Computational design of protein function Loren Looger Hellinga lab

1. Allowable structures for proteins, DNA, small molecules Progesterone

2. Pseudo-geometric potential electrostatics H-bonds sterics solvation

Pretty much like CHARMM...

Hydrogen bonds, too... anchor r H A  D  -8 · { } · ·

Area-based solvation energy P P P = polar H = hydrophobic H H H

Electrostatic potential     is a function of atom-type pair & protein environment. Parameterized to fit experimental data.

3. Algorithm for choosing best structure(s) from all available

Protein (PBP) scaffolds Complementary Surface Construction: Ligand coordinates Protein Poly-alanine PCS Rotational ligand ensemble Docking grid Force field Placed Fixed ligand Side-chain rotamers Evolved Ranked PCS Experiments Periplasmic Binding Protein (PBP) scaffolds Molten zone Evolving zone Fixed zone

Metabolites Neurotransmitters Drugs Kd = 2 µM Kd = 6 µM Kd = 2 nM [L-lactate] (µM) 0.5 1 40 80 F x Kd = 2 µM 52.5 100 Kd = 6 µM [serotonin] (µM) 12.5 25 Kd = 2 nM [TNT] (nM) 150 300 Kd = 4 nM [5-fluorouracil] (nM) 50 [MTBE] (µM) 0.25 Kd = 45 nM [PMPA] (µM) TNT RDX MTBE D-lactate L-lactate 5-fluorouracil ibuprofen PMPA~soman serotonin dopamine & Pollutants Explosives Chemical Threats

QSAR Results for binding affinities for L-lactate & TNT Receptors -8 -6 log Kd (obs) -4 -2

L-lactate designs GBP QBP RBP 1 100µM 10 100mM 0.1 HBP ABP

The use of QSARs in the predictions improves the designs: D-lactate GBP QBP RBP 1 100µM 10 100mM 0.1 HBP ABP

Construction of biological sentinels for chemical threats and pollutants modulation binary expression [inducer]

Unicellular sentinels for chemical threats and pollutants - + - + TNT Ribose Lactate MTBE 5 Fluoro-uracil

Dose Response of TNTa Signaling IPTG 0 mM TNT 100 mM 2,4-DNT 100 mM 2,6-DNT 100 mM 10 mM 1 mM 0.1 mM 0.01 mM 0.001 mM [TNT]

Optically pure enantiomers Absorbance 210nm L D Kdlactate D L none none 200µM 3µM 0.8µM 10µM Racemic mix Optically pure enantiomers Wt Gbp Immobilized receptors L-Lac.G1 D-Lac.G1 Fraction #

Computational design of ligand-binding sites Strategy #2: predefined geometries { l, w1, w2, q1, q2, q3 }n geometrical description of essential features in the complementary surface Complementary surface construction (1010-10200 rotamers) Site 1 Site 2 +... Calculation #2 Complementary surface construction (PCS + SCS) +... Site 1 Site 2 Combinatorial search (108 sequence 1012 rotamers) Calculation #1 Initial placement of PCS on scaffold backbone Design scaffold coordinates side-chain rotamer library Pairwise of atomic interactions

Triose phosphate isomerase chemistry

Acknowledgements Mary Dwyer Jeff Smith Shahir Rizk