BCH2333_61-combined
Hierarchy of Protein Structure
Primary Structure
Linear sequence of amino acids in a polypeptide.
Sequence is written from N-terminus to C-terminus.
Nomenclature: Use suffix ‘-yl’ for amino acids preceding the C-terminal.
Example: ARL = L-alanyl-L-arginyl-L-leucine.
Primary structure dictates all higher levels of protein structure due to interactions among side chains.
Secondary Structure
Regular repeating structures formed by hydrogen bonds between backbone atoms.
Common types include alpha helices and beta sheets.
Local structures are stabilized by non-covalent interactions, primarily hydrogen bonds.
Tertiary Structure
The overall 3D shape of a single polypeptide chain.
Formed by the packing of secondary structural elements and interactions among side chains.
Hydrophobic interactions play a significant role in driving the folding process.
Quaternary Structure
Complexes formed by the assembly of multiple polypeptide subunits.
Stability is achieved through non-covalent interactions.
Amino Acids and Protein Sequence
The primary structure of proteins is determined by gene sequences or mass spectrometry.
Average molecular weight of an amino acid is about 110 Da, allowing for estimation of protein length based on its molecular weight.
Proteomics and Mass Spectrometry
Proteome: Complete set of proteins expressed by a cell, can vary based on conditions.
Proteomics: The study of the proteome, often utilizing mass spectrometry for protein identification.
Basic workflow involves separation (chromatography), followed by mass spectrometry to analyze fragments and identify sequences.
Chromatography Techniques
Types of Chromatography:
Affinity Chromatography:
Uses specific binding properties to purify proteins.
Example: Hexahistidine tag binds nickel.
Ion Exchange Chromatography:
Based on charge properties of proteins, requires knowledge of isoelectric point.
Size Exclusion Chromatography:
Separates proteins based on size; larger proteins elute faster than smaller ones.
Protein Analysis Techniques
SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis):
Coats proteins with a negative charge allowing separation based on size.
Used to visualize protein purity and estimate molecular weights by comparing with standards.
Secondary Structure Definitions
Alpha Helix:
Right-handed coil with 3.6 amino acids per turn.
Stabilized by hydrogen bonds between N-H of one amino acid and C=O of another.
Beta Sheet:
Comprises strands running in parallel or antiparallel fashion with hydrogen bonds stabilizing the structure.
Loop regions can connect these strands.
Ramachandran Plot:
Visual representation of allowed dihedral angles for backbone configurations (phi and psi).
Factors Affecting Protein Stability
Thermodynamics of Folding:
Gibbs Free Energy (ΔG) influences stability; negative ΔG indicates a favorable folding process.
Disulfide Bonds:
Form in oxidizing environments and can stabilize protein structure.
Intrinsically Disordered Regions (IDRs)
Regions within proteins without a stable 3D structure, enriched in certain amino acids (e.g., ARG, GLU, LYS).
Functional in cellular signaling and can adopt structure when interacting with binding partners.
Key Protein Functionalities
Protein modules or domains can independently fold and perform distinct functions.
Many proteins contain a variety of domains, allowing for functional diversity and regulation.
Techniques for Measuring Protein Stability and Structure
Circular Dichroism Spectroscopy (CD):
Measures differences in light absorption due to secondary structure.
Used to monitor folding/unfolding transitions by assessing changes in ellipticity at specific wavelengths.
Summary of Concepts in Class
Ability to describe protein structure hierarchy.
Understanding relationships between amino acid sequence, structure, and function.
Competency in techniques like mass spectrometry and chromatography for protein analysis.
Knowledge of how environmental factors can influence protein folding and stability.