Connect with us

Tech

Exploring the role of protein barcodes and Next-Generation Protein Sequencing™ for protein conformation high-throughput mapping

Published

on

Exploring the role of protein barcodes and Next-Generation Protein Sequencing™ for protein conformation high-throughput mapping

Proteins can adopt a range of specific conformations, some of which significantly contribute to the pathology of many diseases. Protein misfolding and the subsequent aggregation have been associated with neurodegenerative conditions, including Alzheimer’s, Parkinson’s, and Huntington’s disease, as well as non-neurological diseases such as cataracts, certain categories of atherosclerosis, short-chain amyloidosis, and cancers.

Image Credit: Corona Borealis Studio/Shutterstock

Misfolded proteins can appear as small aggregates, soluble monomers, or large insoluble inclusion bodies. While the fundamental biophysical mechanisms that cause cytotoxicity are not fully understood, some hypotheses include disruption of cell membranes, stress on cellular quality control and protein degradation systems, and aberrant protein-protein interactions. 

A deeper understanding of the toxic effects and clearance of misfolded proteins could develop novel strategies for the prevention and treatment of several diseases. Shedding light on non-native, transient, and disordered protein conformations has proved a significant challenge using current experimental techniques. However, in this application, crystallization is not possible, which is a method most frequently used to define the structure of proteins.

To tackle this problem, Eugene Serebryany, Ph. D., and his team at Stony Brook University in New York have developed an advanced approach to systematically identify, stabilize, trap, and purify native and non-native protein conformations created in vitro or in vivo and link them directly to molecular, organismal, or evolutionary phenotypes.

Dr. Serebryany and his team have been tackling the limitations associated with traditional investigational methods for the study of misfolded proteins and how Next-Generation Protein Sequencing (NGPS) on the Quantum-Si Platinum® instrument and protein barcodes are well positioned to revolutionize this area of study.

The focus of the research

Dr. Serebryany’s team’s main area of interest is the biophysics of protein misfolding in vivo and the mechanisms of misfolding-associated diseases. They are also concerned with the discovery and pharmacological targeting of physiologically relevant non-native protein conformations and protein engineering in environments currently deemed inaccessible.

Their lab is currently addressing fundamental questions regarding the exploration of proteins’ conformational space and how they can be trapped in different conformational states. The goal is to map what each of those conformational states does.

To facilitate the relevant research areas, Dr. Serebryany and his team are developing new experimental techniques, such as high-throughput disulfide scanning (HTDS) of protein conformations. This technique facilitates the mapping of conformational landscapes to phenotypic landscapes in vivo.

The inspiration to develop HTDS

Disulfide bonds between cysteine residues in proteins create conformational constraints and help preserve protein stability. Protein disulfide isomerase is an enzyme located in the endoplasmic reticulum (ER) in cells and the periplasm of bacteria that catalyzes the formation and rupturing of disulfide bonds as proteins fold.

Proteins can assume the correct, fully folded state through this catalytic process. The same enzyme also collects misfolded proteins found in the ER. Disulfide scanning mutagenesis is a well-defined approach for determining how particular double-cysteine variants of a protein form intramolecular disulfide bonds.

The method is generally applied when testing structural models of proteins. Throughput remains limited, raising problematic issues as double-cysteine variants significantly increase with longer polypeptide lengths. Scanning an entire protein has never been easy, which is why Dr. Serebryany and his team developed HTDS. This qualitatively new approach allows researchers to map conformational landscapes onto phenotypic landscapes.

How HTDS works

The Stony Brook-based researchers adapted disulfide mapping, which, as Dr. Serebryany explains, “[it is] a process where you put a pair of cysteine residues somewhere in the structure of a protein. If the residues are close together, under some conditions they will form a disulfide bond; if they are far apart, they won’t.”

To determine which disulfides trap chromatographically resolvable conformers, the researchers developed a deep sequencing method for double-cysteine variant libraries of proteins that simultaneously pinpoint both Cys residues within each polypeptide with reasonable accuracy.

