Biology Chapter 5: Proteins Primary Structure

Proteins and Their Functions

• Proteins are essential in biological processes, acting as catalysts, regulators, structural components, and in transport and immunity.
• Techniques like mass spectrometry allow paleobiologists to analyze proteins in ancient bones, aiding species identification and insights into early human agriculture.

Primary Structure of Proteins

• The primary structure consists of the amino acid sequence in polypeptide chains; proteins with multiple chains have a combination of polypeptides.
• The total possible sequences for polypeptides with n residues are 20^n, highlighting extreme diversity; e.g., a 100-residue protein has approximately 1.27x10^130 unique sequences.
• Proteins typically have a minimum of 40 residues to achieve a functional conformation; smaller chains are termed peptides.
• Titin, the largest known protein, comprises 35,213 residues and plays a vital role in muscle fiber structure.

Diversity in Protein Composition

• Polypeptides generally range from 100 to 1000 residues, with the average size varying across different proteins.
• Composition of proteins is not uniform; the most frequent amino acids in proteins include Leu, Ala, Gly, Val, Glu, and Ser, while Trp, Cys, Met, and His are among the rarest.

Genetic Engineering and Protein Production

• Genetic engineering manipulates genes in host cells to produce recombinant proteins for research and medicine.
• Proteins in engineered organisms may form aggregates called inclusion bodies, which require careful purification to prevent damage.

Protein Purification Techniques

• Proteins should be dissolved in buffered solutions to maintain stable pH, typically purified around 0°C to slow enzymatic degradation.
• Storage under inert gases or at very low temperatures (e.g., -80°C) maintains protein integrity.
• Several assays are used to quantify proteins: enzyme assays, immunoassays, and techniques like RIA and ELISA which utilize antibodies for detection.

Chromatography Methods

• Ion exchange chromatography separates proteins based on charge via anion and cation exchangers (DEAE for anions and CM for cations).
• Gel filtration chromatography, or size exclusion chromatography, separates proteins based on size, with larger proteins eluting first.
• Affinity chromatography utilizes specific binding properties of proteins to purify them based on affinity to immobilized ligands.

Electrophoresis for Protein Separation

• Electrophoresis enables separation of proteins based on charge and size, primarily using Polyacrylamide gel electrophoresis (PAGE).
• Proteins typically move towards the positive electrode, and their bands can be visualized using stains or radioactive detection methods (autoradiography).
• SDS-PAGE provides molecular mass data for proteins, facilitating the analysis of protein subunits.

Proteolytic Enzymes

• Trypsin, a highly specific endopeptidase, cleaves peptide bonds post Arg and Lys, playing a critical role in protein digestion.
• Exopeptidases hydrolyze terminal amino acids, and both types are essential in protein processing.

Determining Amino Acid Sequencing

• Edman degradation sequencially removes N-terminal residues from peptides using Edman's reagent (PITC), allowing for controlled amino acid analysis.
• Mass spectrometry, particularly electrospray ionization mass spectrometry (ESI), is the primary technique for protein characterization; it measures ion mass-to-charge ratios to deduce molecular weights.

Conclusion

• Understanding proteins' structures, functions, and purification techniques is central to biochemistry and molecular biology, with implications in research, biotechnology, and medicine.

Protein Functions and Analysis

• Proteins are essential for biological processes, acting as catalysts, regulators, structural components, and playing roles in transport and immunity.
• Mass spectrometry helps paleobiologists analyze ancient proteins in bones to identify species and deduce early farming practices.

Primary Structure of Proteins

• Primary structure consists of the amino acid sequence of a polypeptide chain, which can be single or multiple chains.
• The number of possible sequences for a protein of n residues is 20ⁿ; for example, a protein with 100 residues has approximately 1.27 x 10¹³⁰ unique polypeptide chains.
• A polypeptide must have a minimum of 40 residues to fold into a functional shape; proteins typically range from 100 to 1000 residues.
• Titin, the largest known polypeptide, has 35,213 residues and is key for muscle fiber structure.
• Proteins can be single polypeptides or multisubunits, with insulin being an example of a protein synthesized as a single chain and later cleaved.

Genetic Engineering and Protein Production

• Cloning involves inserting specific genes into organisms to produce recombinant proteins, crucial for research and medicine.
• Engineered organisms may sequester foreign proteins in inclusion bodies within cells.

Protein Purification Methods

• To avoid irreversible damage during purification, consider:
  • Using buffers that maintain stable pH.
  • Performing purification at near-0°C temperatures.
  • Adjusting pH and temperature to inactivate degradative enzymes.
  • Minimizing foaming and maintaining protein concentration.
  • Storing proteins under nitrogen or frozen at -80°C.

Protein Quantification Techniques

• Assays to quantify proteins include:
  • Enzymes that produce detectable products (colored or fluorescent).
  • Coupled enzymatic reactions that yield easily quantified substances.
  • Immunoassays with antibodies binding specifically to proteins.
  • Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) for detection and quantification of antigens.

Chromatography Techniques

• Affinity Chromatography: Uses ligands to bind specific proteins, allowing for their purification from a mixture.
• Ion Exchange Chromatography: Separates proteins based on charge interactions with cation or anion exchangers.
• Gel Filtration (Size Exclusion) Chromatography: Separates molecules by size, allowing larger proteins to elute faster than smaller ones.

Electrophoresis

• Electrophoresis separates molecules based on charge and size, with polyacrylamide gel electrophoresis (PAGE) relying on gel filtration and electric field mobility.
• Proteins are negatively charged at high pH and migrate toward the positive electrode.
• Visualization techniques include staining and autoradiography for radioactive proteins; immunoblotting can detect specific proteins.

Protein Sequencing

• Protein sequencing is vital for understanding structure, function, evolutionary relationships, and mutations.
• Frederick Sanger first determined the sequence of bovine insulin, using a method involving dinitrophenol (DNP) labeling for N-terminal amino acid identification.
• Basic sequencing involves fragmenting the protein, determining peptide sequences, and reconstructing the full sequence from overlapping fragments.

Key Steps in Protein Sequencing

• Reduce disulfide bonds to separate polypeptide chains.
• Use chemical or enzymatic methods to break polypeptides into smaller peptides.
• Determine sequences of peptide fragments.
• Reconstruct the full sequence from overlapping data.
• Eventually, endopeptidases may be employed for cleaving longer peptide chains that cannot be sequenced directly.

Explore the primary structure of proteins and their pivotal roles in biological processes. This quiz will cover topics such as protein function in cellular metabolism, transport, and immunity, as well as techniques used in paleobiology to analyze ancient proteins. Test your knowledge of the importance of proteins in early human farming practices.