from the Stephen White laboratory at UC Irvine

Features at a Glance (v 3.0)

MPEx can be used to explore both α-helical and β-barrel membrane proteins.

General Layout and Functions

The MPEx window is divided into two subwindows: By default, the left-hand plot panel shows the Hydropathy Analysis plot. Different analysis or data display modes can be selected with the tabs at the top of the plot panel. These include "Hydropathy Analysis", "β-Barrel Analysis", and "Buffer Overlays". An analysis plot is calculated and displayed automatically when a sequence is entered. Each time a modification is made to the sequence, the plot is automatically recalculated.

The right-hand dark-blue control panel is for setting parameters used in computing or displaying a data plot. A window at the top of the control panel provides summary information, including (for hydropathy analysis) the hydrophobicity scale in use, residue at cursor, direction of partitioning, and free energy of transfer of the amino acids within the window. Similar windows may appear in this region for other analysis modes.

Just above the plot panel is the buffer control bar, which permits plots to be stored in buffers, manipulated, and compared.

File loading, printing, and many other features are controlled through the menu bar appearing at the top of the window.

The blue cursor in the plot panel allows the central residue of a sliding window to be identified. The residue at the cursor is indicated in a text box at the top of the Plot Panel. The cursor may be moved along the sequence either by using the left and right arrow keys or the mouse.

Marking Interesting Residues (menu bar)

Residues of particular interest (e.g. aromatic) can be marked on the analysis plot. Use MPExToolBox->Mark Residues.

Marking and Measuring TM Segments Selected by User (menu bar)

A user may mark or measure any region s/he wishes. Use MPExToolBox->Mark->Measure User Segments .

Output from MPEx (menu bar)

All output is controlled by the MPExFile menu. The whole window, including the control panel or just the plot panel, can be printed directly (set your printer to landscape orientation). The content of the Results Window can be saved as a plain ASCII text file. Analysis plot data can be saved in a tab-delimited format suitable for direct import into plotting/graphing packages such as Origin™ or Excel™.

Storing Analysis Plots in Buffers (buffer control panel)

Any analysis plot, including Control Panel settings, etc., can be stored in a buffer using the buffer control panel above the plot region. Various plots stored in the buffers can be compared by selecting the Buffer Overlays tab at the top of the plot panel. Individual buffer contents can be loaded into the main workspace using the Load # menu on the buffer control panel menu bar. Overlay plot display characteristics can be modified from the reconfigured parameter control panel menu on the right-hand side. Different data sets can be selected for comparison using the tabs at the bottom of the plot panel.

Hydropathy Analysis Principles and Overview

MPEx hydropathy analysis is based upon and incorporates principles of membrane protein stability determined in our laboratory [see White & Wimley (1999) Annu. Rev. Biophys. Biomolec. Struct. 28:319-365]. The experiment-based Wimley-White whole-residue hydrophobicity scales are used in the plots. The Octanol scale (Oct) measures a residue's free energy of transfer from water to the bilayer hydrocarbon core. For TM segments, the thermodynamic cost of transferring H-bonded peptide bonds (CONH) into the hydrocarbon core of the membrane is a major determinant of stability. We have recently shown that the Octanol scale describes well the total energetics of TM helix stability, including the cost of dehydrating the H-bonded peptide bonds. Tests with the MPtopo 3D_helix set of membrane proteins indicate that TM segments can be identified with high accuracy. See Jaysinghe et al. (2001) J. Mol. Biol. 312:927-934. MPEx, however, allows the CONH cost to be modified for illustrative purposes. The Interface scale (IF) measures a residue's free energy of transfer within an unfolded polypeptide chain from water to a phosphocholine bilayer interface. Both scales identify those regions of a peptide chain most likely to prefer association with membranes. The Oct-IF scale is useful because it identifies segments that tend to prefer a transbilayer helix conformation relative to an unfolded interfacial location. This is useful for examining the refolding of toxins, e.g. diphtheria toxins, on membranes.

We have constructed a database of membrane proteins of known topology (MPtopo), accessible from MPEx, that is useful for comparing the hydropathy behavior of proteins of unknown topology with the behavior of similar proteins of known topology.

Hydrophobicity Scales (control panel)

The choices are Wimley-White Octanol, Interface, or Octanol-Interface whole-residue scales, as noted above. As discussed elsewhere ( whole-residue hydrophobicity scales), a major issue in all hydrophobicity scales is the exact cost of partitioning the H-bonded peptide bonds of alpha-helices into the membrane. Although we are now reasonably certain of this value (see above), MPEx allows the cost to be modified by means of the Set (delta) CONH Value in the MPExToolbox menu. It consists of two buttons: Plus (+) raises the energetic cost of transferring the CONH groups into the membrane, making the transfer less favorable; Minus (-) lowers the cost, making transfer more favorable. The net effect is to shift the plot relative to the hydropathy ΔG = 0 line. The charge state of Asp, Glu, and His can be accounted for using Change Charge at Cursor.

