2014 undergraduate research forum poster
TRANSCRIPT
Matthew KimA, Kristen Procko* and Stephen F. Martin‡
A = Synthesis and Biological Recognition, * = Research Educator, ‡ = Principal Investigator
The main goal of this study is to determine the thermodynamics of protein-‐
ligand interactions to the stream’s target protein, the mouse major urinary protein-‐I (MUP-‐I), which functions in the protection, transport, and slow release of pheromones. Studying MUP-‐I can introduce a more clear understanding of the unorthodox non-‐classical hydrophobic effect, in which the protein-‐ligand interaction is driven enthalpically as opposed to entropically. This sparked interest in structurally changing various ligands in order to study the thermodynamic behavior arising from their interactions to MUP-‐I. An analogue of a high-‐binding ligand, 2-‐sec-‐butyldihydrothiazole (SBT), was synthesized. It was predicted that because of the analog’s unique structure, an increase in the enthalpic stabilization could occur, leading to a higher binding afRinity than that of SBT.
General characteristics: § Functions in the protection, transport, and slow release of pheromones § Single α-‐helix and eight β-‐sheets § Forms hydrophobic barrel § Active sites hydrated with two water molecules § Exhibits hydrophobic effect
Important active site characteristics: § Hydrogen bond network consists of Phe56, Leu58, and Tyr138 § Van der Waals interactions with multiple residues in the active site:
§ Leu72, Val100, Phe108, Leu121, leu123, and Leu134
• Ligand is normally introduced incrementally into a solution of MUP-‐I; the heat released upon binding is recorded.
• Free energy is determined through values derived from the Gibbs free energy equation, ΔG = ΔH – TΔS = -‐RT(lnKa).
• Change in free energy (ΔG), which is inRluenced by the change in enthalpy (ΔH) and change in entropy (ΔS), reveals the driving forces for a particular ligand’s binding to the active site of MUP-‐I.
S
N
Leu72
Leu134
Phe108
Val100
Ala121
HO
HO H O
H
O
H O
Leu58
Tyr138
Leu123
NH
NH
Phe56
Abstract1
Mouse Major Urinary Protein – I1,3,4
Observed Trends4 Isothermal Titration Calorimetry (ITC)2,3
ITC Data
Acknowledgements
References: 1Timm, D.E.; Baker, L.J.; Mueller, H.; Zidek, L.; Novotny, M.V. Structural basis of pheromone binding to mouse major urinary protein (MUP-‐I). Protein Sci. 2001, 10, 997-‐1004. 2Pierce, M.M.; Raman, C.S.; Nall, B.T. Isothermal Titration Calorimetry of Protein-‐Protein Interactions. Methods 1999, 19, 213-‐221. 3Homans, W.S. Water, water everywhere – except where it matters? Drug discovery today 2007, 12, 13-‐14. 4Sharrow, S.D.; Novotny, M.V.; Stone, M. J. Thermodynamic analysis of binding between mouse major urinary protein-‐I and the pheromone 2-‐sec-‐ butyl-‐4,5-‐dihydrothiazole. Biochemistry 2003, 42, 6302-‐6309. 5Jin, Sarah. The University of Texas, Austin, TX. 4,5-‐Dihydro-‐2-‐phenylthiazole, 2010. 6Azarm, Kristopher. The University of Texas, Austin, TX. Elucidating the Thermodynamics of Binding of 2-‐(Pyridin-‐3-‐yl)-‐4,5-‐dihydrothiazole, 2011.
N S HS
H2N
DBHReflux, 7 min. 100 oC
N S
N
4 5 6
Synthetic Route
S
N
S
N
1 2
S
N N
3
Past Studies5,6