L3 - Proteins

objectives

Understandings:

• Proteins are polymers of 2-amino acids, joined by amide links (also known as peptide bonds).
• Amino acids are amphoteric and can exist as zwitterions, cations and anions.
• Protein structures are diverse and are described at the primary, secondary, tertiary and quaternary levels.
• A protein’s three-dimensional shape determines its role in structural components or in metabolic processes.
• Most enzymes are proteins that act as catalysts by binding specifically to a substrate at the active site.
• As enzyme activity depends on the conformation, it is sensitive to changes in temperature and pH and the presence of heavy metal ions.
• Chromatography separation is based on different physical and chemical principles.

Applications and skills:

• Deduction of the structural formulas of reactants and products in condensation reactions of amino acids, and hydrolysis reactions of peptides.
• Explanation of the solubilities and melting points of amino acids in terms of zwitterions.
• Application of the relationships between charge, pH and isoelectric point for amino acids and proteins.
• Description of the four levels of protein structure, including the origin and types of bonds and interactions involved.
• Deduction and interpretation of graphs of enzyme activity involving changes in substrate concentration, pH and temperature.
• Explanation of the processes of paper chromatography and gel electrophoresis in amino acid and protein separation and identification.

Guidance:

• The names and structural formulas of the amino acids are given in the data booklet in section 33.
• Reference should be made to alpha helix and beta pleated sheet, and to fibrous and globular proteins with examples of each.
• In paper chromatography the use of Rf values and locating agents should be covered.
• In enzyme kinetics Km and Vmax are not required.

Forming Peptides

• Amino acids are joined together by a condensation reaction.
• The amino group (-NH2) from one amino acid loses a hydrogen ion (H+) and the carboxyl group (-COOH) from another amino acid loses a hydroxyl group (-OH-), directly bonding the nitrogen to the carbon in an amide linkage, also known as a peptide bond.
• The other product formed is water (H-OH). The number of water molecules removed is always one less than the number of amino acids joined together.
• When two amino acids are joined, the product is referred to as a dipeptide.
• Three amino acids form a tripeptide; multiple amino acids form a polypeptide.
• It isn't until the polypeptide chain takes on a three-dimensional shape that it can be called a protein.

• When a polypeptide chain is formed, one end of the linear chain still retains the amino group, referred to as the N terminal, while the other retains the carboxyl group, referred to as the C terminal.
• The precise sequence of amino acids is important for the overall structure and function of the protein.

Protein Structure

• Proteins can contain anywhere from 20 amino acids up to 30,000 amino acids and can even include multiple polypeptide chains intricately folded together.
• If a single amino acid is missing or incorrect, it can affect the structure and function of the entire protein.
• The smallest protein to be identified is from the saliva of the Gila monster (a type of venomous lizard native to Mexico) and contains only 20 amino acids.
• The largest protein identified is titin, a protein in human muscle, that has over 30,000 amino acids.
• The structure of proteins is broken down into four levels: primary, secondary, tertiary and quaternary.

Primary (1°) level of structure

• The primary level of structure for proteins is the sequence of amino acid residues bonded together in the polypeptide chain by covalent bonds.
• The sequence is coded by genetic material, and a mistake in the genetic code can result in the wrong amino acid sequence, which may result in a protein that cannot perform its function.
• This sequence can be represented with the short names of the amino acids in sequence and shows no level of intermolecular forces/bonding:

Secondary (2°) level

• The secondary level of structure includes the local coiling or folding of the polypeptide chain.
• When the polypeptide chain is formed, it can take on two different, distinct structures known as an alpha helix (coiled structure) or a beta sheet, also known as a beta pleated sheet (accordion-style folding).
• The structures are held together by Hydrogen Bonds from distant amino acids.

a-Helix

• An alpha helix is a coiling of the backbone of the polypeptide chain, where the R groups extend outward from the spiral coil.
• A typical helix contains around 10 amino acid residues, making about three turns, but can be as short as 4 or as long as 40 amino acid residues.
• An alpha helix is held together by strong Hydrogen Bonds between distant carboxyl and amino groups.

B-Pleated Sheets

• Beta sheets contain alternating linear folds of an extended backbone, making a pleated formation, similar to an accordion or paper fan.
• This structural arrangement allows beta sheets from different, distant parts of the polypeptide chain to align together, held by hydrogen bonds.
• The beta sheets align with opposite orientation, allowing hydrogen bonding to maximise the interaction.
• Beta sheets typically form in sections anywhere from 3 to 10 amino acid residues in length.

Tertiary (3°) level

• The tertiary level of structure includes the three-dimensional globular structure of the protein, using the interactions from the disulfide bridges, alpha helices and beta sheets.
• The polypeptide will fold to also allow for better solubility in an aqueous environment, with the more polar R groups on the exterior and the more nonpolar R groups on the interior of the molecule.

• Additional intermolecular forces of attraction between distant R groups can also influence the shape of the polypeptide.
• Polar R groups, especially those with hydrogen bonding capabilities, can have strong interactions with other polar R groups, while nonpolar R groups will attract other nonpolar R groups.
• For acidic and basic R groups that have undergone ionisation, there can also be electrostatic forces of attraction, which are much stronger than the intermolecular forces of uncharged R groups.

• The tertiary level of structure also includes the presence of disulfide bridges between cysteine amino acid residues, also known as SS bridges or simply disulfide.
• When two amino acids containing a thiol group (-SH) are joined, the sulfur atoms can form a covalent bond through an oxidation reaction that releases two hydrogen ions and two electrons.
• Cysteine, with an R group of -CH2SH, can form a disulfide bridge with another cysteine, as shown in the diagram below.
• Not all cysteine amino acid residues will go on to form disulfide bridges.
• One other amino acid, methionine (R group -CH2CH2SCH3), also contains sulfur, but not a thiol group (-SH), so it is not capable of forming disulfide bridges.
• The disulfide bridges play a role in the final structure of the protein, as the bridges make the molecule twist or bend in order for the distant cysteine residues to come in contact with each other.

Quaternary (4°) level

• The final level of protein structure is the combination of more than one polypeptide chain.
• Some proteins consist of a single chain, while others consist of multiple chains.
• Some proteins even have other species, such as transition metal ions, as part of their structure.
• The forces of attraction holding the separate polypeptide chains together are intermolecular forces, not chemical bonds.
• For example, the protein haemoglobin, which is responsible for carrying oxygen and carbon dioxide in the bloodstream, is made up of four polypeptide chains and four iron ions (Fe2+/Fe3+) held on by a small molecule called haem.
• The four chains, the four molecules of haem and the four iron ions must be folded and combined correctly in order for the protein to function.

Summary