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    Membrane-Induced pK(a) Shifts in wt-pHLIP and Its L16H Variant

    Year: 2018

    Journal: J. Chem. Theory Comput., Volume 14, JUN, page 3289–3297

    Authors: Vila-Vicosa, Diogo; Silva, Tomas F. D.; Slaybaugh, Gregory; Reshetnyak, Yana K.; Andreev, Oleg A.; Machuqueiro, Miguel

    Organizations: Fundacao para a Ciencia e a Tecnologia [SFRH/BPD/110491/2015, UM/MULTI/00612/2013, PTDC/QEQ-COM/5904/2014]; General Medical Sciences of the National Institutes of Health [R01GM073857]

    The pH (low) insertion peptides (pHLIPs) is a family of peptides that are able to insert into a lipid bilayer at acidic pH. The molecular mechanism of pHLIPs insertion, folding, and stability in the membrane at low pH is based on multiple protonation events, which are challenging to study at the molecular level. More specifically, the relation between the experimental pK of insertion (pK(exp)) of pHLIPs and the pK(a) of the key residues is yet to be clarified. We carried out a computational study, complemented with new experimental data, and established the influence of (de)protonation of titrable residues on the stability of the peptide membrane-inserted state. Constant-pH molecular dynamics simulations were employed to calculate the pK(a) values of these residues along the membrane normal. In the wt-pHLIP, we identified Asp14 as the key residue for the stability of the membrane-inserted state, and its pK(a) value is strongly correlated with the experimental pK(exp) measured in thermodynamics studies. Also, in order to narrow down the pH range at which pHLIP is stable in the membrane, we designed a new pHLIP variant, L16H, where Leu in the 16th position was replaced by a titrable His residue. Our results showed that the L16H variant undergoes two transitions. The calculated pK(a) and experimentally observed pK(exp) values are in good agreement. Two distinct pK(exp) values delimit a pH range where the L16H peptide is stably inserted in the membrane, while, outside this range, the membrane-inserted state is destabilized and the peptide exits from the bilayer. pHLIP peptides have been successfully used to target cancer cells for the delivery of diagnostics and therapeutic agents to acidic tumors. The fine-tuning of the stability of the pHLIP inserted state and its restriction to a narrow well-defined pH range might allow the design of new peptides, able to discriminate between tissues with different extracellular pH values.
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