Protein Structure and Function

Define Amino acids

monomeric building blocks of proteins. Small organic molecule containing an amino group and a carboxyl group.

Define peptide

a short chain of amino acids linked by peptide bonds.

Define polypeptide

A longer chain of amino acids joined via peptide bonds.

Define primary structure

The full amino acid sequence of a protein, where amino acids are linked together by covalent peptide bonds to form the polypeptide chain.

Define secondary structure

The local interactions between stretches of a polypeptide chain, forming structures including α-helices and β-pleated sheets.

• α-Helix: A coiled structure stabilized by hydrogen bonds.

• β-Sheet: A sheet-like structure formed by hydrogen bonds between strands.

• Turns/Loops: Non-regular structures that connect secondary structural elements.

Define tertiary structure

Folding of the α-helices and β-pleated sheets into a 3D structure

Define quaternary structure

A protein structure consisting of several tertiary structure subunits.

Define hydrogen bonds

Bonds between polar groups, critical for stabilizing secondary, tertiary, and quaternary structures

Define disulfide bonds

Covalent bonds between cysteine residues that stabilize the protein’s tertiary or quaternary structure

Define electrostatic interactions

Interactions between charged amino acid side chains (e.g., lysine, glutamate)

Define van de waals forces

Weak attractions between atoms in close proximity, contributing to

protein stability

Define molecular chaperone

proteins that assist during protein folding, preventing misfolding and the formation of aggregates.

Define native state

The fully folded, functional form of a protein

Define protein denaturation

the disruption and possible destruction of both the secondary and

tertiary structures. Depending on the type of disruption it can be reversible (chemical disruption) or irreversible (heat).

Define protein aggregation

Clustering of misfolded proteins, often associated with the pathology of diseases such as Alzheimer’s Disease.

Define protein domain

a conserved part of a given protein sequence and tertiary structure that can function and exist independently of the rest of the protein chain. Usually, they are responsible for a particular function or interaction

Define entropy

is a concept from thermodynamics that measures the degree of disorder or randomness in a system. In relation to protein folding, it is a measure of the number of possible ways to arrange the protein

6 functions of proteins

• Structural proteins

• Secreted structural proteins

• Molecular motors

• Intracellular proteins

• Secreted proteins

• Membrane bound proteins

Describe structural proteins

Define intracellular proteins

Cytosolic Signalling molecules- transfer signals from the plasma membrane to the nucleus

Example of secreted structural proteins

Collagen- cartilage

Example of molecular motors

DNA helicase – opens the DNA double helix during DNA synthesis\

Examples of membrane bound proteins

Receptors – recognition sites for specific molecules that transmit signals from outside to inside the cell

Mechanisms of protein variation

• Single nucleotide polymorphisms (SNPs)

• Alternative splicing

• Post-translational modifications

Amino acid structure

Define polar and non-polar

Polar = an unequal sharing of electrons between atoms

non-polar = an equal sharing of electrons between atoms

Where do essential amino acids come from?

come from food

How is a peptide bond made?

• Amino acids are held together by peptide bonds generated by ribosomal peptidyl transferase

• Peptide bonds are formed by a condensation reaction to form a covalent bond

• The amino acids are joined in a genetically determined sequence

Is the amino group positive or negative?

Positive

Is the carboxyl group positive or negative?

Negative

True or false - A single polypeptide can only have one secondary structure

false

B pleated sheet - parallel and anti-parallel

Describe Alpha-helix

• Polypeptide backbone folded into a spiral (5-20aa in length)

• Shape forms due to hydrogen bonds forming between carbonyl groups and an amino group 4aa along the chain

• 3.6 amino acids per turn

• Side chains face outwards

Describe interactions between the phospholipid bilayer and alpha helix

• Hydrophobic side chains interact with the hydrophobic hydrocarbon tails of the phospholipids

• The hydrophilic backbone is shielded from the lipid environment of the membrane

• Hydrophilic parts of the backbone form hydrogen bonds between carbonyl groups and amino groups

Describe beta pleated sheet

• flat structure (sheet)

• laterally packed β strands

• each β strand 5-8 amino acids

• Shape forms due to hydrogen bonds forming between carbonyl groups and an amino group of a neighbouring chain

• side chains extend above and below β-sheet

3 other secondary structures

1. Turns

2. Loops

3. Random coils

Describe turns

are 3-5 amino acids long forming a sharp bend redirecting polypeptide backbone

Describe loops

have hydrophilic residues and are found on the surface of proteins

Describe random coils

are polypeptide chains with random configuration

3 types of tertiary structure

1. Myoglobin

2. TNF alpha

3. Barrels

Describe barrels

first and last strand form hydrogen bonds forming a barrel

Describe beta barrells

Beta barrels often found in bacterial membrane proteins and some proteins in human mitochondria; they often allow ions to diffuse across membranes.

• 2 or 3 α-helices wind around each other forming a coiled coil.

• Hydrophobic amino acids line up together where the helices meet. This provides structural strength to the protein.

• Examples are keratin and collagen

Side chain interactions

• The peptide backbone is very flexible and allows rotation and folding.

• This is stabilised by weak non-covalent bonds (hydrogen bonds when a H sandwiched between 2 electron attracting atoms), electrostatic reactions (between charged groups) and van der Waals forces (when molecules (atoms) are very close to each other (due to fluctuating electrical charge)).

