The Patient: Proteins

A typical human cell has as many as How many different proteins does a hu- 10,000 different proteins. They carry out man cell have? What do proteins do? most of the activities of the cell performing many different functions. What are the functions of proteins? Signalling Transport Catalysis Movement Structure Regulation Defence What are some different types of proteins? - Structural proteins - Transcription factors - Enzymes - Membrane transporter/channels - Storage and transport proteins - Signalling molecules and receptors - Antibodies - Motor proteins Keratins and collagens give mechanical support and elasticity to cells, tissues, and organs. What are some examples of structural proteins and what do they do? Cytoskeletal proteins like actin and microtubules give the cell its three-dimensional structure and allow movement of organelles in the cell. What do transcription factors do? regulate cell activities by switching genes on and off. Involved in gene expression and alters structure of DNA, such as bending DNA helix. What do enzymes do? accelerate the rate of metabolic reactions. determine what molecules enter and leave the cell, allow selective permeability of membranes. An example is potassium ion channels, which have 4 mul1 / 12 tipass subunits, and a central selectiviWhat do membrane transporters do and ty pore (which only lets potassium ions what is an example? pass through). store essential molecules and transport them across the body. For example, ferritin which has 24 subunits and can transWhat do storage and transport proteins port up to 4500 iron atoms, and haemodo, and what are some examples? globin which has 4 subunits (4 haem groups with iron attached), can transport 4 oxygen molecules. communication between and within cell, binding and regulation of other proteins. An example is insulin (6 subunits coorWhat do signalling molecules and recep- dinated by a central zinc cation) and the tors do, and what is an example? insulin receptor which is specific. These can create a signalling pathway - cascade, to produce a response. In this case, this is glucose uptake. What do antibodies do and what is an example? immune response to invasion by viruses, bacteria, allergens, and other toxins (antigens). For example, immunoglobulin G has 4 peptide chains - 2 heavy chains and 2 light chains. There are constant regions and then there are variable regions which are specific to molecules. transport of molecules and organelles across the cell, membrane fusion/fission, endo/exocytosis, cell contraction/motility. For example, kinesin uses energy to What do motor proteins do and what are move along a microtubule, and myosin some examples? slides along an actin filament. The common structure of motor proteins is a catalytic core (head), a neck and a coiled-coil. DNA undergoes transcription to form and synthesise pre-RNA. Splicing of introns created mRNA which is exported 2 / 12 out of the nucleus and translated using tRNA (according to the genetic code) into What is the central dogma of molecular amino acids (polypeptide chains). Foldbiology (from genes to protein)? ing of the amino acid chain creates a protein. Have a central alpha carbon, amino group, carboxyl group, hydrogen and a What is the structure of an amino acid? side chain (R group). They are charged at physiological pH values. What are the different types of side chains of amino acids? - Polar (hydrophilic) charged or non-charged - Non-polar (hydrophobic) How does amino acid distribution in a protein differ? Amino acid distribution depends on the protein environment. Cytosolic proteins have mostly polar, hyHow are amino acids distributed in cy- drophilic amino acids on the surface, with tosolic proteins? mostly hydrophobic amino acids on the inside. Membrane proteins have mostly hyHow are amino acids distributed in memdrophobic amino acids on the surface, brane proteins? whereas polar amino acids on the inside. - Glycine has a hydrogen atom side chain; it is a very small molecule which can create local flexibility. - Proline has a side chain linked to the amino group which creates a ring, it therefore has a very rigid structure. It is What are some examples of non-polar called a helix breaker because it induces amino acids? local bends into the sequence. - Cysteines can form disulphide bonds (they have a SH group). These are very strong covalent bonds that can strengthen the protein. There are 7 other non-polar amino acids. Can be charged (lysine, arginine and histidine which are positive, aspartic acid 3 / 12 and glutamic acid which are negative) or non-charged (serine, threonine, tyrosine, What are some examples of polar amino asparagine, glutamine) at physiological acids? pH levels. Involved in ionic bonds and enzymes. Formed by post-translational modifications at the Golgi. They can have different functions based on their modification. How are modified amino acids formed? For example, hydroxylysine (involved in What are some examples of modified stabilisation of collagen) can be formed amino acids? from lysine (addition of a hydroxy group) and phosphoserine can be formed from serine (addition of a phosphate group). How are amino acids joined to form a protein? What is one of the largest polypeptides in humans? Amino acids are linked by