Salt Bridge Formation (control panel)

The effect of salt bridges on hydropathy plots can be investigated using the Set Salt Bridge feature. This is also available in the menu bar under MPExToolBox->Set Salt Bridges.

Two modes of Operation: Scan and Locate ( control panel)

Locate uses an algorithm based upon a 19 AA sliding window to select the regions most likely to be TM segments, based upon the selected hydrophobicity scale and other parameters (see below). MPEx ignores the window length parameter in the Locate mode. Scan produces a hydropathy plot using the selected sliding-window length and places red horizontal markers to indicate regions that favor membrane locations.

Amino Acid Substitutions (control Panel)

A sequence may be changed by selecting a residue with the cursor and using Change Residue To. This feature is especially useful for looking at the effect of residue mutations on hydropathy plots.

β-Barrel Analysis

Selecting the β-Barrel Analysis tab at the top of the plot panel reconfigures MPEx to do a screening analysis for the identification of β-barrel membrane proteins. The program is based upon Wimley (2002), Protein Science11:301-312. The screening algorithm uses amino acid composition and architecture of β-barrel membrane proteins of known structure to make predictions of TM β-strands, connecting β-hairpin loops, and the number of likely TM β-strands.

Scores are assigned for the likelihood of the entire sequence representing a β-barrel motif membrane protein (the β-barrel Sequence Score), the number of predicted β-strand peaks. and for selected windows representing two potential β-strand regions and the connecting β-hairpin loop. A β-barrel sequence score greater than 2 is a strong indicator that a sequence represents a β-barrel protein. These values are displayed in the summary window at the top of the Control Panel. The window scores and predicted TM and hairpin regions for the entire sequence, as well as any known TM regions, are displayed in the plot panel. See the full documenation for detailed information about MPEx.

Controls provided by the β-barrel control panel are similar to those in the Hydropathy Analysis control panel, including a summary window, the ability to send the cursor to a specific amino acid sequence position, visibility of the available data plot sets, setting the length of the transmembrane and hairpin window sizes, and changing and restoring residues within the sequence.

Other Goodies! (menu bar)

MPEx Totalizer is a tool for calculating the hydropathy of peptides. It allows the state of the N- and C-termini to be accounted for (acetylation, amidation, etc.). Totalizer also produces helical wheel plots, calculates and marks the hydrophobic moment, and shows the direction of the moment.

The MPtopo database, accessible from MPExToolBox->Search MPtopo, can be searched using a MySQL server. Proteins identified from a search can be loaded directly into MPEx.

Saving sessions. Your entire MPEx session can be saved and reloaded later in order to resume projects after an interruption. See MPExFile->Save Session and MPExFile->Restore Session on the Toolbar menu.

Brief Instructions (or See Complete Documentation )

Enter Protein Sequence (Required)

The minimum requirement for the proper operation of MPEx is a protein sequence. You have several choices: Using the MPExFile->Read File option, you may (1) read in a file from the lab's MPtopo database of membrane proteins of known topology, (2) enter or paste a sequence of interest into a sequence input window, or (3) obtain a sequence from the Swiss-Prot or PIR database (use FASTA format). The sequence should be in the conventional UPPERCASE ONE LETTER CODE designations for the 20 common amino acids. All lowercase letters and other characters will be discarded by MPEx.

Hydropathy Analysis: Choose Parameters

The control panel on the right side of the main MPEx window or the MPExToolBox menu allow you to select parameters that determine the analysis plot results** (see below).

For hydropathy analysis, you may change/select the following parameters:

• Select Wimley-White Hydrophobicity Scale on the Control Panel (default: Oct).
• Select visibility of Hydrophobic Moment data plot.
• Select Mode:Scan or Locate (default: Locate).
• Sliding-Window Size on control panel (use an odd number of amino acids. Default is 19 AA). Scan Mode Only.
• Change the charged state of a specific D, E, or H residue using Change Charge. MPEx assumes pH = 7. The default state of H is neutral.
• Change an amino acid in the sequence to another by using Change Residue To .
• Form salt bridges using Set Salt Bridge.

