Abstract
Single molecule protein sequencing would tremendously impact in proteomics and human biology and it would promote the development of novel diagnostic and therapeutic approaches. However, its technological realization can only be envisioned, and huge challenges need to be overcome. Major difficulties are inherent to the structure of proteins, which are composed by several different amino-acids. Despite long standing efforts, only few complex techniques, such as Edman degradation, liquid chromatography and mass spectroscopy, make protein sequencing possible. Unfortunately, these techniques present significant limitations in terms of amount of sample required and dynamic range of measurement. It is known that proteins can distinguish closely similar molecules. Moreover, several proteins can work as biological nanopores in order to perform single molecule detection and sequencing. Unfortunately, while DNA sequencing by means of nanopores is demonstrated, very few examples of nanopores able to perform reliable protein-sequencing have been reported so far. Here, we investigate, by means of molecular dynamics simulations, how a re-engineered protein, acting as biological nanopore, can be used to recognize the sequence of a translocating peptide by sensing the “shape” of individual amino-acids. In our simulations we demonstrate that it is possible to discriminate with high fidelity, 9 different amino-acids in a short peptide translocating through the engineered construct. The method, here shown for fluorescence-based sequencing, does not require any labelling of the peptidic analyte. These results can pave the way for a new and highly sensitive method of sequencing.
Highlights
The “sequence–structure–function” paradigm for proteins embodies one of the biggest scientific discoveries about the functioning of Life
To ascertain whether this kind of construct can act as a suitable adaptive nanopore, we investigated its interaction with short poly-peptides that are translocating along its axis, using molecular dynamics (MD) simulations
By exploiting molecular dynamics simulations and machine learning, we showed that a properly designed protein construct can act as a nanopore able to recognize the “shape” of individual amino-acids composing a polypeptide that translocates through the pore
Summary
The “sequence–structure–function” paradigm for proteins embodies one of the biggest scientific discoveries about the functioning of Life. The DNA sequence in each gene contains the information necessary to build proteins, via a mechanism where specific triplets of nucleic acids code for the different amino-acids. In addition to this golden rule for translation, proteins’ fundamental building blocks can be altered by alternative splicing or post-translational modifications. The large amount of molecules required to satisfy the detection limits of standard mass spectrometers make the analysis of low concentration proteins extremely challenging. To overcome these limitations and to pursue the final aim of single-cell analysis, during the last few years single-molecule protein sequencing methods started to emerge [4].
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