Chad Torgerson and Dr. Joshua Price, Department of Chemistry and Biochemistry
Introduction
Proteins are complex molecules that have the potential to treat diseases and illnesses that small molecule drugs cannot. However, a significant problem with protein medications is that proteins tend to have short serum half-lives and can be difficult to store for extended periods of time due to their often unstable nature.1 One widely used strategy to circumvent these problems and increase the serum half-life of protein drugs is to attach polyethylene glycol (PEG) oligomers to amino acid residues (i.e., PEGylation) of protein structures.2 The thermodynamic effects of PEGylation are not always positive, however, and currently difficult to predict.3 In order to better comprehend and predict the effects PEGylation will have on protein stability, we must determine how PEG interacts with the protein and understand the nature of that interaction.
Current theories suggest PEGylation may function either by interacting with amino acid side chains to reduce the solvent accessible surface area (SASA) or via an excluded volume mechanism in which the bulky PEG takes up space and reduces the number of conformational options in the denatured state, thereby pushing equilibrium to favor the native state.1,4 Price et al. has previously shown that the addition of a short four unit PEG at position 19 of a Pin1 WW domain variant, where Ser19 is replaced with an Asn residue (S19N), is thermodynamically stabilizing.1 However, little is known about the mechanism of this stabilization.
I am testing the hypothesis that, in the case of S19N, PEGylation functions by reducing the SASA of S19N through interactions with amino acid side chains by using the program CHARMM to run molecular dynamic simulations that will allow us to predict the most energetically favorable conformations of the native state. Furthermore, I hypothesize that hydrogen bonds between PEG and amino acid side chains are crucial components of the PEGprotein stabilizing interaction.
Materials and Methods Simulation Preparation The crystal structure of the human protein Pin1 (PDB ID: 1PIN) was modified to only contain residues 6 through 34 (the WW domain) using PyMol. The peptide sequence used by Price et al.1 (S19N) was then fit to this WW domain structure using the program Swiss-PdbViewer 4.0.4 and a four unit PEG was then added to S19N (S19NPEG) using GaussView 5.0. Various starting conformations for S19NPEG were then obtained using Vconfand will be minimized using either/or both Gaussian 03 and CHARMM and then placed in an appropriately sized water box in preparation for dynamics simulations. The water box will also contain magnesium and chloride ions at a 50 μM concentration that will be used to both neutralize the overall charge of the system and more adequately represent the experimental conditions used by Price et al.1
Simulation Details
Simulations are being carried out using the program CHARMM (version 37b1). PEG parameters utilized for these simulations are from the CHARMM force field (FF) developed by Vorobyov et al.,5 ion parameters are from the CHARMM FF developed by Beglov et al.,6 and protein residue parameters utilized are from the C36 FF parameters.7 The TIP3P model was used for the water.8
Scripts have been written that will allow the protein to be heated from 0 K to 400 K, equilibrated (maintaining a constant temperature using a Nosé-Hoover thermostat) at 400 K for a few ns, and then cooled back down to 300 K where it will be again equilibrated (maintaining a constant temperature by again using a Nosé-Hoover thermostat) this time at 300 K. All stages of dynamics will be carried out under period boundary conditions using an image cutoff distance of 14 Å, at constant pressure using the PCONS keyword, and using a time step of 1 fs. All hydrogen-carbon bonds are held constant during simulations using the SHAKE command and electrostatic interactions for the system are calculated using the Particle Mesh Ewald summation technique (κ = 0.34, order = 4).
Results and Discussion
Although our simulations have not yet been completed, preliminary experimental data obtained by our lab suggest that the stabilizing effects observed upon PEGylation of S19N cannot be explained via an excluded volume mechanism alone. These prelimanry results also suggest that a large portion of the stabilizing effects that occur upon PEGylating of S19N at position 19 are due to hydrogen bonds forming between PEG and side chains nearby in the native state. Therefore, I expect that our simulations will confirm these preliminary results and show hydrogen bonds forming between the PEG oxygen and amino acid side chain hydrogen bond donators near to position 19 in the native state.
However, hydrogen bonds of surface residues are rarely important by themselves because the water surrounding the protein can create those same hydrogen bond contacts, and, in fact, water tends to create better hydrogen bond contacts. Thus, although hydrogen bonds are likely forming, it seems apparent that there must other interactions occurring between PEG and the protein which are equally important. Determining the nature of these other interactions is paramount to the realization of our lab’s long term goal of developing clear guidelines for how PEGylation can be used to enhance the pharmacokinetic properties of PEGylated protein drugs.
Reference
- Price, J. L.; Powers, E. T.; Kelly, J. W., ACS Chem. Biol. 2011, 6 (Copyright (C) 2011 American Chemical Society (ACS). All Rights Reserved.), 1188-1192.
- Frokjaer, S.; Otzen, D. E., Nature Reviews Drug Discovery 2005, 4 (4), 298-306; Jevsevar, S.; Kunstelj, M.; Porekar, V. G., Biotechnology Journal 2010, 5 (1), 113-128.
- So, T.; Ueda, T.; Abe, Y.; Nakamata, T.; Imoto, T., Journal of Biochemistry 1996, 119 (6), 1086-1093.
- Meng, W.; Guo, X. L.; Qin, M.; Pan, H.; Cao, Y.; Wang, W., Langmuir 2012, 28 (46), 16133 16140; Pandey, B. K.; Smith, M.; Torgerson, C.; Lawrence, P. B.; Matthews, S. S.; Watkins, E.; Lambert, M.; Prigozhin, M. B.; Price, J. L., Bioconjugate Chem., Ahead of Print.
- Price, J. L.; Powers, E. T.; Kelly, J. W., ACS Chem. Biol. 2011, 6 (Copyright (C) 2011 American Chemical Society (ACS). All Rights Reserved.), 1188-1192.
- Vorobyov, I.; Anisimov, V. M.; Greene, S.; Venable, R. M.; Moser, A.; Pastor, R. W.; MacKerell, A. D., Journal of Chemical Theory and Computation 2007, 3 (3), 1120-1133.
- Beglov, D.; Roux, B., Journal of Chemical Physics 1994, 100 (12), 9050-9063.
- MacKerell, A. D.; Bashford, D.; Bellott, M.; Dunbrack, R. L.; Evanseck, J. D.; Field, M. J.; Fischer, S.; Gao, J.; Guo, H.; Ha, S.; Joseph-McCarthy, D.; Kuchnir, L.; Kuczera, K.; Lau, F. T. K.; Mattos, C.; Michnick, S.; Ngo, T.; Nguyen, D. T.; Prodhom, B.; Reiher, W. E.; Roux, B.; Schlenkrich, M.; Smith, J. C.; Stote, R.; Straub, J.; Watanabe, M.; Wiorkiewicz-Kuczera, J.; Yin, D.; Karplus, M., Journal of Physical Chemistry B 1998, 102 (18), 3586-3616; MacKerell, A. D.; Feig, M.; Brooks, C. L., Journal of the American Chemical Society 2004, 126 (3), 698-699.
- Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L., Journal of Chemical Physics 1983, 79 (2), 926- 935.