The coiled-coil motif is present in proteins from all kingdoms of life. Its structure is based on a repeating sequence of 7 amino acids with hydrophobic residues at positions 1 and 4, which folds into an alpha-helix. Two, or more, alpha-helices wind around each other based on hydrophobic interactions forming the coiled-coil. Structural variations include length, deviations from the canonical form based on the heptad repeat, as well as the orientation and number of alpha-helices. They are involved in a wide variety of cellular processes including vesicle tethering and signal transmission along their length. In order to transmit signal, the protein must be able to dynamically rearrange its structure.
An outstanding example of a coiled-coil that needs to rearrange its structure to perform its function is the early endosomal tether EEA1, which has been shown to increase its flexibility upon binding to the active form of the small GTPase Rab5. That conformational change generates an entropic collapse that brings the ends of the protein closer to each other. Nevertheless, the recycling from the more flexible state to its original extended conformation was not addressed. Herein, the entropic collapse mechanism was further studied and the full EEA1 cycle between extended and flexible states described. In addition to these studies, other coiled-coil proteins were assessed to determine if they also experience a binding-induced entropic collapse.
One of the strategies to investigate the entropic collapse mechanism was to compare the adhesive forces along the two alpha-helices of the EEA1 dimer in its extended and flexible conformations. To this end, an experiment was designed to unwind the dimer using optical tweezers, a force-spectroscopy method that uses a highly focused laser beam to manipulate microscopic objects. Each EEA1 monomer was attached to a distinct DNA piece using a site-specific enzymatic reaction. The DNA pieces were linked to two optically trapped micron-sized beads. And the distance between the optical traps increased to unwind the EEA1.
A second strategy to investigate the entropic collapse was to evaluate EEA1 dynamics in solution using dual color fluorescence cross-correlation spectroscopy (dcFCCS). EEA1 C-termini was labeled with two different fluorophores. Fluctuations on fluorescent intensities caused by the dyes crossing a confocal volume were recorded over time. Based on an analysis of these fluctuations, a conformational change in EEA1 from semi-flexible to flexible upon addition of active Rab5 was described. This is in agreement with the previously reported entropic collapse. More importantly, EEA1 was shown to cycle between semi-flexible and flexible states by adding Rab5:GTP and waiting for the GTP to hydrolyse.
To determine whether other proteins experience a binding-induced entropic collapse, coiled-coil proteins that share structural and functional similarities with EEA1 were evaluated. Rotary shadowing EM images of the target protein alone and binding with its suspected allosteric effector were compared. It was found that ELKS, a coiled-coil protein involved in vesicle trafficking, undergoes an increase in flexibility upon binding with the active form of Rab6. Thus, hinting that the entropic collapse may indeed be a general mode of action for at least a sub-group of long coiled-coil proteins.
Overall, the major contributions of this thesis are to describe the full entropic collapse cycle on EEA1 and to show a second example of a coiled-coil protein experiencing a binding induced flexibility increase.:List of Figures
List of Tables
List of Equations
List of Abbreviations
1 Introduction
1.1 EEA1 as an endosomal tether
2 Materials and Methods
2.1 Materials
2.2 Methods
2.2.1 Sub-cloning
2.2.2 Protein expression and purification
2.2.3 Protein-protein binding assays
2.2.4 Electron microscopy
2.2.5 Analysis of electron microscopy
2.2.6 Generation of DNA handles for protein-DNA conjugates
2.2.7 Adding SortaseA recognition site to EEA1
2.2.8 Protein-DNA conjugation3
2.2.9 Sample preparation for optical tweezers
2.2.10 Dual color labeling of EEA1
2.2.11 Fluorescence cross-correlation spectroscopy
2.2.12 Generation of dsDNA for dcFCCS calibration
2.2.13 RabGTPase nucleotide loading
2.2.14 Liposome preparation
2.2.15 MCBs preparation
3 Unwinding EEA1 coiled-coil domain
3.1 Introduction
3.1.1 Optical tweezers for EEA1 unwinding
3.1.2 SortaseA-catalysed ligation
3.2 Aims
3.3 Results
3.3.1 Optimization of SortaseA-catalysed ligation
3.3.2 Formation of EEA1-DNA handle conjugate
3.3.3 EEA1 unwinding experiments
3.4 Discussion
4 EEA1 entropic collapse is recyclable
4.1 Introduction
4.1.1 Advantages of dcFCCS vs FCS
4.1.2 Requirements for dcFCCS measurements
4.1.3 dcFCCS for end polymer dynamics analysis
4.2 Aims
4.3 Results
4.3.1 System preparation and dcFCCS calibration
4.3.2 Labelling of EEA1
4.3.3 Comparing FCS vs dcFCCS
4.3.4 EEA1 entropic collapse shown by dcFCCS
4.3.5 EEA1 flexibility change is recyclable
4.4 Discussion
5 Entropic collapse as a general mechanism
5.1 Introduction
5.2 Aims
5.3 Results
5.3.1 ELKS increases its flexibility upon binding active Rab6
5.3.2 p115-GM130 complex observed by rotary shadowing EM
5.4 Discussion
6 Conclusions and outlook
References
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:78699 |
| Date | 05 April 2022 |
| Creators | Soler Blasco, Joan Antoni |
| Contributors | Zerial, Marino, Grill, Stephan, Rao, Madan, Technische Universität Dresden, Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG) |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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