• They are all weak bonds but when many occur simultaneously that can hold the structure together. Stability of the protein is determined by how many of these bonds form.

Describe disulphide bonds

• Tertiary structure can be stabilised with covalent cross linkages. The most common is a disulfide bond between cysteines that are next to each other in the folded structure. They are formed by an enzyme in the endoplasmic reticulum.

• They give strength but do not alter the shape.

• Typically present in excreted proteins

• Di-sulphide bonds are covalent bonds often seen in extracellular proteins e.g in saliva, they don’t often occur in cytosolic proteins due to reducing agents in the cytosol.

• Needed where proteins may face pH changes.

• Performed by enzymes in the endoplasmic reticulum - protein disulphide isomerase family (PDI).

Describe protein domains

• Small proteins may fold into a single globular molecule.

• Larger polypeptides may fold into two or more domains, each with their own 3D shape, separated by short linker chains.

• Each domain of a protein may play a distinctive role in its function e.g. spanning the plasma membrane (receptor) or DNA-binding (transcription factors)

• Domains often have a distinctive sequence motifs that enable their function to be predicted.

How is the quaternary structure formed?

hydrophobic interaction

What does haemoglobi consist of?

• Haemoglobin is formed of 2 alpha subunits and 2 beta subunits

• Binding of oxygen to one of the non-protein haem groups causes a conformational change that allow oxygen to bind more readily to the other heme groups

negatively charge carboxyl

• Glutamic acid

• Aspartic acid

Positively charged n group

Lysine

Arginine

histidine

Protein denaturation

• Proteins can be unfolded (denatured) by adding solvents (e.g. acids, bases) that disrupt the non-covalent interactions.

• The protein loses its secondary and tertiary structures.

• If the solvent is removed the protein will refold (renature) to the same shape as it started with.

• Heat disrupts non-covalent interactions denaturing the albumin proteins which then aggregate and form new disulphide bonds altering the structure.

What is a folding funnel?

• Proteins spontaneously fold into a three-dimensional conformation of the lowest free energy.

• The folding process is energetically favorable. It releases heat and increases the disorder in the universe

Define energy and entropy

• Entropy is a measure of disorder or randomness (number of possible ways to arrange the protein)

• Energy is the potential energy available (due to movement, rotations and formation of bonds between atoms)

Describe molecular chaperones

• Molecular chaperones – proteins that bind to the partially folded polypeptide chains and assist them in folding

• Two classes include heat shock proteins and chaperonins

• Chaperones: do not change the final 3D structure (native structure) but they do speed up the folding process, prevent protein aggregation and reduce non-productive intermediates

• Called heat shock proteins as their expression is increased at elevated temperatures

• Molecular chaperones are needed to help keep some proteins from misfolding on their way down the folding funnel, preventing them from straying into deep free energy wells (misfolded states).

• Chaperones will bind the unfolded protein as it is made, shielding hydrophobic amino acids during the folding process.

Describe Post translational modifications (PTM)

• A processing event resulting from proteolytic cleavage or the covalent addition of a modifying group.

• Over 200 PTMs have been characterised.

• PTMs modulate the function of most proteins by altering their activity state, localization, turnover, and interactions with other proteins.

Describe phosphorylation

Addition of a phosphate to specific amino acids regulating the activity of the protein

Describe glycosylation

Addition of carbohydrates to specific sites on the protein

N-linked glycans attached to a nitrogen of asparagine or arginine side-chains. N-linked glycosylation requires participation of a special lipid called dolichol phosphate.

O-linked glycans attached to the hydroxyl oxygen of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline side-chains, or to oxygens on lipids such as ceramide

phosphoglycans linked through the phosphate of a phosphoserine.

Describe ubiquitination

Addition of ubiquitin can target the protein for destruction by the proteasome

Describe cystic fibrosis

• Inherited disease of the secretory glands

• DNA mutations that alter the conformation of a Cl- channel

• Pulmonary obstruction due to thickened mucus in the small airways causing recurrent bacterial infections

• Over 800 mutations detected

• 70% of patients share mutation at aa508

CFTR

• All symptoms due to a single DNA mutation which causes the deletion of the amino acid Phenylalanine at position 508

• Mutant CTFR protein becomes stuck in the endoplasmic reticulum leading to reduced chloride conductance out of the cells

• ATC and ATT both code for isoleucine but the phenylalanine becomes deleted.

• Lumacaftor is a small molecule drugs that can bind to and stabilise the protein of the Phe508 deletion gene mutation, meaning more CFTR can be trafficked to the cell surface.

• AD accounts for 60% to 70% of dementia diagnoses

• Most cases are sporadic, caused by a combination of genetic, lifestyle and environmental factors, with <1% of cases classed as familial AD, which is inherited

• The disease is characterised by progressive memory loss and cognitive decline

• Misfolding of amyloid beta causing amyloid beta plaques and tau aggregates causing intracellular neurofibrillary tangles (NFTs)

Misfolded proteins cause neuronal cell death

• The build-up of amyloid beta plaques and NFTs cause irreversible neuronal cell death

• Initial damage is in the areas of the brain associated with memory formation

• Over time the damage becomes more widespread leading to shrinkage of the brain

• Neuronal cell death is thought to be via necroptosis, a type of regulated cell death similar to necrosis.