Introduction

Mechanosensitive channels are critical proteins that act as cellular “emergency release valves,” allowing cells to survive sudden changes in external pressure. The mechanosensitive channel of large conductance (MscL) is a key model for understanding how cells sense and respond to membrane tension. However, a scientific controversy arose when crystal structures of MscL from different bacterial species suggested different architectures: a five-subunit (pentameric) structure for M. tuberculosis MscL and a four-subunit (tetrameric) structure for a truncated version of S. aureus MscL. This study aimed to resolve this discrepancy by determining the channel’s true structure within its natural environment, the living cell membrane.

Research Objective

The primary goals of this NASA-supported research were to:

• Determine the true oligomeric state (i.e., the number of protein subunits) of the S. aureus MscL (SaMscL) channel in its native cellular environment (in vivo).
• Investigate whether truncating the channel’s C-terminal domain, as was done for a previous crystal structure study, alters its oligomeric state in vivo.
• Clarify whether the observed tetrameric structure was a natural state or an artifact of the experimental methods used for protein crystallization.

Key Findings

• SaMscL is a pentamer in vivo: Using a disulfide-trapping technique in live bacteria, the study showed that both full-length and C-terminally truncated SaMscL channels assemble almost exclusively as five-subunit complexes (pentamers).
• Truncation does not alter structure in vivo: The specific C-terminal truncation (Δ95) that previously yielded a tetrameric crystal structure was confirmed to form a stable pentamer in the native cell membrane.
• Tetrameric structure is a detergent artifact: The data strongly suggest that the previously observed tetramer was not a native state but an artifact caused by the detergent (LDAO) used to extract and solubilize the protein for crystallization.
• Functional similarity confirmed: Electrophysiology experiments showed that both full-length and truncated channels were functionally similar in native membranes, further supporting that they share the same fundamental structure.
• Broad consensus for pentameric state: This work, combined with previous studies on MscL from E. coli and M. tuberculosis, solidifies the scientific consensus that MscL channels are pentamers across different bacterial species.

Methodology

• Organisms: S. aureus MscL protein (full-length and various truncated versions) was expressed in E. coli bacteria.
• Experimental Conditions: The study was a ground-based experiment conducted under standard laboratory conditions to investigate fundamental protein biophysics.
• Key Techniques: The primary method was an in vivo disulfide-trapping assay, where cysteine mutations were introduced to chemically lock the protein complex in its native state within the cell membrane. The resulting complexes were analyzed using SDS-PAGE and Western blotting. Patch-clamp electrophysiology was used to measure channel function and confirm its activity in native membranes.

Importance for Space Missions

Mechanosensitive channels like MscL are fundamental to how cells sense and respond to physical forces, including osmotic pressure, gravity, and fluid shifts—all critical factors affecting astronaut health. This study provides the correct structural model (pentamer) for a key mechanosensor, which is essential for accurately predicting how bacterial and potentially human cells will respond to the mechanical stresses of spaceflight. Furthermore, it highlights a critical methodological pitfall: that techniques used to prepare proteins for analysis can create misleading artifacts. This underscores the need for in vivo validation of biological findings, a crucial principle for designing robust and reliable space biology experiments.

Knowledge Gaps & Future Research

While this study resolves a key question, it also points to new areas for investigation:

• The precise molecular mechanism by which different detergents can alter the structure of membrane proteins remains unclear.
• While the C-terminal domain does not dictate oligomeric assembly in vivo, its specific role in channel gating, regulation, or interaction with other cellular components needs further investigation.
• Further research is needed to understand the potential for structural plasticity in MscL channels under different environmental conditions relevant to space, such as altered gravity or radiation.

Results

This research definitively resolves a significant controversy in the field of mechanosensation by demonstrating that the SaMscL channel is a pentamer in vivo. The findings correct the scientific record, showing that a previously reported tetrameric structure was an artifact of experimental preparation. By establishing the true native structure of this paradigm mechanosensitive channel, this work provides a solid foundation for future studies on cellular responses to mechanical stress, a key area of interest for understanding biology in space.