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ClickIn: a flexible protocol for quantifying mitochondrial uptake of nucleobase derivatives.

ABSTRACT: There is an increasing interest in targeting molecules to the mitochondrial matrix. Many proteins are naturally imported through the translocase complexes found in the outer and inner mitochondrial membranes. One possible means for importing molecules is therefore to use a mitochondrial pre-protein as a vector and assess what forms of molecules can be attached to the pre-protein as cargo. A major difficulty with this approach is to ensure that any chimaeric molecule does indeed access the mitochondrial matrix and does not merely associate with the mitochondrial membranes. We have recently demonstrated that click chemistry can be used both to demonstrate convincingly mitochondrial import of a peptide-peptide nucleic acid conjugate and also to quantify the mitochondrial uptake for specific synthetic conjugates. We now report an adaptation of the synthesis to facilitate simple quantification of multiple molecules and hence to calculate the efficiency of their mitochondrial import.

SUBMITTER: Hoogewijs K

PROVIDER: S-EPMC5311907 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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ClickIn: a flexible protocol for quantifying mitochondrial uptake of nucleobase derivatives.

Hoogewijs Kurt K James Andrew M AM Smith Robin A J RA Abendroth Frank F Gait Michael J MJ Murphy Michael P MP Lightowlers Robert N RN

Interface focus 20170401 2

There is an increasing interest in targeting molecules to the mitochondrial matrix. Many proteins are naturally imported through the translocase complexes found in the outer and inner mitochondrial membranes. One possible means for importing molecules is therefore to use a mitochondrial pre-protein as a vector and assess what forms of molecules can be attached to the pre-protein as cargo. A major difficulty with this approach is to ensure that any chimaeric molecule does indeed access the mitoch ...[more]

PMID: 28382203

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Project description:Alkylureas display hydrocarbon and amide groups, the primary functional groups of proteins. To obtain the thermodynamic information that is needed to analyze interactions of amides and proteins with nucleobases and nucleic acids, we quantify preferential interactions of alkylureas with nucleobases differing in the amount and composition of water-accessible surface area (ASA) by solubility assays. Using an established additive ASA-based analysis, we interpret these thermodynamic results to determine interactions of each alkylurea with five types of nucleobase unified atoms (carbonyl sp2O, amino sp3N, ring sp2N, methyl sp3C, and ring sp2C). All alkylureas interact favorably with nucleobase sp2C and sp3C atoms; these interactions become more favorable with an increasing level of alkylation of urea. Interactions with nucleobase sp2O are most favorable for urea, less favorable for methylurea and ethylurea, and unfavorable for dialkylated ureas. Contributions to overall alkylurea-nucleobase interactions from interactions with each nucleobase atom type are proportional to the ASA of that atom type with proportionality constant (interaction strength) ?, as observed previously for urea. Trends in ?-values for interactions of alkylureas with nucleobase atom types parallel those for corresponding amide compound atom types, offset because nucleobase ?-values are more favorable. Comparisons between ethylated and methylated ureas show interactions of amide compound sp3C with nucleobase sp2C, sp3C, sp2N, and sp3N atoms are favorable while amide sp3C-nucleobase sp2O interactions are unfavorable. Strongly favorable interactions of urea with nucleobase sp2O but weakly favorable interactions with nucleobase sp3N indicate that amide sp2N-nucleobase sp2O and nucleobase sp3N-amide sp2O hydrogen bonding (NH···O?C) interactions are favorable while amide sp2N-nucleobase sp3N interactions are unfavorable. These favorable amide-nucleobase hydrogen bonding interactions are prevalent in specific protein-nucleotide complexes.

Dataset Information

ClickIn: a flexible protocol for quantifying mitochondrial uptake of nucleobase derivatives.

Publications

ClickIn: a flexible protocol for quantifying mitochondrial uptake of nucleobase derivatives.

Similar Datasets

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