PEGylation of protein side chains has been used for more than 30 years to enhance the pharmacokinetic properties of protein drugs. However, there are no structure- or sequence-based guidelines for selecting sites that provide optimal PEG-based pharmacokinetic enhancement with minimal losses to biological activity. We hypothesize that globally optimal PEGylation sites are characterized by the ability of the PEG oligomer to increase protein conformational stability; however, the current understanding of how PEG influences the conformational stability of proteins is incomplete. Here we use the WW domain of the human protein Pin 1 (WW) as a model system to probe the impact of PEG on protein conformational stability. Using a combination of experimental and theoretical approaches, we develop a structure-based method for predicting which sites within WW are most likely to experience PEG-based stabilization, and we show that this method correctly predicts the location of a stabilizing PEGylation site within the chicken Src SH3 domain. PEG-based stabilization in WW is associated with enhanced resistance to proteolysis, is entropic in origin, and likely involves disruption by PEG of the network of hydrogen-bound solvent molecules that surround the protein. Our results highlight the possibility of using modern site-specific PEGylation techniques to install PEG oligomers at predetermined locations where PEG will provide optimal increases in conformational and proteolytic stability.
Protein PEGylation is an effective method for reducing the proteolytic susceptibility, aggregation propensity, and immunogenicity of protein drugs. These pharmacokinetic challenges are fundamentally related to protein conformational stability, and become much worse for proteins that populate the unfolded state under ambient conditions. If PEGylation consistently led to increased conformational stability, its beneficial pharmacokinetic effects could be extended and enhanced. However, the impact of PEGylation on protein conformational stability is currently unpredictable. Here we show that appending a short PEG oligomer to a single Asn side chain within a reverse turn in the WW domain of the human protein Pin 1 increases WW conformational stability in a manner that depends strongly on the length of the PEG oligomer: shorter oligomers increase folding rate, whereas longer oligomers increase folding rate and reduce unfolding rate. This strong length dependence is consistent with the possibility that the PEG oligomer stabilizes the transition and folded states of WW relative to the unfolded state by interacting favorably with side-chain or backbone groups on the WW surface.
PEGylation, or addition of poly(ethylene glycol) chains to proteins, is widely used to improve delivery in pharmaceutical applications. Recent studies suggest that stabilization of a protein by PEG, and hence its proteolytic degradability, is sequence-dependent and requires only short PEG chains. Here we connect stabilization by short PEG chains directly to the structural dynamics of the protein and PEG chain. We measured the stability of human Pin1 WW domain with PEG-4 at asparagine 19 for a full mutant cycle at two positions thought to influence PEG-protein interaction: Ser16Ala and Tyr23Phe. We then performed explicit solvent molecular dynamics simulations on all PEGylated and PEG-free mutants. The mutant cycle yields a nonadditive stabilization effect where the pseudo-wild type and double mutant are more stabilized relative to unPEGylated proteins than are the two single mutants. The simulation reveals why: the double mutant suffers loss of β-sheet structure, which PEGylation restores even though the PEG extends as a coil into the solvent. In contrast, in one of the single mutants, PEG preferentially interacts with the protein surface while disrupting the interactions of its asparagine host with a nearby methionine side chain. Thus, PEG attachment can stabilize a protein differentially depending on the local sequence, and either by interacting with the surface or by extending into the solvent. A simulation with PEG-45 attached to asparagine 19 shows that PEG even can do both in the same context.
Experimental and Theoretical Investigation of the Scope of Enantioselective Ketone Allylations Employing Nakamura's Allylzinc-Bisoxazoline Reagent. -The allylation of asymmetric ketones generally proceeds with good yields and low to moderate enantiomeric excess. A computational study rationalizes the results, and the ee-values can be predicted with reasonable accuracy, although entropic factors in combination with small energy differences limit the precision. The importance of both local and global control elements in successful reactions is shown. Perhaps the most useful aspect of chiral allylating agents (I) is their high reactivity, which is consistent with the low computed activation barriers. -(JOHNSON, A. G.; LOERTSCHER, B. M.; MOECK, A. R.; MATTHEWS, S. S.; ESS*, D. H.; CASTLE, S. L.; Bioorg. Med. Chem. Lett. 21 (2011) 9, 2706-2710, http://dx.
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