peptide (amide) bonds, between the carboxyl and amino group. This creates the linear polypeptide chain. Proteins usually contain 50 or more amino acids which start with a N-terminus and end with a C-terminus. One of the largest polypeptides in humans is the muscle protein titin (27,000 amino acids). What are amino acid units called when they are in a polypeptide chain? Residues Almost all peptide bonds are in trans conformation, which is very stable as side chains can have steric repulsion if they What conformation of peptide bond is are next to each other. more stable? What amino acid is an ex- However, for proline, the two configuraception to this? tions have similar energies, due to the side chain ring connecting to the nitrogen of the amino group. The two conformations are the same. Peptide bonds have a partial double bond character and can exist as two resonance structures. 4 / 12 There is no rotation around the peptide How can peptide bonds have resonance bonds, only around the alpha carbon. structures? - Read from the amino terminus to the carboxyl terminus - Encoded by the nucleotide sequence of What is the primary structure of a pro- DNA tein? - Unique characteristic of the protein - Alteration of even a single amino acid can cause disease - Stabilised by peptide bonds Result of local folding (rearrangement of residues) of the polypeptide chain and formation of hydrogen bonds between C=O and N-H groups in the backbone. What is the secondary structure of a proA hydrogen bond is formed between the tein? oxygen on the carboxyl group and the hydrogen on the nitrogen group. This can create an alpha helix, beta sheet, beta turns, and loops. - Alpha helix - 310 helix - Pi helix - Beta sheet and turns - Loops What are the different types of secondary structures? 5 / 12 What is the alpha helix structure? The alpha helix is the most common type of helix. It has 3.6 residues (amino acid molecules) per turn. Hydrogen bonds between the carbonyl group (C=O) of residue X and the N-H group of residue X+4. It has a clockwise (right-handed) rotation. It has a dipole moment corresponding to a partial positive charge at the N-terminus, and a partial negative charge at the C-terminus. Side chains extend radially from the alpha helix core. What is the 310 helix? The 310 helix occurs mostly at the ends of alpha helices. There are 3 residues per turn and hydrogen bonds form between residue X and X+3. It is called 310 because the hydrogen bond closes a loop of 10 atoms, containing 3 residues. They are more flexible regions than alpha helices. What is the pi helix? The pi helix is very rare and occurs mostly at the ends of alpha helices. There are 4.4 residues per turn and there are hydrogen bonds between residue X and X+5. What is the beta sheet structure? In a beta sheet, peptide chains are fully extended in a pleated shape. It is stabilised by hydrogen bonds between C=O and N-H groups of the backbone (flanking regions). Side chains project above and below the sheet. 6 / 12 What are parallel beta sheets? two polypeptide strands (flanking regions) running in the same direction. There are non-linear hydrogen bonds which make it less stable. Parallel beta sheets with less than five parallel strands are rare. What are antiparallel beta sheets? two polypeptide strands run in opposite directions. There are well-oriented hydrogen bonds which make it strong and stable. What are beta turns? Beta turns (reverse turns) reverse the direction of the polypeptide chain. In standard turns, the C=O group of residue X is bonded to the N-H group of residue X+3. Most turns contain at least one residue of glycine or proline. Beta turns are stabilised by hydrogen bond. It stabilises antiparallel beta sheets. What are loops? Loops are various lengths, irregular shape and very flexible. They connect other elements of secondary structure. Combinations of a few secondary structure elements with a specific geometWhat is the super secondary structure? ric arrangement. These often perform a What are some examples? particular function such as binding metal ions. For example, a helix-loop-helix motif and zinc finger motif. What is the tertiary structure of a protein? What is an example? Overall three-dimensional structure of a protein molecule. It is the spatial relationship of the secondary structure elements to one another. For example, a lysozyme or an alpha+beta fold (five helices that are three alpha and two intermediate alpha or 310, and three stranded antiparallel beta sheets). 7 / 12 This structure is stabilised by bonds and interactions between the side chains, such as hydrogen bonds, ionic bonds, How is the tertiary structure stabilised? disulfide bridge between cysteines, hydrophobic (nonpolar) and van der Waals interactions. What is the quaternary structure of a protein? What is an example? Spatial arrangement of subunits in a protein composed of more than one polypeptide chain. It is stabilised by interactions between side chains (like tertiary structure). Subunits can be identical or different. For example, neurotrophin-4 has two identical subunits and haemoglobin has two alpha and two beta subunits. What are protein domains? spatially distinct fragments of a polypeptide chain. Proteins are organised in functional/structural units that may be connected by a flexible segment of polypeptide chain called domains. What is the difference between a domain A single polypeptide chain can form sevand subunit? eral domains but only one subunit. What percentage of eukaryotic proteins have multiple domains? 