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PK(a) values for side-chain carboxyl groups of a PGB1 variant explain salt and pH-dependent stability.

ABSTRACT: Determination of pK(a) values of titrating residues in proteins provides a direct means of studying electrostatic coupling as well as pH-dependent stability. The B1 domain of protein G provides an excellent model system for such investigations. In this work, we analyze the observed pK(a) values of all carboxyl groups in a variant of PGB1 (T2Q, N8D, N37D) at low and high ionic strength as determined using (1)H-(13)C heteronuclear NMR in a structural context. The pK(a) values are used to calculate the pH-dependent stability in low and high salt and to investigate electrostatic coupling in the system. The observed pK(a) values can explain the pH dependence of protein stability but require pK(a) shifts relative to model values in the unfolded state, consistent with persistent residual structure in the denatured state. In particular, we find that most of the deviations from the expected random coil values can be explained by a significantly upshifted pK(a) value. We show also that (13)C backbone carbonyl data can be used to study electrostatic coupling in proteins and provide specific information on hydrogen bonding and electrostatic potential at nontitrating sites.

SUBMITTER: Lindman S

PROVIDER: S-EPMC1697841 | biostudies-literature | 2007 Jan

REPOSITORIES: biostudies-literature

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pK(a) values for side-chain carboxyl groups of a PGB1 variant explain salt and pH-dependent stability.

Lindman Stina S Linse Sara S Mulder Frans A A FA André Ingemar I

Biophysical journal 20061013 1

Determination of pK(a) values of titrating residues in proteins provides a direct means of studying electrostatic coupling as well as pH-dependent stability. The B1 domain of protein G provides an excellent model system for such investigations. In this work, we analyze the observed pK(a) values of all carboxyl groups in a variant of PGB1 (T2Q, N8D, N37D) at low and high ionic strength as determined using (1)H-(13)C heteronuclear NMR in a structural context. The pK(a) values are used to calculate ...[more]

PMID: 17040982

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Project description:New molecular motifs that can act as pH-regulating triggers for amphiphilic, pH-sensitive block copolymers are investigated. Inspired by the mechanism of action of pH-indicators, such as methyl orange, and natural amino acids, we designed these copolymers where either 4-Amino-4'-dimethylaminoazobenzene, AZB (pKa 3.4, an amine derivative of methyl orange), isoleucine, Ile (pKa 2.37 for carboxylic acid), or a statistical mixture of both were appended as side chains to the hydrophobic block to act as pH-triggers. These new side chain motifs were identified with an aim to enhance the self-assembling properties of the block copolymers in terms of particle size and stability, drug encapsulation, and release. As the parent polymer, poly (ethylene) glycol-block- poly (carbonate) (PEG-b-PC) of number average molecular weight 12.1 kDa was used. We observed that PEG-b-PC block copolymers, when engineered with AZB or Ile-type of pH-regulators appended as side chains to PC blocks, formed self-assembled, spherical nanoparticles with hydrodynamic diameters ranging from 114 to 137 nm depending on copolymer composition. Critical aggregation concentrations (CAC) of the block copolymers were found to be governed by the type and content of side chains. We explored the use of these newly designed block copolymer assemblies as drug carriers using gemcitabine (GEM) as a model cytotoxic drug generally used for pancreatic ductal adenocarcinoma (PDAC). We showed that AZB and Ile decorated copolymeric nanocarriers were able to encapsulate GEM at 13.8-28.8 % loading content and release the drug in a pH-dependent pattern. Drug-loaded nanocarriers showed cellular entry into PDAC cells in vitro and were found to exert cytotoxicity against these cells. Neither the block copolymers bearing AZB or Ile-type pH-responsive triggers, nor their self-assembled nanoparticles showed any cytotoxicity at usable concentrations, thereby reflecting the potentials of these molecular motifs for designing stimuli-responsive drug delivery nanosystems.

Dataset Information

PK(a) values for side-chain carboxyl groups of a PGB1 variant explain salt and pH-dependent stability.

Publications

pK(a) values for side-chain carboxyl groups of a PGB1 variant explain salt and pH-dependent stability.

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