# The Structural basis of Protein Biogenesis

> **NIH NIH R01** · BOSTON UNIVERSITY MEDICAL CAMPUS · 2020 · $387,750

## Abstract

Protein translocation and biogenesis machines
Abstract
Cells are compartmentalized by membranes and macromolecules must cross these barriers. The
SecY/Sec61 complex and the Nuclear Pore Complex (NPC) mediate transport across single and double
membranes, respectively. Secretory and membrane proteins are translocated by the bacterial ribosome-
SecY complex and by the 80S-Sec61 complex. The NPC is a gateway for proteins and RNPs to cross the
nuclear envelope and is comprised of a spoke ring complex and a central transporter.
 Aim 1- We will elucidate the mechanism of co-translational translocation by determining structures of
ribosome-SecYEG complexes with a defined nascent chain, to give novel views of early translocation
intermediates. To this end, stalled translocation intermediates are trapped by cross-linking in E. coli, purified
and reconstituted in nanodisks with native E. coli lipids. Frozen-hydrated particles are then imaged with a
Volta Phase Plate to give informative maps after multi-body refinement. Active complexes will be compared
to the ground state channel to provide a detailed view of this stepwise process. This work may also provide
insights into the positive inside rule for membrane protein topology. An N-terminal, 2-helix bundle in SecE of
medically-relevant enterobacteria, such as E. coli (O157:H7), may provide a novel antibiotic target, once we
understand its role in translocation. Our pipeline will provide a path forward to study Signal Sequence
Peptidase interactions with SecYEG, membrane protein integration and the role of non-canonical signal
sequences in translocation. Finally, a structure of the TRAP complex will help us to understand how nascent
chains with weaker signal sequences may be translocated more efficiently by the ribosome-Sec61 complex.
 Aim 2- We recently determined a first-pass, domain structure of the yeast NPC, which revealed the
functional anatomy of this translocation machine. A composite 3D map will now be determined at higher
resolution with single particle cryo-EM and/or state-of-the-art tomographic sub-volume averaging. The new
3D map will be combined with a large database of nucleoporin (Nup) crosslinks, ~25 published Nup domain
structures, and a structure of the Nup84 complex, to create a multi-scale model of the NPC with Integrative
Modeling. This in turn, will provide insights into Nup roles in assembly, pore stabilization, transport, and
evolution of the NPC. Three-dimensional classification will reveal the density distribution of FG Nups in the
central transporter, which will guide Brownian Dynamics simulations to dissect the transport mechanism.

## Key facts

- **NIH application ID:** 9973766
- **Project number:** 2R01GM045377-24A1
- **Recipient organization:** BOSTON UNIVERSITY MEDICAL CAMPUS
- **Principal Investigator:** CHRISTOPHER W AKEY
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $387,750
- **Award type:** 2
- **Project period:** 1991-01-01 → 2024-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9973766

## Citation

> US National Institutes of Health, RePORTER application 9973766, The Structural basis of Protein Biogenesis (2R01GM045377-24A1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9973766. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*