Optical tools can be used to activate biological functions, but with current methods the effects are slow to appear, and sustained effects require continuous light activation. As a result, these light-activation tools provide limited control of fast biological processes and can lead to toxicity in cells and organisms.
Although light is a well-established tool for control of bond breakage, it is less firmly established for the control of specific bond formation in complex environments.
A team at Tampere University worked with researchers at the University of Cambridge and the University of Pittsburgh to develop a way to use visible light to control irreversible protein binding. The new optical technique for fast, irreversible protein conjugation could be especially valuable in processes where a short initial signal leads to long-term changes in cell or tissue function. Examples include the regulation of gene expression during stem cell differentiation and the activation of immune cells in viral infections.
The researchers built on their previous work with proteins to develop a system for the rapid, light-activated control of protein bond formation. Their “protein superglue” is a peptide/protein pair called SpyTag003/SpyCatcher003 that exhibits fast, irreversible binding. Based on an engineered protein, the SpyTag003/SpyCatcher003 peptide/protein pair allows the modular assembly of complex protein structures.
To achieve optical control of the protein superglue, the researchers looked beyond the 20 amino acids constituting human proteins. Using modified protein synthesis machinery from archaebacteria, they incorporated a light-reactive, unnatural amino acid into the SpyCatcher003 protein to make the protein photoreactive. The amino acid was strategically placed to block the peptide/protein pairing until it was activated by light.
In experiments, the researchers showed a uniform, specific reaction in cell lysate upon light activation.
“A short pulse of light was enough to trigger the rapid and efficient formation of the irreversible peptide/protein complex, both in the test tube and in living cells,” said Mark Howarth, a professor at the University of Cambridge. “Importantly, the activation only took place with specific wavelengths of light, making it possible to combine protein control with live-cell fluorescence microscopy.”
After validating their approach to optically controlling irreversible protein coupling, the researchers applied the technique to the covalent reconstitution of a talin protein that was split in half. The researchers used light to activate the talin — a central adhesion protein — inside living cells.
Optical control of talin reconstitution allowed the researchers to probe the timescale of the initial adhesion complex formation. By tracking the timing of protein recruitment into the adhesion complex, the team could determine a timeline of the events leading to the formation of the adhesion complex, and the hierarchy of the recruitment of key components for cell adhesion.
Cell-matrix adhesions — large protein complexes consisting of hundreds of different proteins — are highly dynamic. “Their dynamic structure and vast complexity make cell adhesions difficult to study,” Tampere University professor Vesa Hytönen said. “The details of how cell-matrix adhesions initially form and how they react to different stimuli have remained largely unknown.”
Researcher Rolle Rahikainen said that the team observed an immediate cell response after activating the talin protein with a short pulse of light. “We got very excited when we first realized how well the system worked in controlling complex cellular processes, such as the formation of adhesion and cell spreading.”
The findings demonstrate the potential of the light-activated protein superglue for investigating complex cellular processes. The results could also lead scientists to a more comprehensive understanding of the complex structure and function of adhesion.
The modular, Lego brick-like structure of the system makes it applicable to the study and control of diverse cellular functions. The precise, irreversible assembly of biological building blocks has many applications, from biomaterials to vaccines.
Beyond adhesion, SpyCatcher003 could be used for the photocontrol of biomolecules. The robust cellular response, initiated in seconds, opens possibilities for spatiotemporal control of highly dynamic intracellular and extracellular processes.
Bio Photonics Research Award
Visit: biophotonicsresearch.com
Nominate Now: https://biophotonicsresearch.com/award-nomination/?ecategory=Awards&rcategory=Awardee
#MeatAnalysis #FluorescenceTech #FoodQuality #FoodSafety #SpectroscopyInFood #MeatAuthentication #RapidDetection #FoodScience #MeatFreshness #MolecularDetection #FoodIndustryInnovation #NonDestructiveTesting #FoodMonitoring #SpectroscopyApplications #QualityControl #AdvancedSpectroscopy #MeatSpoilageDetection #FoodIntegrity #SmartFoodTesting #RealTimeAnalysis #FoodAuthenticity #FoodSafetyInnovation #SpectroscopyResearch #NextGenFoodSafety #InnovativeFoodScience,
Although light is a well-established tool for control of bond breakage, it is less firmly established for the control of specific bond formation in complex environments.
