Biochemistry: A Short Course 5

The Proteome: A Functional Representation of the Genome

Introduction to the Proteome

The proteome is the complete set of proteins expressed by a genome, cell, tissue, or organism at a certain time. Unlike the genome, which is relatively constant, the proteome is dynamic and varies with cell type, developmental stage, and environmental conditions.

• Definition: The proteome includes all proteins, their modifications, functions, and interactions.

• Significance: Understanding the proteome is essential for elucidating cellular processes and disease mechanisms.

• Comparison: Genome = static, DNA-based; Proteome = dynamic, protein-based.

Protein Purification and Characterization

Purification of Proteins

Purifying proteins is a fundamental step in studying their structure and function. Proteins can be separated from complex mixtures based on their physical and chemical properties.

• Solubility: Proteins can be precipitated by changing salt concentration (salting out).

• Size: Gel filtration chromatography (size-exclusion) separates proteins by molecular size.

• Charge: Ion-exchange chromatography separates proteins based on net charge.

• Binding Affinity: Affinity chromatography uses specific interactions between a protein and a ligand attached to a matrix.

• SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis separates proteins by mass under denaturing conditions.

• Isoelectric Focusing: Separates proteins based on their isoelectric point (pI) in a pH gradient.

Immunological Techniques

Immunological methods use antibodies to detect, quantify, and purify proteins with high specificity.

• Enzyme-Linked Immunosorbent Assay (ELISA): Quantifies proteins using antibody-antigen interactions and enzyme-linked detection.

• Western Blot: Detects specific proteins separated by SDS-PAGE using antibodies.

• Monoclonal Antibodies: Highly specific antibodies produced from a single clone of cells, useful for purification and detection.

Determination of Primary Structure

Amino Acid Composition and Sequencing

Determining the primary structure (amino acid sequence) of a protein is crucial for understanding its function and evolutionary relationships.

• Hydrolysis: Proteins are hydrolyzed to release constituent amino acids.

• Separation: Amino acids are separated by ion-exchange chromatography and quantified by reaction with fluorescamine.

• Edman Degradation: Sequentially removes one amino acid at a time from the N-terminus for identification.

• Cleavage of Large Proteins: Large polypeptides are cleaved at specific sites using chemical or enzymatic reagents to generate smaller fragments for sequencing.

• Mass Spectrometry: Determines protein mass, identity, and sequence by analyzing peptide fragments.

Applications: Sequence data reveal evolutionary relationships, functional domains, and disease-causing mutations.

Case Study: Affinity Chromatography of Chymotrypsin

Principle and Application

Affinity chromatography exploits specific binding interactions between a protein and a ligand attached to a stationary matrix. This method allows for highly selective purification.

• Example: Purification of chymotrypsin using a Sepharose matrix with a covalently attached inhibitor (D-tryptophan methyl ester).

• Process: The enzyme binds to the inhibitor on the matrix; it is then eluted by changing pH (e.g., with 0.1 M acetic acid, pH 3).

• Detection: Protein elution is monitored by measuring absorbance at 280 nm (proteins absorb UV light at this wavelength).

Experimental Controls and Validation

• Control: Sepharose matrix alone does not bind chymotrypsin, confirming specificity.

• Active Site Binding: Use of diisopropylphosphofluoridate (DIFP), which covalently modifies the active site, shows that binding to the column is via the active site.

• Result: Only a small amount of DIFP-modified enzyme binds, confirming the mechanism.

Significance: Affinity chromatography is a powerful tool for purifying enzymes and studying their active sites.

Problem-Solving: Peptide Structure Determination

Case Study: Circular Peptide Antibiotic

A peptide antibiotic from a prokaryote was analyzed using several biochemical techniques to determine its structure.

• Acid Hydrolysis: Yields equal amounts of specific amino acids, indicating composition.

• Carboxypeptidase Treatment: No hydrolysis occurs, suggesting no free C-terminus.

• Fluorescamine Reaction: No modified N-terminal amino acid detected, indicating no free N-terminus.

• Partial Hydrolysis and Chromatography: Yields overlapping dipeptides and tripeptides, allowing sequence reconstruction.

• Molecular Weight: Approximately 1100 Da, suggesting a decapeptide (10 amino acids, average ~110 Da each).

Conclusion: The peptide is circular, with no free N- or C-terminus. Sequence is deduced by overlapping fragments.

Example Table: Overlapping Peptide Fragments

Fragment	Type
L-F	Dipeptide
F-I-M-L	Tetrapeptide
V-M	Dipeptide
V-M-L	Tripeptide
F-I-V	Tripeptide
I-V-M	Tripeptide

Sequence Construction: Overlapping fragments suggest a circular sequence such as L-F-I-V-M-L-F-I-V-M.

Summary Table: Protein Purification Methods

Method	Basis of Separation	Example/Application
Differential Centrifugation	Size, density	Cell fractionation
Salting Out	Solubility	Precipitation of proteins
Dialysis	Size (semipermeable membrane)	Buffer exchange, desalting
Gel Filtration Chromatography	Size	Separation of proteins by molecular weight
Ion-Exchange Chromatography	Charge	Separation by net charge
Affinity Chromatography	Binding affinity	Purification using specific ligand
High-Pressure Liquid Chromatography (HPLC)	Various (size, charge, hydrophobicity)	High-resolution separation
SDS-PAGE	Mass (denatured proteins)	Protein analysis
Isoelectric Focusing	Isoelectric point (pI)	Separation by charge in pH gradient
2D Electrophoresis	pI and mass	Complex protein mixtures

Key Equations and Concepts

• Isoelectric Point (pI): The pH at which a protein has no net charge.

• SDS-PAGE Separation: Proteins migrate according to mass: $d = \frac{1}{\sqrt{M}}$ (where d = distance migrated, M = molecular mass)

• Average Amino Acid Mass: $\text{Average mass} \approx 110 \text{ Da per residue}$

Additional info: The notes also reference the importance of knowing both the amino acid composition and the sequence for understanding protein function, evolutionary relationships, and disease mechanisms. Circular peptides are a unique structural class found in some prokaryotes and can be resistant to enzymatic degradation due to the lack of free termini.