Self-assembling artificial proteins (SAPs) have gained enormous interest in recent years due to their applications in different fields. Synthesis of well-defined monodisperse SAPs is accomplished predominantly through genetic methods. However, the last decade witnessed the use of few chemical technologies for that purpose. In particular, micelle-assisted protein labeling technology (MAPLabTech) has made huge progress in this area. The first generation MAPLabTech focused on site-specific labeling of the active-site residue of serine proteases to make SAPs. Further, this methodology was exploited for labeling of N-terminal residue of a globular protein to make functional SAPs. In this study, we describe the synthesis of novel SAPs by developing a chemical method for site-specific labeling of a surface-exposed cysteine residue of globular proteins. In addition, we disclose the synthesis of redox- and pH-sensitive SAPs and their systematic self-assembly and dis-assembly studies using complementary biophysical studies. Altogether these studies further expand the scope of MAPLabTech in different fields such as vaccine design, targeted drug delivery, diagnostic imaging, biomaterials, and tissue engineering.

/pdf/rational-design-of-self-assembling-artificial-proteins-25jx9w2zlx.pdf

Rational Design of Self-assembling Artificial Proteins Utilizing a Micelle-Assisted Protein Labeling Technology (MAPLabTech): Testing the Scope

Non-invasive, real-time, longitudinal imaging of protein functions in living systems with unprecedented specificity is one of the critical challenges of modern biomedical research. Despite several advancements, it is estimated that nearly 35% of the human proteome is not completely characterized. Therefore, the development of new technologies is imperative for shining more light on so-called “dark proteomes”. Towards that goal, here we report a platform fusion technology called activity-based protein profiling-bioluminescence resonance energy transfer (ABPP-BRET). This method provides an opportunity to study the post-translational modification of a target protein in real-time in living systems in a longitudinal manner with a high spatio-temporal resolution. This semi-synthetic BRET biosensor method is used for target engagement studies and further for inhibitor profiling in live cells. The simplicity of this method coupled with the critical physical distance dependent BRET read-out turned out to be a powerful method, thus pushing the activity-based protein profiling technology to the next level.

Development of Non-invasive ABPP-BRET Platform Technology Enable Real-Time Detection of Post-Translational Modification and Target Engagement with Absolute Specificity in Living Systems

Understanding cellular signaling flow is required to comprehend living organisms. Various live cell imaging tools have been developed but challenges remain due to complex cross-talk between pathways and response heterogeneities among cells. We have focused on multiplex live cell imaging for statistical analysis to address the difficulties and developed simple multiple fluorescence imaging system to quantify cell signaling at single-cell resolution using Förster Resonance Energy Transfer (FRET)-based chimeric molecular sensors comprised of fluorescent proteins and dyes. The dye-fluorescent protein conjugate is robust for a wide selection of combinations, facilitating rearrangement for coordinating emission profile of molecular sensors to adjust for visualization conditions, target phenomena, and simultaneous use. As the molecular sensor could exhibit highly sensitive in detection for protease activity, we customized molecular sensor of caspase-9 and combine the established sensor for caspase-3 to validate the system by observation of caspase-9 and -3 dynamics simultaneously, key signaling flow of apoptosis. We found cumulative caspase-9 activity rather than reaction rate inversely regulated caspase-3 execution times for apoptotic cell death. Imaging-derived statistics were thus applied to discern the dominating aspects of apoptotic signaling unavailable by common live cell imaging and proteomics protein analysis. Adopted to various visualization targets, the technique can discriminate between rivalling explanations and should help unravel other protease involved signaling pathways. 

/pdf/live-imaging-of-apoptotic-signaling-flow-using-tunable-1hkqmep4.pdf

Live imaging of apoptotic signaling flow using tunable combinatorial FRET-based bioprobes for cell population analysis of caspase cascades

Self‐assembling artificial proteins (SAPs) have gained enormous interest in recent years due to their applications in different fields. Synthesis of well‐defined monodisperse SAPs is accomplished predominantly through genetic methods. However, the last decade has witnessed the use of a few chemical technologies for this purpose. In particular, micelle‐assisted protein labeling technology (MAPLabTech) has made huge progress in this area. The first generation MAPLabTech focused on site‐specific labeling of the active‐site residue of serine proteases to make SAPs. Further, this methodology was exploited for labeling of N‐terminal residue of a globular protein to make functional SAPs. In this study, we describe the synthesis of novel SAPs by developing a chemical method for site‐specific labeling of a surface‐exposed cysteine residue of globular proteins. In addition, we disclose the synthesis of redox‐sensitive SAPs and their systematic self‐assembly and disassembly studies using size‐exclusion chromatography. Altogether these studies further expand the scope of MAPLabTech in different fields such as vaccine design, targeted drug delivery, diagnostic imaging, biomaterials, and tissue engineering.

Rational Design of Self‐Assembling Artificial Proteins Utilizing a Micelle‐Assisted Protein Labeling Technology (MAPLabTech): Testing the Scope **

Self-assembly of a monomeric protease to form a multi-subunit protein complex "proteasome" enables targeted protein degradation in living cells. Naturally occurring proteasomes serve as an inspiration and blueprint for the design of artificial protein-based nanoreactors. Here we disclose a general chemical strategy for the design of proteasome-like nanoreactors. Micelle-assisted protein labeling (MAPLab) technology along with the N-terminal bioconjugation strategy is utilized for the synthesis of a well-defined monodisperse self-assembling semi-synthetic protease. The designed protein is programmed to self-assemble into a proteasome-like nanostructure which preserves the functional properties of native protease.

A Universal Chemical Method for Rational Design of Protein-Based Nanoreactors*.

Imaging of an active protease with an exquisite specificity in the presence of highly homologous proteins within a living cell is a very challenging task. Herein, we disclose a new method called “A...

Development of Activity-Based Reporter Gene Technology for Imaging of Protease Activity with an Exquisite Specificity in a Single Live Cell

Self-assembly of a monomeric protease to form a multi-subunit protein complex “proteasome” enables targeted protein degradation in living cells. The naturally occurring proteasomes serve as an inspiration and blueprint for the design of artificial protein-based nanoreactors. Here we disclose a general chemical strategy for the design of proteasome-like nanoreactors. Micelle-assisted protein labeling (MAPLab) technology along with the N-terminal bioconjugation strategy is utilized for the synthesis of a well-defined monodisperse self-assembling semi-synthetic protease. The designer protein is programmed to self-assemble into a proteasome-like nanostructure which preserves the functional properties of native protease.

https://www.biorxiv.org/content/biorxiv/early/2021/03/01/2021.03.01.433350.full.pdf

Punita Bathla

Papers

A Universal Chemical Method for Rational Design of Protein-Based Nanoreactors*.

Development of Activity-Based Reporter Gene Technology for Imaging of Protease Activity with an Exquisite Specificity in a Single Live Cell

A Universal Chemical Method for Rational Design of Protein-based Nanoreactors