{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/121370"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/121370","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Dynamics and equilibria of weak protein interactions in cells","abstract":"Submission published under a 24 month embargo labeled 'U of I Access', the embargo will last until 2025-08-01","abstract_html":"Submission published under a 24 month embargo labeled 'U of I Access', the embargo will last until 2025-08-01","abstract_has_math":false,"creators":["Wang, Yuhan"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Biophysics & Quant Biology","degree_department":null,"school":null,"contributors":["Gruebele, Martin","Murphy, Catherine J","Selvin, Paul R","Kraft, Mary L"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2023,"date_issued":"2023-08","date_published":"2023-08","updated_at":"2026-07-22T22:24:57Z","subjects":["Protein Interaction","Protein Folding","Protein Diffusion","Microtubule Assembly","Cell Volume Modulation","Live Cell Imaging","Fluorescence Microscopy","Minflux"],"languages":["en","eng"],"rights":["Copyright 2023 Yuhan Wang"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://hdl.handle.net/2142/121370","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Gruebele, Martin","Murphy, Catherine J","Selvin, Paul R","Kraft, Mary L"]},{"key":"dc:creator","label":"Author","values":["Wang, Yuhan"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2023-08","2023-07-14"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biophysics & Quant Biology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Protein Interaction","Protein Folding","Protein Diffusion","Microtubule Assembly","Cell Volume Modulation","Live Cell Imaging","Fluorescence Microscopy","Minflux"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en","eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2023 Yuhan Wang"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://hdl.handle.net/2142/121370"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Submission published under a 24 month embargo labeled 'U of I Access', the embargo will last until 2025-08-01","The student, Yuhan Wang, accepted the attached license on 2023-07-14 at 12:30.","The student, Yuhan Wang, submitted this Dissertation for approval on 2023-07-14 at 13:10.","This Dissertation was approved for publication on 2023-07-14 at 16:24.","DSpace SAF Submission Ingestion Package generated from Vireo submission #19697 on 2023-12-04 at 17:18:47","When transitioning protein studies from in vitro to in cell, one of the major challenges is the dynamic and complex nature of the cellular environment. Cells contain a large network of weak interactions, including crowding, sticking and quinary interactions. Recent advancements in imaging techniques and computational power have revealed the significance of these weak, transient interactions in biological processes. To gain a better understanding of them, we studied weak interaction from various perspectives, including domain-domain interaction, protein-protein dimerization, protein-RNA interaction, and multi-protein interaction. Chapter 1 provides an overview of weak interactions in cells and explores the factors of the cellular environment that influence them. Chapter 2 focuses on the impacts of cell volume change on different ensembles of the proteins in cell. In this project, a cell volume modulation was designed and conducted. By measuring the response of FRET-labeled kinase, we proposed that cell volume change affects the protein stability and as well as compacts or expands the unfolded states. Unfolded proteins, lacking rigid and well-defined tertiary structure, are highly sensitive to the variation of cell volume. Chapter 3 delves into how cell environment affects protein dimerization in cells. The binding affinity of tubulin proteins BtubA/B was quantified in cell using microinjection and fluorescent microscopy, revealing that their in-cell binding affinity is ten-fold stronger than in vitro. To mimic the in-cell environment, we modified in vitro conditions and found both crowding and sticking effects contributed to the promotion of cellular binding. A Monte-Carlo-sampling-based simulation model was built to reproduce experimental results. The simulation suggested that sticking facilitates binding by offering interaction surfaces, while crowding enhanced binding by increasing the effective concentration. The formation of microtubule-like structures by BtubA/B were observed in vitro as well as human cells, and we successfully replicated these results in the simulation. Chapter 4 investigates the coupling of protein-RNA diffusion and interaction in cells. We approached this question from the single-molecule level using MINimal photon FLUX (MINFLUX), a super-resolution nanoscope with sub-5 nm spatial resolution and few bleaching effects. The protein-RNA interaction complex U1A-SL2 was employed as our model system. Their diffusion and interaction were quantified in vitro and in cell. Bound and unbound protein/RNA populations were differentiated and spatially located with respect to specific regions in the cell. Chapter 5 examines how multi-protein complexes diffuse as a response to changes in cellular crowding. A setup for Wide-field Intensified Fluctuation Imaging (WIFI) was designed and built to measure protein folding, interaction, and diffusion data from an entire cell simultaneously by analyzing the auto-/cross- correlation of intensity fluctuations. To manipulate the intercellular crowding, an osmotic pressure modulation system is connected to WIFI controlling cell volume. The diffusion coefficients of Hepatitis B virus (HBV) capsid were quantified in regular and high crowding conditions within cells. The result exhibited significant agreement with single-molecule diffusion results from MINFLUX tracking. In summary, this thesis aims to deepen our understanding of weak interactions in cellular environments and their implications for protein folding, interactions, and diffusion. The research employed a combination of experimental techniques, such as spectroscopy, fluorescent microscopy at ensemble level and single-molecule level, as well as computational modeling to study protein dynamics and equilibria."