Time-lapse structural analysis of biomolecular processes

• Applicant: Professor Dr. Harald Schwalbe
• Subject Area: Structural Biology
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 451906961

The project aims at atomic-scale time-lapse imaging of two biomolecular processes - co-transcriptional folding of riboswitch RNA and assembly of apoferritin nanocage. Riboswitch folding is part of genetic regulatory network. DNA transcription, mRNA self-cleavage, splicing and translation can all be modulated, providing long-term and/or tight control of gene expression. Riboswitch-based genetic regulation is strictly essential for all uni- and simple multicellular organisms, allowing efficient adaptation to complex and/or dynamic environments including specific development phases. The detailed understanding of riboswitch-based genetic regulation can have an immediate implementation for the broad range of anthropogenic applications, agricultural, environmental, urban, medical, industrial, forensic, etc., including, e.g., development of new antibiotics, small molecule detection and biodegradation of organic waste. Apoferritin is the primary protein responsible for the intracellular iron uptake and storage, ubiquitous in all domains of life. It is the key regulatory element for the iron metabolism that is designed to maintain iron in the soluble non-toxic form. Due to its (i) high structural stability, (ii) physiological relevance and non-toxicity, (iii) ability to pass the body barriers, (iv) ability to carry encapsulated drug molecules, (v) ability to acquire new properties by surface functionalization, and (vi) easy expression and purification, apoferritin is an attractive smart tool for biomedical applications. The detailed understanding of apoferritin nanocage assembly can revolutionize disease treatment strategies, broaden the list of the applicable therapeutic agents, increase their efficiency and notably simplify nanoparticle engineering. Atomic-scale time-lapse imaging will be realized using nuclear magnetic resonance spectroscopy (NMR), transmission electron cryo-microscopy (ECM) and the novel method of time-resolved pre-steady-state mixing-and-sampling – nanosecond hyperquenching (NHQ). This method currently stands out in the field, outperforming all time-resolved methods for 3D imaging. On one hand, it is applicable to any biological function of interest; on the other – it provides the access to the entire micro-milli-second time domain – the natural timescale in structural biology. NHQ provides three orders of magnitude gain in time resolution vs current methods. Using NHQ, the biological function can be triggered by nanosecond mixing and/or large temperature jump (LTJ). Time-resolved structural intermediates can be captured by nanosecond hyperquenching in liquid cryogen at 77 K. The 3D structures of these intermediates can be analyzed using cryogenic solid-state magic-angle-spinning (cryo-MAS) NMR, electron cryo-microscopy (ECM) as well as as well as (pulsed) EPR techniques.

• DFG Programme: New Instrumentation for Research