Pull-down menus from the menu bar also provide several parameter selections for hydropathy analysis:

• Transfer free energy from 'water to bilayer' or 'bilayer to water' under MPExToolBox->Set partitioning from... (default: bilayer to water). Hydrophobic peaks are positive when 'bilayer-to-water' is chosen.
• Set the charged state of all D, E, or H residues on plot panel using MPExToolbox->Select Residue State (default: D & E charged, H neutral).
• Mark locations of specific amino acids on the hydropathy plot under MPExToolBox->Mark Residues (default: none highlighted).
• Mark TM segments of your choice with a horizontal marker, using MPExToolBox->User TM Segments, by either dragging the cursor or entering residue numbers in windows.

β-Barrel Analysis: Choose Parameters

As for Hydropathy Analysis, the control panel allows you to select parameters that determine the β-barrel analysis plot results. The β-barrel analysis control panel allows selection of the following parameters:

• Select visibility of β-Strand Scores plot data and β-Hairpin Scores plot data.
• Set TM-Window Length sets the length of the two transmembrane sliding windows (default is 10 AA).
• Set Hairpin Window Length sets the length of the hairpin window between the two transmembrane windows (default is 5 AA).
• Change an amino acid in the sequence to another by using Change Residue To.

Several pull-down menus from the menu bar also affect β-barrel analysis:

• MPExToolBox->Mark Residues, as above.
• MPExToolBox->User TM Segments, as above.

The Mark Residues feature allows you to identify "interesting" amino acids, including charged residues that are titratable near physiological pH (D, E, and H), non-titratable basic residues (K and R) that reveal topology via the so-called "positive inside rule," and aromatic residues (F, W, and Y) known to prefer membrane surface locations in membrane proteins of known 3D structure.

**When you select a sequence and enter or change your parameters, the data analysis plot will be displayed automatically. The data analysis plot will be updated automatically whenever a parameter is changed.

Hydropathy Analysis Results

The hydropathy profile of the entered sequence will be plotted in the graph window. The black curve is the actual profile; the superimposed green curve is a smoothed version of the profile.

Regions that have a favorable free energy of transfer determined in Scan Mode are indicated with horizontal red bars. In Locate Mode, the red bars indicate MPEx's best guess about putative TM segments. These favorable regions, the AA sequences of these regions, and their total transfer free energies are listed in the Results Window. If MPtopo database files are being used, the known TM segments are marked by blue bars. User-designated TM segments are shown by rust-colored bars.

A hydrophobic moment plot for the current sequence can be displayed by selecting the HF Moment checkbox under Plot Visibility on the Control Panel.

The Residues in Window box on the Control Panel shows the amino acids contained within the sliding-window, centered on the residue at the cursor. If the residue at the cursor is D, E, or H, checking the Change Charge box on the Control Panel will change the charge state. In the Residues in Window box on the Control Panel, D, E, or H indicates charged, while d, e, or h indicates neutral residues.

β-Barrel Analysis Results

In β-barrel analysis, two plots can be displayed. β-strand scores are shown in violet, and β-hairpin scores are shown in tan. These plots can be selected individually on the control panel under Plot Visibility.

Regions of the β-strand score plot having y-axis values between 2 and 6 are indicated with horizontal red bars, the range in which most membrane-spanning β-strands are found.

Regions of the β-hairpin score plot having y-axis values greater than 6 are indicated with horizontal green bars. These segments are likely β-hairpin regions of the AA sequence.

As for hydropathy analysis results, β-barrel predicted transmembrane and hairpin regions of the AA sequence are listed in the Results Window. If MPtopo database files are being used, the known TM segments are marked by blue bars. User-designated segments are shown by rust-colored bars.

The Residues in Windows boxes on the Control Panel show the amino acids contained in the sliding window that represent a hairpin region of the AA sequence wherein the cursor is centered, flanked by two transmembrane regions.

For additional information, please refer to the complete documentation.

About MPEx

MPEx uses several public Java packages and classes that we are pleased to acknowledge.

MPEx is signed by a certificate issued by Thawte, Inc.

MPEx is available as a Web Start application

MPEx is available to run as a Web Start application. (What is Web Start?)

MPEx Is No Longer Available as a Java Application

The Web Start application (see just above) offers all of the advantages of the old standalone installation, plus automatic updates.

"Odd" Behavior

If you experience buggy behavior when using MPEx, or if certain functionalities are not working, please consult the list of known incompatibilities, system requirements, and tested platforms. We have made every effort to test MPEx for stability but cannot guarantee its performance on all platforms and browser combinations. We do not take responsibility for system crashes caused by running MPEx. NOTE: This section is out of date. It will be revised as compatibilities, etc. are tested on other platforms.

---