A team at Tampere University worked with researchers at the University of Cambridge and the University of Pittsburgh to develop a way to use visible light to control irreversible protein binding. The new optical technique for fast, irreversible protein conjugation could be especially valuable in processes where a short initial signal leads to long-term changes in cell or tissue function. Examples include the regulation of gene expression during stem cell differentiation and the activation of immune cells in viral infections.
The researchers built on their previous work with proteins to develop a system for the rapid, light-activated control of protein bond formation. Their “protein superglue” is a peptide/protein pair called SpyTag003/SpyCatcher003 that exhibits fast, irreversible binding. Based on an engineered protein, the SpyTag003/SpyCatcher003 peptide/protein pair allows the modular assembly of complex protein structures.
To achieve optical control of the protein superglue, the researchers looked beyond the 20 amino acids constituting human proteins. Using modified protein synthesis machinery from archaebacteria, they incorporated a light-reactive, unnatural amino acid into the SpyCatcher003 protein to make the protein photoreactive. The amino acid was strategically placed to block the peptide/protein pairing until it was activated by light.
In experiments, the researchers showed a uniform, specific reaction in cell lysate upon light activation.
“A short pulse of light was enough to trigger the rapid and efficient formation of the irreversible peptide/protein complex, both in the test tube and in living cells,” said Mark Howarth, a professor at the University of Cambridge. “Importantly, the activation only took place with specific wavelengths of light, making it possible to combine protein control with live-cell fluorescence microscopy.”
After validating their approach to optically controlling irreversible protein coupling, the researchers applied the technique to the covalent reconstitution of a talin protein that was split in half. The researchers used light to activate the talin — a central adhesion protein — inside living cells.
Optical control of talin reconstitution allowed the researchers to probe the timescale of the initial adhesion complex formation. By tracking the timing of protein recruitment into the adhesion complex, the team could determine a timeline of the events leading to the formation of the adhesion complex, and the hierarchy of the recruitment of key components for cell adhesion.
Cell-matrix adhesions — large protein complexes consisting of hundreds of different proteins — are highly dynamic. “Their dynamic structure and vast complexity make cell adhesions difficult to study,” Tampere University professor Vesa Hytönen said. “The details of how cell-matrix adhesions initially form and how they react to different stimuli have remained largely unknown.”
Researcher Rolle Rahikainen said that the team observed an immediate cell response after activating the talin protein with a short pulse of light. “We got very excited when we first realized how well the system worked in controlling complex cellular processes, such as the formation of adhesion and cell spreading.”
The findings demonstrate the potential of the light-activated protein superglue for investigating complex cellular processes. The results could also lead scientists to a more comprehensive understanding of the complex structure and function of adhesion.
The modular, Lego brick-like structure of the system makes it applicable to the study and control of diverse cellular functions. The precise, irreversible assembly of biological building blocks has many applications, from biomaterials to vaccines.
Beyond adhesion, SpyCatcher003 could be used for the photocontrol of biomolecules. The robust cellular response, initiated in seconds, opens possibilities for spatiotemporal control of highly dynamic intracellular and extracellular processes.
Bio Photonics Research Award
Visit: biophotonicsresearch.com
Nominate Now: https://biophotonicsresearch.com/award-nomination/?ecategory=Awards&rcategory=Awardee
#MeatAnalysis #FluorescenceTech #FoodQuality #FoodSafety #SpectroscopyInFood #MeatAuthentication #RapidDetection #FoodScience #MeatFreshness #MolecularDetection #FoodIndustryInnovation #NonDestructiveTesting #FoodMonitoring #SpectroscopyApplications #QualityControl #AdvancedSpectroscopy #MeatSpoilageDetection #FoodIntegrity #SmartFoodTesting #RealTimeAnalysis #FoodAuthenticity #FoodSafetyInnovation #SpectroscopyResearch #NextGenFoodSafety #InnovativeFoodScience,
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