Uncovering the Binding Partners of PARP15: From Biophysical Characterization to Proteomic Discovery
(2025) KEMR30 20251Department of Chemistry
- Abstract
- Introduction: Discovering and characterizing protein interaction partners can provide valuable insights into key functions. It can also enable the structure determination of small and flexible proteins by providing rigidity through the formation of protein complexes.
Background: To date, there are no protein structures of full-length PARP15 in the protein data bank (PDB). Utilizing binding partners to reduce the flexibility of PARP15 could enable structural determination.
Aim: The aim of the thesis is to discover protein partners that can bind and form stable complexes with PARP15, and to study the interaction using various biochemical and biophysical methods.
Method: Biophysical (fluorescence polarization, mass photometry) and... (More) - Introduction: Discovering and characterizing protein interaction partners can provide valuable insights into key functions. It can also enable the structure determination of small and flexible proteins by providing rigidity through the formation of protein complexes.
Background: To date, there are no protein structures of full-length PARP15 in the protein data bank (PDB). Utilizing binding partners to reduce the flexibility of PARP15 could enable structural determination.
Aim: The aim of the thesis is to discover protein partners that can bind and form stable complexes with PARP15, and to study the interaction using various biochemical and biophysical methods.
Method: Biophysical (fluorescence polarization, mass photometry) and biochemical (western blot, pull-down, LC-MS/MS) methods are used to characterize the binding of 2 potential partner proteins. Moreover, a pull-down assay is used to find binders in human keratinocyte lysate.
Results: Many potential binders were identified through a pull-down assay and LC-MS/MS analysis. Additionally, dissociation constants (Kd) for PARP15 interactions with two kinases (MAPK8, PKM2), and for the homodimerization of two PARP15 constructs, were determined.
Conclusion: The generated lists of potential partners can be explored in future research. (Less) - Popular Abstract
- The function of a protein is not only dictated by its amino acid sequence, but also by its three-dimensional structure in the native environment. Although modern tools like AlphaFold allow researchers to predict molecular structures, the layouts of complex or highly flexible proteins are harder to predict. Sometimes, the binding of a partner protein can reduce the overall flexibility, allowing scientists to determine a more accurate structure using a variety of techniques.
In this work, potential binding partners of poly-(ADP-ribosyl) polymerase 15 (PARP15) are analyzed using both chemical and physical methods. Through a molecular fishing expedition, an assortment of proteins are pulled out of human skin cell lysate – or cell juice –... (More) - The function of a protein is not only dictated by its amino acid sequence, but also by its three-dimensional structure in the native environment. Although modern tools like AlphaFold allow researchers to predict molecular structures, the layouts of complex or highly flexible proteins are harder to predict. Sometimes, the binding of a partner protein can reduce the overall flexibility, allowing scientists to determine a more accurate structure using a variety of techniques.
In this work, potential binding partners of poly-(ADP-ribosyl) polymerase 15 (PARP15) are analyzed using both chemical and physical methods. Through a molecular fishing expedition, an assortment of proteins are pulled out of human skin cell lysate – or cell juice – using two different bait proteins. These proteins were attached to magnetic beads, which act as the fishing rod. Once stripped from the beads, the binding proteins were identified using conventional analytical methods.
Conclusively, a processed list of several proteins that potentially bind PARP15 is presented, along with insights into the interactions of PARP15 with two other proteins. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9195585
- author
- Sjölin, Herman LU
- supervisor
- organization
- course
- KEMR30 20251
- year
- 2025
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- Assay, Binding, Kinases, PARP15, Pull-down, biochemistry
- language
- English
- id
- 9195585
- date added to LUP
- 2025-06-12 11:45:21
- date last changed
- 2025-06-12 11:45:21
@misc{9195585,
abstract = {{Introduction: Discovering and characterizing protein interaction partners can provide valuable insights into key functions. It can also enable the structure determination of small and flexible proteins by providing rigidity through the formation of protein complexes.
Background: To date, there are no protein structures of full-length PARP15 in the protein data bank (PDB). Utilizing binding partners to reduce the flexibility of PARP15 could enable structural determination.
Aim: The aim of the thesis is to discover protein partners that can bind and form stable complexes with PARP15, and to study the interaction using various biochemical and biophysical methods.
Method: Biophysical (fluorescence polarization, mass photometry) and biochemical (western blot, pull-down, LC-MS/MS) methods are used to characterize the binding of 2 potential partner proteins. Moreover, a pull-down assay is used to find binders in human keratinocyte lysate.
Results: Many potential binders were identified through a pull-down assay and LC-MS/MS analysis. Additionally, dissociation constants (Kd) for PARP15 interactions with two kinases (MAPK8, PKM2), and for the homodimerization of two PARP15 constructs, were determined.
Conclusion: The generated lists of potential partners can be explored in future research.}},
author = {{Sjölin, Herman}},
language = {{eng}},
note = {{Student Paper}},
title = {{Uncovering the Binding Partners of PARP15: From Biophysical Characterization to Proteomic Discovery}},
year = {{2025}},
}