Single-molecule fluorescence reveals sequence-specific misfolding in multidomain proteins

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• Protein misfolding is associated with numerous debilitating diseases.
• Multidomain proteins constitute 75% of the eukaryotic proteome, yet mechanisms preventing interdomain misfolding remain unclear.
• Low sequence identity between linked domains is hypothesized to prevent misfolding.

Purpose of the Study:

• To investigate interdomain misfolding events in tandem immunoglobulin domains from titin.
• To quantify the stability of misfolded protein states.
• To determine the role of sequence identity in preventing protein misfolding.

Main Methods:

• Single-molecule Förster resonance energy transfer (smFRET) was employed to detect and quantify misfolding events in vitro.
• Tandem immunoglobulin domains with varying sequence identities were analyzed under native conditions.
• Coarse-grained molecular simulations were used to model misfolded structures.

Main Results:

• Approximately 5.5% of identical domain molecules misfolded during in vitro refolding, forming a stable misfolded state.
• Tandem domains with 42% sequence identity exhibited misfolding, while those with 24% sequence identity did not.
• Molecular simulations predicted domain-swapped structures consistent with experimental smFRET data.

Conclusions:

• Protein misfolding involves specific, sequence-dependent domain-swapping mechanisms.
• Diversifying sequence identity between neighboring domains is an effective evolutionary strategy to prevent misfolding in multidomain proteins.
• Understanding these mechanisms is crucial for addressing protein misfolding-related diseases.