Notes of a CUHK Medic: MEDF 1012 Lecture 1

Notes of a CUHK Medic: MEDF 1012 LECTURE 1 Proteins: The Biomolecules of Life

Christopher Chan

Creative Commons 2.0 – Author: Christopher Chan

LECTURE 1: Proteins – The Biocatalysts of Life

An Introduction to Proteins The Role of Proteins in Life • Proteins are a class of highly vital molecules for organic life

o They carry out and/or assist in causing chemical reactions that are involved in processes of life, such as respiration

o Haemoglobin is a protein that is extremely essential to the process of respiration, as it is the main oxygen-‐carrying component in erythrocytes

The Structure of Proteins – An Overview • Proteins are chains of amino acids folded and collated in stable conformations (physical transformations)

o The chains of amino acids in a protein are usually arranged in different levels of order • In increasing order of size, they are:

o Primary Structure § A basic amino acid sequence of the protein, always counted from the N-‐terminal to the C-‐

terminal o Secondary Structure

§ Regular, larger structures consisting of multiple primary structures held together by hydrogen bonds

§ May form Alpha-‐helices or Beta-‐sheets/strands § Proteins may contain one type of Secondary Structure more than another

o Tertiary Structure § Groups of secondary structures (Alpha or Beta) held together by side-‐chain interactions

such as: • Hydrophobic interactions • Hydrogen bonds • Disulphate bonds

o Quaternary Structure § Bigger groups containing one or more Tertiary Structures and additional subunits and

peptide sequences – the final form of a folded protein § Allows for regulatory sites that modulate protein function by allosteric factors

• E.g. Oxygen binding causes quaternary structure of haemoglobin to change • This because ionic interaction between secondary structures are broken • Quaternary structure allows oxygen binding to be regulated

• The surface of a protein is also usually covered by polar amino acid molecules; variations in surface amino acids can sometimes have disastrous effects

The Sickle Cell Mutation • The mutation of codon 6 in the β-‐globin chain of a haemoglobin molecule changes glutamic acid into valine (hydrophobic amino acid)

o This substitution of a hydrophobic amino acid molecule in the β2 chain creates a knob on the surface of deoxygenated haemoglobin

o This fits into a hydrophobic binding site in a β1 chain of a different molecule o Once these different molecules interact, the haemoglobin molecules will come together, forcing the

erythrocyte into a sickle cell shape Fitting Form to Function • The shape of a protein is largely determined by its function

o Haemoglobin has its distinctive shape with pockets to enable it to carry Oxygen more effectively o Enzymes in the digestive tract have their shapes to aid in catalyzing decomposition reactions of the

digestive system


The Building Blocks of Proteins – Amino Acids • Amino acids are the monomer units from which peptide sequences (and consequently, proteins) are formed

o Each protein contains different Amino Acids • In the human body, there are up to 20 different amino acids that form various proteins • Each Amino Acid Molecule consists of:

o A chiral centre Carbon o A basic Amino group o An acidic Carboxyl group o A Hydrogen atom o An R group side chain that varies depending on the Amino Acid

• Because of the chiral centre, optical isomers exist; • In the human body, only the L isomer exists Typical Structure of an Amino Acid: How Different Side Chains form Different Amino Acids:


Polarity of Amino Acids:


Identifying Proteins and their Constituents: The Absorbance Spectra • All chemicals take in certain wavelengths of UV light • The amount of light absorbed by these chemicals is then plotted against increasing wavelength of UV light

o This is known as the chemicals’ Absorbance Spectra • By contrasting the spectra of an unknown protein versus spectra of different amino acids, its composition can be identified through the identification of its individual constituents, as shown below:

• It can be clearly seen from the above graph, that tryptophan and tyrosine are both constituent amino acids of the protein albumin, because of the similar peak at around wavelength 270 nm • Constituents and other proteins can be identified in a similar manner


