STUDENT VERSION Put your understanding of binding thermodynamics to the test in this simple game: Predict which ligand binds more potently! You can find the answers online, but what’s the fun in that?
Warm-up: Small hydrophobic binding site This artificial cavity was created by mutating a leucine side chain to alanine in T4 lysozyme, which was chosen simply because it expresses and crystallizes easily. Before you start: 1) What are the 3 letter and 1 letter codes for alanine and leucine? 2) How many carbon atoms in each of those side chains? 3) What other amino acid has the same mass as leucine, and in what way does it differ? 4) How the heck do ligands get in there? From the standpoint of thermodynamics (i.e., not kinetics), do we care?
LIGAND 1
LIGAND 2
benzene (181L)
ethylbenzene (1NHB)
benzene (181L)
p-xylene (187L)
benzene (181L)
isobutylbenzene (184L)
ANSWER
REASONING
Hint: Can you learn anything from visualizing the B-factors (measure of local disorder, to be discussed more later in the course)?
A little more complicated: Introducing a polar side chain Using the preceding mutant, an additional methionine to glutamine substitution was introduced around the artificial pocket. 1) What are the 3 letter and 1 letter codes for methionine and glutamine? 2) How many “heavy” (non-hydrogen) atoms in each side chain? 3) What other amino acid has the most similar molecular mass to glutamine; what are its 3 letter and 1 letter codes, and how are its properties different? 4) What other amino acid has the most similar physico-chemical properties to glutamine; what are its 3 letter and 1 letter codes, and how is its chemical structure different? 5) Methionine is one of two amino acids with a sulfur atom; what is the other one, its 3- and 1letter codes, and how do the properties differ?
LIGAND 1
LIGAND 2
phenol (1LI2)
3-chlorophenol (1LI3)
phenol (1LI2)
2-aminophenol (no structure)
2-fluoroaniline (1LGW)
2,4difluoroaniline (no structure)
ANSWER
REASONING
Challenge: Charged, semi-open binding site This cavity is considerably more complex. The mutation is from a tryptophan to glycine (residue 191). [Original paper describing the CCP W191G mutant: http://www.ncbi.nlm.nih.gov/ pubmed/?term=7972020.] Before you begin: 1) What are the 3- and 1-letter codes for tryptophan? What properties make it unique? Can the side chain form hydrogen bonds? 2) What are the 3- and 1-letter codes for glycine? With no side chain at all, glycine has different backbone (phi,psi) propensities than all other amino acids; describe in what way. As such, in what ways is a mutation to alanine different than a mutation to glycine? 3) A structure of the wild-type (not mutated) protein is in the PDB (2CYP), as well as a structure of the apo (no ligand bound) mutated protein (1CPE). Do any major conformational changes occur upon deleting the tryptophan side chain? How about when benzimidazole binds to the newly created cavity (1RYC)? 4) Mutating tryptophan to glycine does not change the net charge of the protein. However, it does greatly increase the accessibility (to small molecules) of a charged side chain which then lines the artificial cavity … a) You guessed it, what are its 3- and 1-letter codes? b) What is the nominal pKa of this amino acid (i.e., the pH at which the side chain would be expected to be 50% protonated, if it were just floating around in solution)? c) How do its properties change with pH? d) What other amino acid has the most similar physico-chemical properties? e) In this protein, the amino acid side chain makes a hydrogen bonding interaction … with what amino acid (3- and 1-letter codes)? [This amino acid in turn coordinates the iron in the heme co-factor.] More challenging: how might this hydrogen bonding interaction change the properties of the charged side chain?
LIGAND 1
LIGAND 2
phenol (2AS3)
aniline (1AEE)
indazole (no structure)
benzimidazole (1RYC)
3,4dimethylthiazole (1AED)
2,3,4trimethylthiazole (1AC4)
ANSWER
REASONING
