Question

You have cloned the gene for a human erythrocyte protein, which you suspect is a membrane protein. You deduce the amino acid sequence of the protein from the nucleotide sequence of the gene. From this sequence alone, how would you evaluate the possibility that the protein is an integral protein? Suppose the protein proves to be an integral protein with one transmembrane segment. Suggest biochemical or chemical experiments that might allow you to determinewhether the protein is oriented with the amino terminus on the outside of the cell or on the inside of the cell.

Short answer

Use hydropathy plots and transmembrane prediction tools to identify integral proteins. For orientation, use antibody-binding assays and protease protection assays.

Step-by-step solution

Analyze Hydrophobicity

Determine if the amino acid sequence contains hydrophobic regions by using a hydropathy plot. High hydropathy indices suggest potential transmembrane helices, indicating the protein could be integral to the membrane. A single transmembrane segment often spans about 20 hydrophobic amino acids.

Predict Transmembrane Segments

Use software tools such as TMHMM, Phobius, or similar, to predict transmembrane segments in the protein sequence. These tools can further confirm the presence and position of transmembrane regions identified in the hydropathy plot.

Experiment to Determine Protein Orientation

To determine if the amino terminus of the protein is outside or inside the cell, employ antibody-binding assays. Design antibodies specific to the amino or carboxyl terminus. If these antibodies bind to intact cells, the corresponding terminus is facing outside.

Use Protease Protection Assay

Permeabilize the cell membrane slightly and treat cells with protease. If the protease digests the extracellular regions, and no digestion occurs on the intracellular side, this can help confirm the orientation of the protein termini.

Key concepts

Hydropathy plot

A hydropathy plot is a graphical representation of the hydrophobic and hydrophilic regions within a protein's amino acid sequence. The plot uses a scale that assigns a hydropathy index to each amino acid. Regions with higher indices are more hydrophobic, while lower indices indicate hydrophilicity. Hydrophobic regions often suggest potential transmembrane domains as these segments prefer the lipid bilayer environment of a membrane.
To create a hydropathy plot:

• Use software tools like Kyte-Doolittle to input the protein sequence.
• Identify regions with a high hydropathy index, which are usually above zero on the plot.
• Look for continuous stretches of about 20 amino acids indicating a potential transmembrane segment.

Detecting these regions gives a preliminary indication that a protein might be membrane-bound.

Transmembrane Segment Prediction

Software tools are pivotal for predicting transmembrane segments in a protein sequence. Tools like TMHMM and Phobius specifically analyze the sequence to identify likely transmembrane regions. These predictions rely on algorithms trained on known membrane proteins and are essential for confirming results from hydropathy plots.
While using transmembrane segment prediction software, you should:

• Input your protein sequence into the tool for analysis.
• Review predictions for the number and position of transmembrane segments.
• Cross-reference these segments with the hydropathy plot to enhance accuracy.

This dual approach offers a robust method to determine a protein's integral status.

Protein Orientation

Determining protein orientation is crucial for understanding its function and interaction within the cell membrane. The orientation refers to whether the amino or carboxyl terminus is inside or outside of the cell. Two main experiments can help determine protein orientation: antibody-binding assay and protease protection assay.
By understanding how a protein is oriented, researchers can infer its possible roles in cellular communication or transport. Knowing which end is exposed to the cell's exterior can also guide further studies on interactions with other molecules or pathogens.

Antibody-Binding Assay

The antibody-binding assay is a specific method used to determine protein orientation. Custom antibodies are raised to target specific termini (amino or carboxyl) of the protein. By adding these antibodies to intact cells, researchers can detect whether the terminus is extracellular based on binding activity.
Steps for an antibody-binding assay include:

• Designing antibodies that bind to the target region of the protein.
• Incubating intact cells with these antibodies.
• Observing for binding under a microscope or using enzyme-linked detection methods.

Binding indicates the terminus is on the outside, revealing critical orientation details about the protein's structure.

Protease Protection Assay

A protease protection assay involves using proteases to degrade accessible protein regions while leaving protected or membrane-embedded parts intact. This method offers insights into protein orientation by determining which parts are exposed to the extracellular environment.
Here's how the assay works:

• The cell membrane is mildly permeabilized to allow protease entry without excessive disruption.
• Protease treatment is applied, digesting regions accessible in the cellular exterior.
• After digestion, protected segments are analyzed, usually by gel electrophoresis or mass spectrometry.

If a terminus resists digestion, it implies that it is likely facing the cytoplasm. With this technique, researchers can gain further understanding of the membrane protein's topology.