This makes it possible to unpack the 3D structural space of a protein into subgroups consisting of one-dimensional sequence space via disulfide cross-links. From there, the sequence can be mapped to establish whether a given pair of cysteine residues can covalently bind to one another. Having already published this method in Molecular Cell in 2023 and as a proof of concept, Dr. Serebryany and his team applied the technique to HdeA, an inherently disordered protein chaperone found in E. coli periplasm.

HdeA impedes the aggregation of periplasmic proteins as E. coli travels through stomach acid en route to the gut. An intramolecular disulfide connects the only two native cysteine residues; when this bond is reduced, the protein’s structure is broken down and obstructs chaperone activity in vitro.

This study demonstrated the feasibility of performing disulfide scanning of an entire protein in a single experiment. Remarkably, it is a consistently successful methodology as it allows researchers to observe the biophysical properties of protein conformations in vitro and the phenotypic consequences in vivo.

It is possible to transform these double cysteine scanning libraries into the host organism, in this case, E. coli or different cell types. Moreover, this grants the ability to determine, at the phenotypic level, which cells possess greater fitness, which have lower fitness, which has different phenotypes that can be sorted, and which disulfide aligns with those phenotypes.

We can also assess the changes in the protein molecule’s biophysical properties in vivo that emerge from forming individual disulfides that trap certain conformations.

The practical impact of protein barcodes

When Dr. Serebryany and his team were developing a proof of concept, they relied on mass spectrometry to run the protein sequencing, which can present serious challenges.

Mass spectrometry is excellent for point mutations, but with disulfide scanning, two mutations must be made at a time in the same molecule, and the location and the sequence of both cysteines in the same polypeptide chain must always be known.

To overcome such a challenge, the team was able to adapt a relatively intuitive type of chemistry that enabled them to cleave proteins precisely at the sites where the cysteines were and then apply mass spectrometry at the intervening peptides to map the termini, and therefore, where the cysteines used to be.

While this method has demonstrated successful outcomes to a certain extent, it still has limitations. The cysteine residues must remain close together in the sequence because mass spectrometry cannot cope with extremely long peptides. Moreover, any intervening cysteines must be eliminated between the two introduced cysteines. Otherwise, a cut between the two introduced cysteines can occur, resulting in a complete loss of all the structural information.

This is where protein barcodes are useful, as they can overcome such limitations and enable the application of this technique to larger, more complex proteins and those with native cysteine residues. This allows researchers to create a library of peptide barcodes attached as fusions to one of the termini. At the end of the experiment, the barcode is simply severed and sequenced.

Barcoding strategies for HTDS

The most practical barcode is as short as possible, and all the barcodes in the library share similar amino acid composition and chemical properties. This means they share the same combination of residues or a few chemically similar combinations, with the residues rearranged in various orders.

This would allow researchers to create an extensive library of barcodes for increased throughput while limiting the likelihood of differential effects on protein structure and folding, and therefore almost eliminating the chances of any artifacts finding their way into the experiment.

Therefore, the barcode sequences should be around four, five, or six residues. This would also depend on the complexity of the library required. Barcodes that share the same amino acid composition can be challenging to sequence using mass spectrometry because they would yield the same single precursor ion.

Using barcodes that demonstrate considerable differences would enhance the chemical heterogeneity of the barcode library; barcodes with varying chemical properties (such as hydrophobicity) have the potential to interfere with the protein structure in various ways.

Furthermore, mass spectrometry requires peptides longer than six residues for accurate sequence determination. As a result, single-molecule peptide sequencing is preferred to mass spectrometry for analyzing libraries of short, chemically similar peptide barcodes.

This led the Stony Brook team to take an interest in Quantum-Si, as it is now leveraging this technology for use with HTDS, opening the door to greater innovation for the team to advance its novel structural technique. Exploring the vast conformational space of proteins via peptide barcoding demanded a new sequencing approach, and Quantum-Si has brought this one step closer.

