Protein Structure - Biochemistry

Committee V

• This section consists of 8 lectures and 1 lab section.
• Topics include proteins, protein structures, buffer systems, feed & fast cycle, and water.
• 10% of the total committee grade is based on lab grades.
• "Lippincott's Illustrated Reviews: Biochemistry 6th Edition" is followed in class and is available in English and Turkish in the library.
• "Experimental Biochemistry for Medical Sciences Students” (T. Yigitbasi, S.S.Erdem, P.Bozaykut) and “Lehningers Principals of Biochemistry" are required for laboratory sections.

Learning Objectives

• Understanding protein structures
• Knowing the importance of primary structure
• Learning about amino acids and interactions, contributing to secondary structure of proteins
• Knowing about Tertiary and quaternary structure of proteins
• Learning about diseases related to protein misfolding

Structure of Proteins

• 20 amino acids form proteins with varying sequences.
• Unlike other organic polymers, proteins have a specific 3D conformation.
• This structure enables specific biological functions.
• The structure that allows the protein to perform its biological function is termed the native fold.
• The native fold is stabilized by numerous favorable interactions within the protein.

Four Levels of Protein Structure

• Primary structure: amino acid sequence.
• Secondary structure: local spatial arrangement of the polypeptide backbone.
• Tertiary structure: overall three-dimensional structure of a polypeptide.
• Quaternary structure: arrangement of multiple polypeptide subunits.

Primary Structure

• Primary structure is the sequence of amino acids.
• Amino acids are linked covalently by peptide bonds.
• A component amino acid in a polypeptide is called is a "residue" or "moiety”.
• Strong acids or bases at high temperatures are required to break peptide bonds.
• By convention, a protein's primary structure starts at the amino-terminal (N) end and finishes at the carboxyl-terminal (C) end.

Effect of Primary Structure Changes

• Abnormal hemoglobin results from single amino acid substitutions.
• Single amino substitutions (changes) can cause hemoglobin to crystallize.
• Crystallization of hemoglobin leads to deformed red blood cells and blood vessel blockages.

Formation of Primary Structure

• Amino acid sequences are read from the N-terminal to the C-terminal end of the peptide.
• Each component in the sequence is called a residue.
• Peptide bonds exhibit partial double-bond characteristics.
• α-amino and α-carboxyl bonds can freely rotate.
• Carbonyl and amide amine groups are involved in hydrogen bonding.

Importance of Primary Structure

• Primary structure helps predict secondary and tertiary structures through sequence comparisons.
• Genetic diseases can arise from abnormal amino acid sequences.
• The molecular mechanism of protein action can be understood by studying primary structure.

Methods of Amino Acid Sequence Determination

• Identification and quantification of amino acids
• End group analysis (Edman degradation)
• DNA sequencing method

Determination of Amino Acid Composition

• Hydrolysis of peptide bonds
• Ion exchange chromatography
• Labeling with ninhydrin
• Analysis with spectrophotometry

Edman Method

• The N-terminal end of the protein is labeled.
• Phenyl-isothiocyanate is used to cleave the peptide bond under mild conditions.

Secondary Structures

• Secondary architecture refers to the local spatial arrangement of a polypeptide's backbone.
• α helix & β sheet are two common regular arrangements.
  • α helix: stabilized by hydrogen bonds between nearby residues.
  • β sheet: stabilized by hydrogen bonds between adjacent segments that may be distant.
• Irregular arrangement of the polypeptide chain is called the random coil.

Secondary Structure Details

• Localized arrangements of adjacent amino acids are formed as the polypeptide chain folds, and produce regular arrangements.
• It consists of α-helices, β-pleated sheets, β-bends (reverse turns), non-repetitive structures, and super-secondary structures (motifs).
• The amide group is rigid and planar, so rotation about the C-N bond is not feasible.
• Rotation can occur about the N-Cα and Cα-C bonds.
• A trans configuration is more stable than a cis configuration for R groups at Cα.

The α Helix

• The alpha-helix is the most common polypeptide helix in nature.
• Keratin (fibrous protein) and myoglobin (globular protein) contain alpha-helices.
• The helical backbone is held together by hydrogen bonds between the backbone amides of amino acids n and n+4.
• Peptide bonds align roughly parallel to the helical axis.
• A right-handed helix has 3.6 residues (5.4 Å) per turn.
• Side chains point outward and are roughly perpendicular to the helical axis.
• The helix's inner diameter is about 4-5 Å, which is too small for anything to fit inside.
• The helix's outer diameter is 10-12 Å.
• The outer diameter fits well into the major groove of dsDNA.
• Residues 1 and 8 align nicely on top of each other.

Sequence Affects Alpha Helix Stability

• Not all polypeptide sequences form α-helical structures.
• Small hydrophobic residues like alanine (Ala) and leucine (Leu) are strong helix formers.
• Proline (Pro) acts as a helix breaker due to the impossibility of rotation around the N-Cα bond.
• Glycine (Gly) acts as a helix breaker because the tiny R-group supports other conformations.
• Charged amino acids (glutamate, histidine, lysine) disrupt the helix structure.
• A large quantity of bulky side chain-bearing amino acids e.g. tryptophan and isoleucine can interfere with the structure.
• Attractive or repulsive interactions between side chains 3–4 amino acids apart can affect formation.

