Question

Predicting Membrane Protein Topology I Online bainformatics tools make hydropathy analysis easy if you know the amino acid sequence of a protein. At the Protein Data Bank (www?rosharg), the Protein Feature View displays additional information about a protein gleaned from other databases, such as Uniprot and SCOP2. A simple graphical view of a hydropathy plot created using a window of 15 residues shows hydrophobic regions in red and hydrophilic regions in blue. a. Looking only at the displayed hydropathy plots in the Protein Feature View, what predictions would you make about the membrane topology of these proteins: glycophorin A (PDB ID 1AFO), myoglobin (PDB ID \(1 \mathrm{MBO}\), and aquaporin (PDB ID 2B6O)? 1507 b. Now, refine your information using the ProtScale tools at the ExpASy bioinformatics resource portal. Each of the PDB Protein Feature Views was created with a UniProt Knowledgebsese ID. For glycophorin \(A\), the UniProtKB ID is P02724; for myoglobin, P02185; and for aquaporin, Q6J819. Go to the ExPASy portal (http://web.expasy orgLprotscale) and select the Kyte \& Doolittle hydropathy analysis option, with a window of 7 amino acids. Enter the UniProtKB ID for aquaporin (Q6JS19, which you can also get from the PDB's Protein Feature View page), then select the option to analyze the complete chain (residues 1 to 263). Use the default values for the other options and click Submit to get a hydropathy plot. Save a GIF image of this plot. Now repeat the analysis using a window of 15 amino acids. Compare the results for the 7 -residue and 15-residue window analyses. Which window size gives you a better signal-to-noise ratio? c Under what circumstances would it be important to use a narrower window?

Short answer

Glycophorin A likely has membrane-spanning regions; myoglobin does not. A 15-residue window plots a better signal-to-noise ratio for aquaporin analysis. Use a narrower window to detect short transmembrane regions.

Step-by-step solution

Understanding the Hydropathy Plot

A hydropathy plot provides insights into the hydrophobic or hydrophilic regions within a protein sequence. Hydrophobic regions are more likely to be found in membrane-spanning segments, while hydrophilic regions are exposed to the aqueous environment.

Analyzing the PDB Hydropathy Plots

Examine the hydropathy plots in the Protein Feature View for each of the proteins listed. For glycophorin A (PDB ID 1AFO), look for prominent hydrophobic regions, which suggest sections that span the membrane. Myoglobin (PDB ID 1MBO) should show mostly hydrophilic sections since it is not a membrane protein. Aquaporin (PDB ID 2B6O) should display alternating hydrophobic and hydrophilic regions, characteristic of channel proteins.

Using ProtScale Tools for Analysis

Visit the ExPASy ProtScale tools page for conducting the Kyte & Doolittle hydropathy analysis. Enter the UniProtKB ID for aquaporin (Q6JS19) and set the analysis window to 7 amino acids first. This will highlight finer details and isolated hydrophobic peaks that might indicate transmembrane regions.

Changing the Window Size

Re-run the ProtScale analysis using a window of 15 amino acids. A larger window smooths out noise and better highlights broad trends, such as longer stretches of hydrophobicity, but decreases the resolution for short hydrophilic segments.

Comparing Window Sizes for Signal-to-Noise Ratio

The plot using a window of 15 amino acids usually offers a clearer distinction between broader hydrophobic and hydrophilic regions, providing a better signal-to-noise ratio than the 7 amino acid window. This helps in identifying prominent membrane-spanning domains.

Circumstances for Using a Narrower Window

A narrower window is useful for detecting rapid changes in hydrophobicity over short stretches of amino acids, which can indicate shorter, less prominent transmembrane sections that might otherwise be blurred in broader analyses.

Key concepts

Membrane Protein Topology

Understanding membrane protein topology is key to revealing how a protein functions within biological membranes. This topology refers to the three-dimensional arrangement of a protein’s regions as they span and interact with the lipid bilayer. Membrane proteins often have both hydrophobic regions, which interface with the lipid tails of the bilayer, and hydrophilic regions, which face the aqueous exterior or interior of a cell.

The primary purpose of analyzing membrane protein topology is to predict how these proteins are situated within the cell membrane. Membrane-spanning regions are typically made up of hydrophobic amino acids arranged in alpha-helices or beta-sheets, facilitating stable insertion into the lipid bilayer. With structures like aquaporins or glycophorins, alternating hydrophobic and hydrophilic stretches indicate boundaries between membrane-spanning regions and aqueous-exposed loops.

Kyte & Doolittle Method

The Kyte & Doolittle method is a widely used algorithm for determining hydropathy plots of proteins. This method assesses the hydrophobicity and hydrophilicity values of each amino acid in a protein's sequence and averages these values over a sliding window of specified length. Typically, window sizes of 7 or 15 residues are used, each revealing different levels of detail.

Smaller windows, like 7 residues, provide high resolution and allow for identification of subtle changes, useful for detecting short, less obvious transmembrane segments. In contrast, a 15-residue window averages the values over a broader range, smoothing out noise and highlighting significant hydrophobic regions that suggest membrane-spanning helices.

When choosing window size, consider the specific structural characteristics of the protein in question. Short transmembrane regions, being less prominent, might require a smaller window to avoid blending in with the noise.

Protein Data Bank (PDB)

The Protein Data Bank (PDB) is an essential resource for exploring the three-dimensional structures of proteins. It contains detailed entries about proteins, including their structural information, hydrophathy plots, and additional valuable features obtained from various databases. By examining PDB data, one can explore the topographical arrangement of proteins and discern their functional domains inside membranes.

For instance, the PDB provides valuable insights on protein structures like glycophorin A, a membrane protein, and myoglobin, a non-membrane protein. Reviewing hydropathy plots within the PDB’s Protein Feature View can reveal regions of strong hydrophobicity, often correlating with segments embedded within membranes. This information aids researchers and students alike in predicting membrane protein topology more effectively.

ExPASy ProtScale tool

The ExPASy ProtScale tool is an online resource that facilitates hydropathy analysis using the Kyte & Doolittle method. This tool allows users to input a protein's UniProt ID and analyze hydrophobic and hydrophilic regions along the sequence based on user-defined parameters, such as sliding window sizes.

With ProtScale, users can seamlessly generate hydropathy plots, visually representing hydrophobic and hydrophilic regions of proteins. For example, analyzing aquaporin with a UniProt ID Q6JS19 using both short (7-residue) and long (15-residue) window sizes, provides distinct insights into the arrangement and extent of membrane-spanning domains.

The tool is particularly helpful for bioinformatics exercises involving prediction and analysis of membrane protein topology, offering a graphical representation of the protein’s interaction with its membrane environment.