HTDS and Next-Generation Protein Sequencing

With new methodologies supported by innovative technologies, the potential becomes rather expansive. This, in turn, would enable several different types of biophysical measurements, including thermodynamic stability, binding affinity to a target, or aggregation propensity, at much greater throughputs than traditional methods.

HTDS stabilizes diverse conformations, which could prove extremely valuable in various applications, such as vaccine design and drug screening.

As described by Dr. Serebryany’s team in its Molecular Cell paper, which was related to the respiratory syncytial virus (RSV) vaccine, stabilizing the wrong conformation of the antigen in the vaccine could lead to tragic consequences. As a result, disulfide engineering was applied to stabilize the correct conformation. The same technique was applied to the SARS-CoV-2 spike protein, but less than 100 disulfides were screened and selected through structural intuition and computational modeling.

HTDS could facilitate screening several thousands of disulfides even before an atomistic structure is available and may expose disulfides with unforeseen practical allosteric effects. HTDS could also lead to progress in treating neurodegenerative and other diseases resulting from protein misfolding.

Consequently, stabilization of misfolded aggregation precursors via disulfide crosslinking could be utilized as protein vaccines. Another captivating application is determining disulfides that can trap non-native cytotoxic conformations. This could allow screening for drugs that select that conformation, potentially leading to a new class of antibiotics that work by induced misfolding of target proteins.

References and further reading

  1. Reynaud, E. (2010) Protein Misfolding and Degenerative Diseases. Nature Education 3(9):28
  2. Li J, et al. (2022) p53 amyloid aggregation in cancer: function, mechanism, and therapy. Exp Hematol Oncol 11, 66. https://doi.org/10.1186/s40164-022-00317-7
  3. Folger, A., and Wang, Y.C. (2021). The cytotoxicity and clearance of mutant huntingtin and other misfolded proteins. Cells 10, 2835. https://doi.org/10.3390/cells10112835.
  4. Serebryany, E, et al. Systematic conformation-to-phenotype mapping via limited deep sequencing of proteins. Molecular Cell 83(11): 1936-1952. https://doi.org/10.1016/j.molcel.202
  5. Killikelly AM, et al. (2016). Pre-fusion F is absent on the surface of formalin-inactivated respiratory syncytial virus. Sci. Rep. 6, 34108. https://doi.org/10.1038/srep34108.
  6. McLellan JS, et al. (2013). Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus. Science 342, 592–598. https://doi.org/10.1126/ science.1243283.

About Quantum-SI

Inspired by Ion Torrent’s success at shrinking next-generation sequencing technology into a benchtop instrument, Jonathan Rothberg founded Quantum-SiTM to bring the same semiconductor technology to protein sequencing with the launch of the Platinum® Next-Generation Protein Sequencer™.

That was in Guilford, CT, back in 2013. Fast forward to today and we now have over 1,000 patents issued and applications pending, plus a groundbreaking single-molecule protein sequencing technology platform, the Platinum.

Along the way, we solved critical challenges around sensitive and unambiguous amino acid detection, blending biology, chemistry, and semiconductor technology to help biologists see what other approaches cannot deliver. We also set the stage for a revolution in how scientists understand biology and build new treatments for disease by making single molecule protein sequencing accessible to every lab everywhere.

We are now entering a new phase of our development as a company. Starting with an initial public offering in June 2021 (QSI on the NASDAQ) and continuing with a new product development and operations facility in San Diego, CA, in 2022, we have entered a period of rapid growth. Through this expansion, we will be able to fuel a new era of biology, the post-genomic era, where biologists accelerate basic scientific insight and biomedical advances through the power of next-generation protein sequencing.


Sponsored Content Policy: News-Medical.net publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of News-Medical.Net which is to educate and inform site visitors interested in medical research, science, medical devices and treatments.

Continue Reading