Experimental phasing for structure determination using membrane-protein crystals grown by the lipid cubic phase...

Area of Science:

• Structural Biology
• Membrane Protein Crystallography
• Biophysics

Background:

• Crystallization of membrane proteins using the lipid cubic phase (in meso) method has increased.
• However, experimental phasing (SAD/MAD) of these proteins remains difficult due to handling challenges.
• The in meso method involves viscous mesophases, complicating protein and crystal manipulation.

Purpose of the Study:

• To address the challenges in experimental phasing of membrane proteins crystallized in meso.
• To report a four-year effort to phase the structure of E. coli diacylglycerol kinase (DgkA).
• To provide a reference for future phasing of membrane proteins crystallized using the lipid cubic phase method.

Main Methods:

• Heavy-atom labeling strategies including pre-labeling, co-crystallization, soaking, and site-specific mercury binding.
• Genetic engineering of single-cysteine mutants for targeted mercury binding.
• Selenomethionine incorporation for phasing.
• Development of an assay to assess cysteine accessibility for mercury labeling.

Main Results:

• Various heavy-atom labeling techniques were attempted for the integral membrane enzyme DgkA.
• Strategies, handling techniques, typical results, and lessons learned for each approach are detailed.
• A novel assay for assessing cysteine residue accessibility in membrane proteins was introduced.

Conclusions:

• Successful experimental phasing of membrane proteins crystallized in meso is achievable with dedicated strategies.
• The reported techniques and assay provide valuable guidance for future structural studies.
• Overcoming handling difficulties in viscous mesophases is key to advancing membrane protein structure determination.