Disruption in posttranslational modifications (PTMs) can lead to the dysfunction of vital biological processes and various diseases. Additionally, the position and chemical structure of PTMs in proteins and peptides are critical in diseases like cancer.
Study: Resolving Isomeric Posttranslational Modifications Using a Biological Nanopore as a Sensor of Molecular Shape. Image Credit: ArtemisDiana/Shutterstock.com
Although mass-spectrometry and immunoassay-based approaches are the advanced methods involved in PTM diagnosis, these technologies are expensive and have limited specificity and selectivity.
An article published in the Journal of American Chemical Society demonstrated the differentiation of human histone H4-derived proteins using protein nanopore. The protein differentiation was based on the positions of methylated and acetylated lysine residues.
Utilizing a protein nanopore eliminated the need for controlled translocation and detected the position PTMs by sensing the shape of entrapped peptides. Furthermore, molecular dynamics simulations revealed that the non-uniform electric field in the nanopore increased its sensitivity toward the molecular structure of peptides. Thus, the molecular shape-sensing by the nanopore offered a label-free and versatile path toward protein detection.
Nanopore Technology Towards PTM Diagnosis
PTMs are covalent processing events that change the properties of a protein by proteolytic cleavage and adding a modifying group, such as acetyl, phosphoryl, glycosyl, and methyl, to one or more amino acids. PTMs play a key role in numerous biological processes by significantly affecting the structure and dynamics of proteins.
Generally, a PTM can be reversible or irreversible. The reversible reactions contain covalent modifications, and the irreversible ones, which proceed in one direction, include proteolytic changes.
PTMs occur in a single type of amino acid or multiple amino acids and lead to changes in the chemical properties of modified sites. PTMs usually are seen in proteins with important structures and functions such as secretory proteins, membrane proteins, and histones.
A nanopore is a nanoscale hole between two electrolytic fluid chambers with an impermeable membrane. Applying a voltage across the chambers results in a steady-state ion current developed across a nanopore. Transient changes in the ion flux across a nanopore can result from the occupation of a macromolecule in the pore, and therefore, monitoring the current across nanopores enables molecular sensing.
Despite the scientific advancements in nanopore technology, harnessing the promising features of nanopores for proteomics is challenging. Recently, polyarginine-based peptides with variation in single terminal amino acid substitutions were differentiated using aerolysin nanopores. Here the nanopore’s trapping and sensing did not restrict the movement of peptide, thus, showing overall molecular volume.
Resolving Isomeric PTMs Using a Biological Nanopore
Utilizing wt-AeL pore in previous works increased the dwell times for peptides of up to 4.45 nanometers in length. A tridecapeptide that included three extra amino acids at the N-terminal direction of the native peptide (H4f.K8-R17) was well accommodated in the pore with extended dwell time compared to the unmodified H4f.K8-R17.
In the present study, the highly sensitive protein pore was used in combination with nanopore recordings based on the high-resolution chip to demonstrate the whole molecule sensing of an entire peptide by nanopore entrapment, that was capable of differentiating PTMs based on their position. This process was devoid of differences in mass or overall volume in human peptide sequences, which is advantageous over previously reported works.
Unlike the previous report that used larger solid-state pores to characterize the nanoparticles and proteins, the present approach could differentiate true positional isomers and was not limited to charge-conferring modifications. Additionally, the all-atom molecular dynamics simulations indicated that the inhomogeneous electric field induced by the conformation of the analyte in the pore conferred the sensitivity of the ionic current to molecular shape.
Thus, the aerolysin nanopore helped in the quantitative analysis of methylation and acetylation of peptides in a position-sensitive manner. This functionalization of peptides helped in differentiating between the peptide forms of equal mass. Here, the discrimination method was driven by the uneven distribution of the electric field, shaped by the pore structure and peptide conformation.
Conclusion
Overall, the present work demonstrated that an engineered aerolysin nanopore could be used to quantitatively analyze the amino acid functionalization (acetylation/methylation) by differentiating among peptides of equal mass.
This differentiation was made based on either duration of resistive pulses or the depth of the block. The non-uniform electric field, determined by the structure of the nanopore and the conformation of the peptide, influenced the differentiation mechanism. The effect of peptide’s conformation in the differentiation process indicates that PTM’s sequence of the mean current value (I/I0) of various lysines was different in all three PTMs analyzed.
The aerolysin nanopore has provided a powerful molecular trap that improved the sensitivity of the variant pore relative to the wild type whose efficiency increased upon the removal of bulky and positively charged R220.
Further increase in differentiation among positional isoforms is anticipated by focusing on reducing instrumental noise and protein engineering to facilitate optimized conformational degrees of freedom of peptides in the nanopore.
Reference
Ensslen, T., Sarthak, K., Aksimentiev, A., Behrends, J. C. (2022). Resolving Isomeric Posttranslational Modifications Using a Biological Nanopore as a Sensor of Molecular Shape. Journal of the American Chemical Society. https://pubs.acs.org/doi/10.1021/jacs.2c06211
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