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Dynamics and equilibria of weak protein interactions in cells"]}]}],"canonical_facts":{"dc:contributor":["Gruebele, Martin","Murphy, Catherine J","Selvin, Paul R","Kraft, Mary L"],"dc:creator":["Wang, Yuhan"],"dc:date":["2023-08","2023-07-14"],"dc:description":["Submission published under a 24 month embargo labeled 'U of I Access', the embargo will last until 2025-08-01","The student, Yuhan Wang, accepted the attached license on 2023-07-14 at 12:30.","The student, Yuhan Wang, submitted this Dissertation for approval on 2023-07-14 at 13:10.","This Dissertation was approved for publication on 2023-07-14 at 16:24.","DSpace SAF Submission Ingestion Package generated from Vireo submission #19697 on 2023-12-04 at 17:18:47","When transitioning protein studies from in vitro to in cell, one of the major challenges is the dynamic and complex nature of the cellular environment. Cells contain a large network of weak interactions, including crowding, sticking and quinary interactions. Recent advancements in imaging techniques and computational power have revealed the significance of these weak, transient interactions in biological processes. To gain a better understanding of them, we studied weak interaction from various perspectives, including domain-domain interaction, protein-protein dimerization, protein-RNA interaction, and multi-protein interaction. Chapter 1 provides an overview of weak interactions in cells and explores the factors of the cellular environment that influence them. Chapter 2 focuses on the impacts of cell volume change on different ensembles of the proteins in cell. In this project, a cell volume modulation was designed and conducted. By measuring the response of FRET-labeled kinase, we proposed that cell volume change affects the protein stability and as well as compacts or expands the unfolded states. Unfolded proteins, lacking rigid and well-defined tertiary structure, are highly sensitive to the variation of cell volume. Chapter 3 delves into how cell environment affects protein dimerization in cells. The binding affinity of tubulin proteins BtubA/B was quantified in cell using microinjection and fluorescent microscopy, revealing that their in-cell binding affinity is ten-fold stronger than in vitro. To mimic the in-cell environment, we modified in vitro conditions and found both crowding and sticking effects contributed to the promotion of cellular binding. A Monte-Carlo-sampling-based simulation model was built to reproduce experimental results. The simulation suggested that sticking facilitates binding by offering interaction surfaces, while crowding enhanced binding by increasing the effective concentration. The formation of microtubule-like structures by BtubA/B were observed in vitro as well as human cells, and we successfully replicated these results in the simulation. Chapter 4 investigates the coupling of protein-RNA diffusion and interaction in cells. We approached this question from the single-molecule level using MINimal photon FLUX (MINFLUX), a super-resolution nanoscope with sub-5 nm spatial resolution and few bleaching effects. The protein-RNA interaction complex U1A-SL2 was employed as our model system. Their diffusion and interaction were quantified in vitro and in cell. Bound and unbound protein/RNA populations were differentiated and spatially located with respect to specific regions in the cell. Chapter 5 examines how multi-protein complexes diffuse as a response to changes in cellular crowding. A setup for Wide-field Intensified Fluctuation Imaging (WIFI) was designed and built to measure protein folding, interaction, and diffusion data from an entire cell simultaneously by analyzing the auto-/cross- correlation of intensity fluctuations. To manipulate the intercellular crowding, an osmotic pressure modulation system is connected to WIFI controlling cell volume. The diffusion coefficients of Hepatitis B virus (HBV) capsid were quantified in regular and high crowding conditions within cells. The result exhibited significant agreement with single-molecule diffusion results from MINFLUX tracking. In summary, this thesis aims to deepen our understanding of weak interactions in cellular environments and their implications for protein folding, interactions, and diffusion. The research employed a combination of experimental techniques, such as spectroscopy, fluorescent microscopy at ensemble level and single-molecule level, as well as computational modeling to study protein dynamics and equilibria."],"dc:format":["application/pdf"],"dc:identifier":["https://hdl.handle.net/2142/121370"],"dc:language":["en","eng"],"dc:rights":["Copyright 2023 Yuhan Wang"],"dc:subject":["Protein Interaction","Protein Folding","Protein Diffusion","Microtubule Assembly","Cell Volume Modulation","Live Cell Imaging","Fluorescence Microscopy","Minflux"],"dc:title":["Dynamics and equilibria of weak protein interactions in cells"],"dc:type":["text"],"thesis:degree_discipline":["Biophysics & Quant Biology"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:24:57Z"}