The Ionization of Amino Acids The Physiological Significance of Amino Acid Molecules • The ionization states of Amino Acids are significant because different charges of amino acids will affect the charges of the proteins they form

o This may cause the function of the protein to be altered in varying ways • At different pH levels, the charge bearing states of enzymes and substrates will vary, affecting the effectiveness of the enzymes

o (Hence, most enzymes will have an optimal working pH) • At different levels of pH, there will be different levels of charge in the amino acids

o At low pH levels (0-‐4), both the amine group and the carboxyl group are protonated (in possession of a H+ ion), leading to a net positive charge

o At medium pH levels (4-‐8), the carboxyl group loses its proton while the amine group still holds theirs, putting the amino acid into an amphoteric state, in a zwitterionic form, with no net charge

§ However, the amino acid is still technically a charged molecule o At high pH levels (8-‐14), neither the amine group nor the carboxyl group have protons, leading to a

net negative charge • The presence of charge at all pH levels makes it necessary for proteins and amino acids to travel through transporters across membranes


The Ionization of Amino Acids The Significance of the pKa and pH Balance • The status of ionization of an amino acid can generally be determined from the relative magnitudes of the pH and pKa of the amino acid molecule

o pH < pKa = amino acid is protonated o pH > pKa = amino acid is deprotonated

• Titration curves can be used to find out the pKa and pH balance, and hence, find out the charge/ionization status of an amino acid, as shown below

• From the above, it should be noted that

o The first point (from left to right) on each graph shows the point of ionization of the carboxyl group, with an overall positive charge

o The mid point (isoionic point) on each graph shows the point where there is no net charge o The last point on the graph shows the point from which there will be a net negative charge

• At different points of ionization (different points of the pH – pKa balance), each amino acid will have a different dominant form, as shown below:


The Bonding and Structure of Amino Acids and Proteins – An In-‐Depth Analysis Formation of Peptide Bonds • The diagram below shows the process by which Peptide Bonds are formed:


The Planarity of Peptide Bonds • The partial (40%) double bond character found in peptide bonds causes resonance, which in turn causes the planarity of the peptide bonds

• As can be seen from the diagram above, except for the R group and Hydrogen atom attached to the chiral carbon, all components are in the same plane


Further Bonding and Structure The Flexibility of the Polypeptide Chain

• As seen from the diagram above, the bonds stemming from the carbon that is NOT part of the peptide bond are able to rotate

o This gives the chain a certain degree of flexibility to twist into various shapes, as illustrated below • However, over-‐rotation may induce forces of extreme attraction or repulsion

o Hence, there are conformations that are more favourable, as illustrated by the following Ramachandran Diagram


Conformations and Higher Order Structures The Ramachandran Diagram • This diagram is a visual representation to illustrate the likelihoods of different types of conformation of amino acid chains and secondary structures

o Conformations with a high likelihood of occurring exist within zones of red o Conformations with relatively lower likelihood of occurring exist within zones of green o Colourless patches indicate likelihoods close to or equivalent to zero

• As illustrated, the types of secondary structures in order of increasing likelihood are:

o Beta (β) strands o Right-‐handed Alpha (α) helix o Left-‐handed Alpha (α) helix (very rare)

• Conformations where both bond rotations have a magnitude of +90° are disfavored • Secondary structures are formed by conformed, polypeptide sequences of sufficient lengths Secondary Structures Beta Sheets • Beta (β) sheets are formed when two Beta (β) strands are aligned in either manner shown below


Secondary Structures (cont’d) Alpha Helices • Alpha helices are formed by highly convoluted polypeptide sequences • The CO group of monomer unit n bonds with the NH group of monomer unit n+4 in an Alpha (α) helix, as shown below

• Refer to Page 1 for information on Tertiary and Quaternary Structures


Special Proteins and Their Significance in the Human Body • Proteins have the ability to carry small molecules including organic and inorganic compounds, or simple molecules like oxygen

o This is particularly significant to the human body as its only form of oxygen transport relies on the protein haemoglobin

