Design and characterization of natural and naturally-inspired helical protein assemblies Open Access
Miller, Jessalyn (Spring 2023)
Abstract
Protein helical assemblies are abundant in nature because they are useful to the cell and because they can form via a great diversity of mechanisms. In this work, multiple such assemblies are examined, from native-context A. tumefaciens T-pili to naturally derived CanA and Hyper2 proteins to ɑReps, which are naturally-inspired but rationally engineered. Nature has designed proteins that are extremely durable against thermal energy and enzymatic activity. Human scientists have developed predictive algorithms to make educated guesses about the tertiary and even quaternary structures of proteins. However, these powerful tools are primarily informed by proteins from organisms that either maintain their own body temperature (e.g., humans) or grow within animals that do (e.g., E. coli). The proteins described in this work do not always fully auto-assemble; instead, their assembly is mediated by lipids or divalent cations, or they are co-assemblies of multiple proteins. In other words, these modeling tools are very good at predicting how thermally unstable proteins fold. But hyperthermostable proteins such as those described in this paper are ostensibly better for designing tools. They are more likely to be correctly folded in a given condition and they will not degrade as quickly. For these reasons, study in thermostable proteins is as valuable as ever for protein design and structural biology in general.
Table of Contents
Abstract
Acknowledgements
Chapter 1 | Introduction: Structures of synthetic helical filaments and tubes based on peptide and peptido-mimetic polymers
Introduction
Structural characterization of synthetic helical filaments
Structures of helical peptide assemblies
Short-peptide assemblies
Cross-β filaments
Cross-β nanotubes
Coiled-coil filaments
Tandem repeat assemblies
Foldamer-based helical assemblies
Conclusions
References
Chapter 2 | Deciphering the rules governing synthetic protein assemblies based on repeat proteins
Background
Results
Stabilizing the assembly by optimizing Brick:Brick binding interface
Kinetics of Brick C crystallization
Changing morphology of the Brick to alter assembly morphology
Thermostability
Stabilizing the interior of the filament
Stabilizing the exterior of the filament
Solubilizing filaments
Capping
Mixing Bricks
Discussion
Materials and Methods
Expression plasmids
Plasmid preparation.
Protein expression and preparation
Filament assembly.
Crystallization rates.
Circular dichroism.
Negative staining sample protocol.
Cryo imaging protocol.
References
Supplemental Information
Chapter 3 | Archaeal DNA-import apparatus is homologous to bacterial conjugation machinery
Introduction
Results
Archaeal conjugation pili are stoichiometric complexes of pilins and lipids
Conjugation pili of Agrobacterium tumefaciens
Different helical symmetries still allow quasi-equivalent interactions
Discussion
Methods
Cultivation of archaeal cells and preparation of pili samples
pED208 pili purification
A. tumefaciens pili purification
Cryo-EM sample preparation and data collection
Data processing and helical reconstruction
Sequence analyses
Mass spectrometry and lipidomics of A. pernix
Electron spray ionization and tandem MS-MS of pED208 F-pilus and pC58 T-pilus lipidomics
Nomenclature.
Lipid extraction.
PLA2 treatment.
Lipid standards.
Lipid spectrum acquisition.
Lipid identification and quantification.
Chemical and physical treatments of pED208 F-pilus and pC58 T-pilus.
Negative stain transmission electron microscopy of treated pED208 F-pilus and pTiC58 T-pilus
Reporting summary
Data availability
References
Acknowledgements
Author contributions
Competing interests
Additional information
Supplementary Information
Chapter 4 | Stacked cyclic peptide assemblies mediated by pyrene stacking
Introduction
Results and Discussion
Materials and Methods
Preparing filaments
Electron Microscopy:
Structural determination
Circular dichroism spectropolarimetry.
Conclusions
References
Chapter 5 | Structure and characterization of self-assembling archaeal cannula from metagenomic data
Introduction
Results
CanA
Hyper2
Materials and Methods
Protein expression
Nanotube assembly
Negative Stain transmission electron microscopy.
Circular dichroism spectropolarimetry.
Cryo-electron microscopy and structural determination.
Atomic force microscopy and quantitative nanomechanical mapping.
Analytical ultracentrifugation.
Discussion
Structural motifs
Role of ion or heat
Outlook
References
Supplemental Information
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