Dataset Information

Bacterial fermentation and isotope labelling optimized for amyloidogenic proteins.

ABSTRACT: We developed a cost sensitive isotope labelling procedure using a fed-batch fermentation method and tested its efficiency producing the ¹⁵ N-, ¹³ C- and ¹⁵ N/¹³ C-labelled variants of an amyloidogenic miniprotein (E5: EEEAVRLYIQWLKEGGPSSGRPPPS). E5 is a surface active protein, which forms amyloids in solution. Here, we confirm, using both PM-IRRAS and AFM measurements, that the air-water interface triggers structural rearrangement and promotes the amyloid formation of E5, and thus it is a suitable test protein to work out efficient isotope labelling schemes even for such difficult sequences. E. coli cells expressing the recombinant, ubiquitin-fused miniprotein were grown in minimal media containing either unlabelled nutrients, or ¹⁵ N-NH₄ Cl and/or ¹³ C-D-Glc. The consumption rates of NH₄ Cl and D-Glc were quantitatively monitored during fermentation and their ratio was established to be 1:5 (for NH₄ Cl: D-Glc). One- and two-step feeding schemes were custom-optimized to enhance isotope incorporation expressing five different E5 miniprotein variants. With the currently optimized protocols we could achieve a 1.5- to 5-fold increase of yields of several miniproteins coupled to a similar magnitude of cost reduction as compared to flask labelling protocols.

SUBMITTER: Vida I

PROVIDER: S-EPMC8085922 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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Bacterial fermentation and isotope labelling optimized for amyloidogenic proteins.

Vida István I Fazekas Zsolt Z Gyulai Gergő G Nagy-Fazekas Dóra D Pálfy Gyula G Stráner Pál P Kiss Éva É Perczel András A

Microbial biotechnology 20210319 3

We developed a cost sensitive isotope labelling procedure using a fed-batch fermentation method and tested its efficiency producing the 15 N-, 13 C- and 15 N/13 C-labelled variants of an amyloidogenic miniprotein (E5: EEEAVRLYIQWLKEGGPSSGRPPPS). E5 is a surface active protein, which forms amyloids in solution. Here, we confirm, using both PM-IRRAS and AFM measurements, that the air-water interface triggers structural rearrangement and promotes the amyl ...[more]

PMID: 33739615

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Project description:The advancement in site-directed spin labeling of proteins has enabled EPR studies to expand into newer research areas within the umbrella of protein-membrane interactions. Recently, membrane remodeling by amyloidogenic and non-amyloidogenic proteins has gained a substantial interest in relation to driving and controlling vital cellular processes such as endocytosis, exocytosis, shaping of organelles like endoplasmic reticulum, Golgi and mitochondria, intracellular vesicular trafficking, formation of filopedia and multivesicular bodies, mitochondrial fusion and fission, and synaptic vesicle fusion and recycling in neurotransmission. Misregulation in any of these processes due to an aberrant protein (mutation or misfolding) or alteration of lipid metabolism can be detrimental to the cell and cause disease. Dissection of the structural basis of membrane remodeling by proteins is thus quite necessary for an understanding of the underlying mechanisms, but it remains a formidable task due to the difficulties of various common biophysical tools in monitoring the dynamic process of membrane binding and bending by proteins. This is largely since membranes generally complicate protein structure analysis and this problem is amplified for structural analysis in the presence of different types of membrane curvatures. Recent EPR studies on membrane remodeling by proteins show that a significant structural information can be generated to delineate the role of different protein modules, domains and individual amino acids in the generation of membrane curvature. These studies also show how EPR can complement the data obtained by high resolution techniques such as X-ray and NMR. This perspective covers the application of EPR in recent studies for understanding membrane remodeling by amyloidogenic and non-amyloidogenic proteins that is useful for researchers interested in using or complimenting EPR to gain better understanding of membrane remodeling. We also discuss how a single protein can generate different type of membrane curvatures using specific conformations for specific membrane structures and how EPR is a versatile tool well-suited to analyze subtle alterations in structures under such modifying conditions which otherwise would have been difficult using other biophysical tools.

Dataset Information

Bacterial fermentation and isotope labelling optimized for amyloidogenic proteins.

Publications

Bacterial fermentation and isotope labelling optimized for amyloidogenic proteins.

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