specific substrates can yield more information about enzyme function.

Enzyme Kinetics Assay
  • Determining kinetic parameters for the enzyme of interest can indicate the best substrate(s) for the enzyme and consequently provide insight into its function within an organism.