Carbohydrate-‐carrying Proteins • These two types of proteins bond to carbohydrates in different ways, creating different characteristics • Only certain types of amino acids can bond with carbohydrates, as illustrated below Glycoproteins • These proteins hold oligosaccharide chains that are attached to the polypeptide side chains as branches • They are integral membrane proteins that play a role in intercellular interactions Proteoglycans • These proteins are heavily glycosylated, containing several straight glycosaminoglycan chains that are directly covalently attached to a core protein • These proteins occur mainly in connective tissue like ligaments or cartilage


Special Proteins and Their Significance in the Human Body (cont’d) Protein Family of Myoglobin and Haemoglobin • They possess similar (but not identical) structure; because of this, they also share similar (but not identical) functions

o Myoglobin stores Oxygen in the skeletal and cardiac muscles as a storage protein o Haemoglobin transports Oxygen around the human body

• Myoglobin binds and releases oxygen at relatively constant rates • Haemoglobin is slow to bind initially, but causes a rapid increase in rate once the first subunit is bound to oxygen

o This is because ionic interactions between secondary structures α and β are broken by the first oxygen molecule, making it possible for the haemoglobin molecule to open up more, opening up more spaces for other oxygen molecules

o This initial “barrier” allows for regulation of oxygen affinity o Affinity is lowered in CO2 rich environments, releasing oxygen to surrounding tissue (Bohr effect)

2,3-‐Biphosphoglycerate (BPG) Affinity • BPG is a chemical that prompts the release of oxygen from haemoglobin by decreasing oxygen affinity • The affinity of haemoglobin to BG is dependent on spatial distribution and physical changes in form

o In oxygen-‐rich environments, when the haemoglobin is forced open by oxygen, the affinity for BPG is increased

§ This eventually prompts release of oxygen into organs that require it o The relationship to oxygen concentration and BPG affinity is proven below:

• Furthermore, it should be noted that Fetal Haemoglobin has lower affinity for BPG in comparison to that of Maternal Haemoglobin

o This is because during pregnancy, the red blood cells in the maternal bloodstream are needed to release oxygen to the fetal red blood cells through diffusion

o This can only be done when Maternal red blood cells have lower affinity for Oxygen


Prion Proteins Invulnerable Protinaceous Infectious Particles • Prion proteins are pathogens that cannot be destroyed • They are known to cause neurodegenerative diseases that are almost always lethal, and do not have cures • They are membrane-‐anchored glycoproteins • They are already present during embryogenesis, and will continue to exist in adult tissues • Highest expression in CNS, and also in cells of immune system • They may have a role in mediating cell adhesion or signalling, but precise function is unclear • No obvious anatomical or developmental defects in prion-‐deficient mice, except subtle abnormalities in neurotransmission and circadian rhythms

Nature of the Deformity • Mutated prion proteins aggregate as a covalent long chain polymer that is insoluble and sticky • This is due to a change of one α helix into a β sheet

o This change transmits protein signals which sets off an exponential chain reaction, transforming healthy prion proteins into the mutated state

• The misfolded/mutated prion protein may also set off a toxic signal that causes neurodegeneration • Aggregation of these misfolded proteins will create “holes” amongst clusters of neurons, found in the brain, spinal cord, and other areas of the CNS

o This structural and physical damage is what causes the neurodegeneration, causing degradation of physical and mental abilities

§ This ultimately results in death • The picture below shows the mutation caused by infectious prion proteins: Transmission of Disease • Because proteins cannot be “killed”, no conventional treatment will render the proteins non-‐infectious • Prion proteins can be transferred via tissue contact, and even through eating the infected tissue

o This is how bovine spongiform encephalopathy was transmitted from sheep to cows in the UK • The highest risk of infection is created when infected proteins come into contact with areas with high prion protein expression (CNS and other areas with high neuron concentration)

Notes of a CUHK Medic: MEDF 1012 Lecture 1

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