Irreversible Misfolding of Diacylglycerol Kinase Is Independent of Aggregation and Occurs Prior to Trimerization and Membrane Association

Abstract

Escherichia coli diacylglycerol kinase (DAGK) is a homotrimeric helical integral membrane protein in which a number of single-site mutations to cysteine are known to promote misfolding. Here, effects of other amino acid replacements have been explored using a folding assay based on the dilution of acidic urea/DAGK stock solutions into detergent/lipid mixed micelles. DAGK with an I110P or I110R mutation in the third transmembrane helix could not be purified because its expression was toxic to the E. coli host, most likely because of severe folding defects. Other mutations at Ile110 enhanced irreversible misfolding to varying degrees that generally correlated both with the polarity of the inserted amino acid and with the degree of protein destabilization. However, the I110W mutant was an exception in that it was highly misfolding prone while at the same time being more stable than the wild-type protein. This contrasts with I110Y, which also exhibited enhanced stability but folded with an efficiency similar to that of the wild type. For most mutants, the critical step leading to irreversible misfolding occurred for monomeric DAGK prior to trimerization and independent of association with mixed micelles. Misfolding of DAGK evidently involves the formation of incorrect monomer tertiary structure. Mutations appear to enhance misfolding by disfavoring the formation of correct structure rather than by directly stabilizing the misfolded state. Finally, when urea-solubilized DAGK was diluted into detergent/lipid-free buffer, it retained a significant degree of folding competency over a period of minutes. This property may be relevant to membrane protein folding in cells under conditions where the usual machinery associated with membrane integration is saturated, dysregulated, or dysfunctional.

Prokaryotic Diacylglycerol Kinase and Undecaprenol Kinase

“…Studies of a series of over 20 mutants revealed that that the overall rate of folding and insertion of DAGK into lipid vesicles correlates well with folding efficiency: mutants that insert and/or folding slowly are more likely to become trapped in misfolded states (59,62–64). Most mutations appear to lower folding efficiency by disfavoring the pathways leading to the folded state rather than by actively favoring misfolding (59).…”

“…Most mutations appear to lower folding efficiency by disfavoring the pathways leading to the folded state rather than by actively favoring misfolding (59). It was observed that there is a generally strong correlation between protein stability (both kinetic and thermodynamic, which seem to be well-correlated for DAGK) and folding efficiency (59,64). By far the most common mutations that results in significantly reduced folding efficiency are mutations that destabilize DAGK (59,64).…”

“…It was observed that there is a generally strong correlation between protein stability (both kinetic and thermodynamic, which seem to be well-correlated for DAGK) and folding efficiency (59,64). By far the most common mutations that results in significantly reduced folding efficiency are mutations that destabilize DAGK (59,64). A second class of misfolding-promoting mutations appears to be rare: amino acid changes that have little effect on the stability of DAGK but that reduce the folding rate and efficiency.…”

An Unfolding Story of Helical Transmembrane Proteins

“…Acidic urea solutions solubilize diacylglycerol kinase (DGK) in a form which spontaneously inserts into lipid vesicles and refolds into an active state 1 Abbreviations: BR, bacteriorhodopsin; SDS, sodium dodecyl sulfate; BO, bacterio-opsin; TM, transmembrane; DGK, diacylglycerol kinase; CD, circular dichroism; BSA, bovine serum albumin; FRET, fluorescence resonance energy transfer; NOE, nuclear Overhauser effect; LM, lauryl maltoside; CHAPSO, 3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate; HF-TAC, C 2 H 5 C 6 F 12 C 2 H 4 -S-poly-Tris-(hydroxymethyl)aminomethane. (27,28). There are a few reports of solubilization of helical membrane proteins by urea (29) or guanidinium (30) under neutral conditions.…”

Phosphatidylglycerol Lipids Enhance Folding of an α Helical Membrane Protein

“…A study of mutant DGK has also shown that mutant protein misfolds as a monomer, prior to trimerisation, when the protein is refolded into mixed detergent/lipid micelles. 22 We are interested to see here whether the folding yield in lipid vesicles can be manipulated by lipid properties and to ascertain conditions that increase the yield from that in DOPC, which is about 50-60%. 17 …”