Beta Sheets

• Beta sheets are commonly formed with two or more polypeptides that are side by side.
• All peptide components participate in hydrogen bonding.
• The planarity of the peptide bond and tetrahedral geometry of the α-carbon creates a pleated sheet-like structure.
• The sheet-like backbone is held together by hydrogen bonds between the backbone amides in different strands.
• Side chains project out of the sheet, alternating in the up and down directions.

Parallel and Antiparallel Beta Sheets

• Parallel or antiparallel orientations of two chains within a sheet are possible.
• Parallel β sheets have strands running in the same direction with bent H-bonds (weaker).
• Antiparallel β sheets have strands running in opposite directions with linear H-bonds (stronger).

Beta Turns

• Beta turns (β-bends) occur where β sheets change direction.
• Beta turns contribute to the globular shape of proteins.
• Turns are stabilized by a hydrogen bond from a carbonyl oxygen to amide proton three residues down the sequence.
• Proline and glycine are frequently found in β turns.
• Beta turns often promote the formation of antiparallel beta sheets, and occur at protein surfaces.
• Involve four successive amino acid residues.

Non-Repetitive Structures

• A considerable a large part of a globular protein's structure may show irregular or unique features.
• They include coils and loops.
• These nonrepetitive secondary structures are not random, but have less regular structure than those of alpha-helices and beta-sheets.

Super Secondary Structures (Motifs)

• Globular proteins combine secondary structure elements such as alpha-helices, beta-sheets, and coils.
• These structural elements create geometric patterns or motifs, connected with loop regions.
• Motifs include beta-alpha-beta, Greek key, beta-meander, and beta-barrel motifs.

Tertiary Structure

• The tertiary structure is a polypeptide's three-dimensional conformation.
• Common characteristics of protein tertiary structure reveal the biological functions and evolutionary origins of proteins.
• The function of a protein requires its tertiary structure, and disrupts activity when disrupted.

Domains

• Polypeptide chains with more than 200 residues typically fold into two or more globular clusters called domains.
• Domains contribute to the functional and 3D structure of proteins.
• Domains have specific functions, such as small molecule binding.
• Domains are structurally independent units with characteristics that resemble small globular proteins.

Interactions Stabilizing Tertiary Structure

• The final shape is determined by bonding interactions between the "side chains" on the amino acids.
• These include hydrogen bonds, ionic bonds, disulphide bridges, and hydrophobic interactions.

Formation of Disulfide Bonds

• Performic acid can break down S-S bonds.
• Non-polar amino acid side chains are typically located in the interior of the polypeptide chain in a reverse arrangement within lipids.

Determination of Tertiary Structure

• Methods used to determine protein structures include:
  • X-ray crystallography
  • Nuclear Magnetic Resonance studies
• Atomic coordinates of most structures are deposited in the Protein Data Bank (PDB).
• Protein data banks allow comparing and analyzing varied protein structures.

Protein Folding

• Interactions of side chains determine how a protein will fold into a three dimensional structure.
• Primary structures form secondary structures which then stabilizes buy the formation of tertiary structures.
• The peptide achieves its fully folded, native (functional) form in the final stage.

Denaturation of Proteins

• When proteins denature, they unfold without hydrolyzing peptide bonds.
• Denaturation can result from heat, organic solvents, strong acids and bases, detergents, and heavy metal ions (Pb).
• Denaturation can be reversible in the right conditions, however, most proteins will not regain their structure and will precipitate.

Chaperons

• For many proteins, folding is a facilitated process that requires specialized proteins called molecular chaperones or heat shock proteins.
• Molecular chaperones interact with polypeptides at different stages of folding.
• Some chaperones bind to hydrophobic regions to prevent folding until synthesis is complete, while others form cage-like structures.
• Partially folded proteins enter the cavity of the chaperons, folds, and are subsequently released.

Quaternary Structure

• Some proteins are composed of two or more polypeptide chains (subunits).
• Quaternary structure refers to the arrangement of these polypeptide chains (subunits).
• Subunits are held together by non-covalent interactions like ionic bonds, hydrogen bonds, and hydrophobic bonds.

Protein Misfolding

• Misfolded proteins can form intracellular or extracellular aggregates, which are associated with a number of diseases.

Amyloid Disease

• Amyloid plaques are associated with the development of Alzheimer's disease.
• Amyloid Beta is an extracellular peptide containing 40-42 amino acids.
• The peptide, when aggregated in a beta-pleated sheet configuration, is toxic.

Prion Disease

• Diseases caused by prion aggregates.

Test Questions and Answers

• Which of the following are known as helix breakers?
  • a) Proline and glycine
• Which of the following bonds does performic acid used to break in proteins? (TUS 2008)
  • D. Disulfide bond
• A structure that has hydrogen bonds between polypeptide chains arranged side by side is a
  • c) ẞ-pleated sheets
• Which of the following disease is seen as a result of accumulation of misfolded protein in tissue? (TUS 2008)
  • E. Amyloidose