Derek P. Ng

Membrane protein misassembly in disease

Helix-helix interactions play a central role in the folding and assembly of integral α-helical membrane proteins and are fundamentally dictated by the amino acid sequence of the TM domain. It is not surprising then that missense mutations that target these residues are often linked to disease. In this review, we focus on the molecular mechanisms through which missense mutations lead to aberrant folding and/or assembly of these proteins, and then discuss pharmacological approaches that may potentially mitigate or reverse the negative effects of these mutations. Improving our understanding of how missense mutations affect the interactions between TM α-helices will increase our capability to develop effective therapeutic approaches to counter the misassembly of these proteins and, ultimately, disease. This article is part of a Special Issue entitled: Protein Folding in Membranes.

Modulation of the Oligomerization of Myelin Proteolipid Protein by Transmembrane Helix Interaction Motifs

Proteolipid protein (PLP) is a highly hydrophobic 276-residue integral membrane protein that constitutes more than 50% of the total protein in central nervous system myelin. Previous studies have shown that this protein exists in myelin as an oligomer rather than as a monomer, and mutations in PLP that lead to neurological disorders such as Pelizaeus-Merzbacher disease and spastic paraplegia type 2 have been reported to affect its normal oligomerization. Here we employ peptide-based and in vivo approaches to examine the role of the TM domain in the formation of PLP quaternary structure through homo-oligomeric helix-helix interactions. Focusing on the TM4 alpha-helix (sequence (239)FIAAFVGAAATLVSLLTFMIAATY(262)), the site of several disease-causing point mutations that involve putative small residue helix-helix interaction motifs in the TM4 sequence, we used SDS-PAGE, fluorescence resonance energy transfer, size-exclusion chromatography, and TOXCAT assays in an Escherichia coli membrane to show that the PLP TM4 helix readily assembles into varying oligomeric states. In addition, through targeted studies of the PLP TM4 alpha-helix with point mutations that selectively eliminate these small residue motifs via substitution of Gly, Ala, or Ser residues with Ile residues, we describe a potential mechanism through which disease-causing point mutations can lead to aberrant PLP assembly. The overall results suggest that TM segments in misfolded PLP monomers that expose and/or create surface-exposed helix-helix interaction sites that are normally masked may have consequences for disease.

Novel Hydrophobic Standards for Membrane Protein Molecular Weight Determinations via Sodium Dodecyl Sulfate−Polyacrylamide Gel Electrophoresis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a universally employed technique that separates proteins on the basis of molecular weight (MW). However, membrane proteins are known to size anomalously on SDS-PAGE calibrated with conventional standards, an issue that complicates interpretation of protein identity, purity, degradation, and/or stoichiometry. Here we describe the preparation of novel polyleucine hydrophobic standards for SDS-PAGE that reduce the average deviation of the apparent MW from the formula MW of natural membrane proteins to 7% versus 20% with commercially available standards. Our results suggest that gel calibration with hydrophobic standards may facilitate the interpretation of membrane protein SDS-PAGE experiments.

Helix-Helix Interactions: Is the Medium the Message?

In this issue of Structure, Zhang and colleagues compare the helix-helix interaction spaces of an extensive database of soluble and membrane proteins. Intriguingly, the resultant clusters show similar helix interaction geometries between the protein classes, differing in detail only by patterns of local interactions and inter-helical distances.

Modulating Transmembrane α-Helix Interactions through pH-Sensitive Boundary Residues

Changes in pH can alter the structure and activity of proteins and may be used by the cell to control molecular function. This coupling can also be used in non-native applications through the design of pH-sensitive biomolecules. For example, the pH (low) insertion peptide (pHLIP) can spontaneously insert into a lipid bilayer when the pH decreases. We have previously shown that the α-helicity and helix-helix interactions of the TM2 α-helix of the proteolipid protein (PLP) are sensitive to the local hydrophobicity at its C-terminus. Given that there is an ionizable residue (Glu-88) at the C-terminus of this transmembrane (TM) segment, we hypothesized that changing the ionization state of this residue through pH may alter the local hydrophobicity of the peptide enough to affect both its secondary structure and helix-helix interactions. To examine this phenomenon, we synthesized peptide analogues of the PLP TM2 α-helix (wild-type sequence (66)AFQYVIYGTASFFFLYGALLLAEGF(90)). Using circular dichroism and Förster resonance energy transfer in the membrane-mimetic detergent sodium dodecyl sulfate, we found that a decrease in pH increases both peptide α-helicity and the extent of self-association. This pH-dependent effect is due specifically to the presence of Glu-88 at the C-terminus. Additional experiments in which Phe-90 was mutated to residues of varying hydrophobicities indicated that the strength of this effect is dependent on the local hydrophobicity near Glu-88. Our results have implications for the design of TM peptide switches and improve our understanding of how membrane protein structure and activity can be regulated through local molecular environmental changes.

Cancer Cells Are Like Weeds: Use of Visual Analogies to Explain Cancer Treatments

Estimates are that more than 50% of adults living in North America have low health literacy. Unfortunately, much of the available health education material is written at a grade level that most people don’t understand. To facilitate understanding, a 3D animation was created to explain cancer treatment options using analogies between cancer cells and weeds. The goal is to create educational material that people of all levels of health literacy can understand and learn from.

Developing a three-dimensional animation for deeper molecular understanding of michaelis-menten enzyme kinetics: Enzyme Kinetics Three-Dimensional Animation

The mathematical models that describe enzyme kinetics are invaluable predictive tools in numerous scientific fields. However, the daunting mathematical language used to describe kinetic behavior can be confusing for life science students; they often struggle to conceptualize and relate the mathematical representations to the molecular phenomena occurring at both macroscopic and microscopic levels. Students with less developed abstract and mathematical thinking skills may benefit from a visual learning approach. The paucity of visual resources for enzyme kinetics makes this a fertile field for developing novel learning media. We discuss developing a three‐dimensional animation aimed at introducing key concepts of Michaelis–Menten enzyme kinetics to undergraduate life science students. This animation uses both realistic and metaphoric depictions of the underlying molecular players, environments, and interactions in enzyme kinetics to contextualize and explain the relationship between the mathematical models and underlying molecular systems. The animation can be viewed at bit.ly/michaelis‐menten.

Modulating Transmembrane α-Helix Interactions through pH

The folding and assembly of α-helical integral membrane proteins into functional molecules is largely mediated by the lateral interactions of its transmembrane (TM) α-helices where sequence motifs often maximize van der Waals packing and/or polar interactions at sites of helix-helix contact. These interactions play a major role in determining the correct formation of tertiary and quaternary structure for these hydrophobic proteins. We have previously shown that a Lys-tagged TM2 α-helix of myelin proteolipid protein (PLP), of parent sequence, KKKK-AFQYVIYGTASFFFLYGALLLAEGF-KKKK, is capable of self-dimerization in the membrane mimetic detergent sodium dodecylsulfate (SDS). In the present work, we use protein gel electrophoresis, circular dichroism, and fluorescence resonance energy transfer to demonstrate that the oligomeric state of this peptide can be modulated by pH. Specifically, as the pH is increased, the peptide shifts from dimer to monomer. Mutational analysis reveals that this functionality is due to the C-terminal glutamic acid. Our overall results emphasize the potential influence of surrounding residues on the interaction strength of TM helix-helix contact sites that mediate oligomerization and may have implications for the control of biologically relevant events involving helix-helix interactions in some membrane proteins.