80% The same domains can appear in more than one protein. many multidomain proteins have arisen during evolution by the fusion of genes What does the domain shuffling theory that encoded separate proteins in an ansuggest? cestor. 8 / 12 How can domains move? Flexible connections between domains allow their movement in relation to each other. They have flexible loops, an example is PcrA DNA helicase which moves to bind to DNA. Domains are not rigid structures; they change their orientation in space. Protein-only enzyme What are the two different types of enApoenzymes have a cofactor which crezymes? ates a holoenzyme. - Prosthetic groups (covalently bound to the protein such as haem in haemogloWhat are some examples of cofactors? bin) - Metal ions (zinc 2+, iron 3+ for example) - Coenzymes (often vitamins) What is the active site? a cleft or groove in the protein, containing residues that bind the substrate and residues that perform the catalytic reaction. The active site is complementary to the substrate in terms of size, shape, charge and hydrophobicity. What is the lock and key model? Proposed by Emil Fischer in 1894 to explain the specificity of enzyme. According to this model, the active site is rigid, and the substrate fits the active site like a key fits a lock. What does the lock and key model not This model cannot explain why some enexplain? zymes act on a large range of substrates. What is the induced fit model? Proposed by Daniel Koshland in 1959. According to this model, the substrate induces a change in the enzyme structure that allow fitting. Enzymes are not rigid but flexible and mould to fit the substrate molecule. This model explains why some enzymes can act on a very large range of substrates. 9 / 12 What are two examples of an enzyme that follows the induced fit model? An example of this model is the enzyme hexokinase which phosphorylates glucose to glucose 6-phosphate. Conformational changes in hexokinase are associated with glucose binding to the active site. Another example is DNA helicase where one of the domains of PcrA DNA helicase rotates during binding to DNA. Phosphofructokinase 2 (PFK2) is a bifunctional enzyme with 2 domains: kiWhat is an example of an enzyme that nase domain and phosphatase domain. has different functions due to multiple They have opposite reactions, the kidomains? nase domain adds a phosphate, whereas the phosphatase domain can remove a phosphate. Why do some enzymes only produce one isomer? Some enzymes produce only one isomer, because the molecule they catalyse are quite symmetrical and it always attaches at the same place - three-point attachment model. Some residues in the active site can only bind to certain groups, so the substrate must bind in the same place/direction each time. What does the three-point attachment model suggest? Two chemically identical groups in the substrate can be distinguished by an enzyme because the substrate binds in the active site at three or more points. How can diseases be caused by proteins? Mutations in protein sequence may cause changes in protein structure leading to disease. Normal red blood cells live 120 days in the bloodstream, but sickled cells die after 10-20 days. Sickled cells cause Why is sickle cell anaemia dangerous? blockages (they aggregate) that deprive organs and tissues of oxygen. There are not enough red blood cells in the 10 / 12 bloodstream to carry oxygen resulting in anaemia. How are sickle cells different in their structure from red blood cells? Single nucleotide substitution in the beta chain of haemoglobin. GAG in normal Hb is changed to GTG in HbS (sickle cell haemoglobin). This changes glutamate (hydrophilic) to valine (hydrophobic). This changes the residue from a positive polar molecule to one that is non-polar. - In deoxygenated HbS, a hydrophobic pocket with phenylalanine and leucine is exposed on the surface. Val on one molecule is attracted to Phe and Leu on How does the amino acid substitution another HbS molecule by hydrophobic cause a distorted shape and aggrega- interactions. tion in red blood cells? - Many HbS molecules aggregate into HbS fibres. - These fibres cause red blood cells to become stiff and assume a distorted shape that can block blood vessels. What are the symptoms of sickle cell anaemia? The patient gets frequent episodes of severe pain, organ damage (due to oxygen deprivation), serious infections, or even stroke. Most common lipid storage disease caused by mutations in the enzyme acid-beta-glucosidase that breaks down What is Gaucher disease and what is it glucosylceramide into glucose and ceramide. caused by? Intracellular accumulation of glucosylceramide occurs, particularly in the spleen, liver, and bone marrow. Some of the known mutations in acid-beta-glucosidase are in a surface How does the mutation in Gaucher disloop that forms part of the active site ease occur? of the enzyme. They appear to stabilise the conformation that limits access to the 11 / 12 active site and therefore these mutations reduce enzyme catalytic activity. 12 / 12