Biochem Midterm I - Oct 11th Flashcards
Describe Proteins
- Covalently linked amino acids in an approx 20 amino acid chain
- AKA polypeptides
- Amino acids connected by peptide bonds between C-N, an Amino-terminal end (NH3) and a carboxyl end (O=C-O)
Describe carbohydrates
- Structure and Composition: Carbohydrates are organic molecules made up of carbon, hydrogen, and oxygen, typically with a hydrogen-to-oxygen ratio of 2:1, resembling that of water (H₂O).
- Types: They are classified into three main categories: monosaccharides (simple sugars), disaccharides (two sugar units), and polysaccharides (long chains of sugar units), each serving different roles in biological processes.
- Functions: Carbohydrates provide energy for living organisms, serve as structural components in cells (like cellulose in plants), and are involved in cell recognition and signalling.
Describe polysaccharides
large, complex carbohydrates composed of long chains of monosaccharide (single sugar) units linked together by glycosidic bonds. These bonds form through dehydration synthesis, resulting in various structures such as starch, glycogen, and cellulose, each serving different functions in living organisms. Their unique configurations contribute to properties like energy storage, structural support, and biological recognition.
Describe esters in the context of lipids
The chemical compounds formed when fatty acids react with alcohol, typically glycerol, through a condensation reaction. This reaction results in the formation of triglycerides, which are the main constituents of body fat in humans and other animals, as well as vegetable fat.
What are the three major types of lipid esters?
- Glycerophospholipid: glycerol ester
- Sphingolipids: sphingosine ester
- Steroids: Derivative of waxes making nonpolar esters w a long chain
Describe the structure of P-glycoprotein
- ATP-binding cassette (ABC)
- Contain two homologous halves, each containing transmembrane segments (x6 per, aka MSD1 and MSD2) and a nucleotide-binding domain (NBD)
- The MSDs form a hydrophobic channel that facilitates the efflux of substrates
- The NBDs interact with ATP to cause conformational changes in the R complex, enabling the transport cycle that effectively expels substrates from the intracellular milieu, thus playing a crucial role in multidrug resistance.
Regarding the two members of the ATP-binding cassette (ABC) superfamily:
____ functions as an efflux pump, expelling drugs and toxins, while ____ acts as a chloride channel regulating ion and water transport in epithelial tissues. Both utilise ATP hydrolysis to drive conformational changes, but ____includes unique regulatory elements; and its dysfunction causes cystic fibrosis, whereas altered ____ function can lead to multidrug resistance in cancer.
P-gp
CFTR
CFTR
P-gp
What are the three types of systems?
- Open: Matter and energy are freely exchanged with the surroundings in an open system
- Closed: Only energy is exchanged with the surroundings in a closed system
- Isolated: Neither matter nor energy is exchanged with the surroundings in an isolated
Describe the first law of thermodynamics
- Energy (E) can neither be created nor destroyed
- Energy can only be converted from one form to another
- q = heat absorbed from surrounding, w = work done to surrounding
- ∆E = EFinal - EInitial = q * w
- Energy has an effect on enthalpy (heat content, H)
- H = E + PV
- If constant pressure:
- ∆H = ∆E + P∆V = q - w + P∆V = qv for the volume change is insignificant in most biological systems
What is the second law of thermodynamics?
- All spontaneous processes in the universe tend toward disorder (entropy, S) in the absence of energy input
- Entropy definition: The degree of randomness of a system
- S = KBlnW
- Where W is the number of energetically equivalent ways and KB is the Boltzmann constant (1.38x10-23 j/K)
Compare exothermic and endothermic enthalpy
- Exothermic
- Reaction releases heat
- Has a -∆H
- Example: wearing a winter coat to keep your body warm during cold weather - Endothermic
- Reaction absorbs heat
- Has a +∆H
- Example: Absorbing heat into your skin from a heated blanket
Describe Gibbs free energy
- The difference between enthalpy (H) and entropy (S) of a system at a given temperature
- G = H - TS
- At equilibrium, ∆G = 0, the rate of formation of products and reactants is equal
Exergonic:
- ∆G < 0
- Seen in forward rxns
- Reaction is favourable and spontaneous
Endergonic:
- ∆G > 0
- Seen in reverse rxns
- Rxn is unfavourable and non-spontaneous
Describe the difference between Gibbs free energy (∆G) and Standard gibbs free energy (∆Gº)
In essence, ΔG is context-specific, while ΔGº provides a standard benchmark for evaluating reactions. Other conditions include:
- pH = 7
- [H2O] = 55.5 M
- Mg2+ = 1 mM
Describe Standard gibbs free energy (∆Gº)
ΔGº (Standard Gibbs Free Energy Change) represents the free energy change under standard conditions (1 M concentrations, 1 atm pressure, and typically 25°C), serving as a reference for comparing reaction favorability.
Describe the relationship between ∆Gº and Keq
Describe coupled reactions
- If a rxn is endergonic, it can be coupled to an exergonic rxn to become overall favourable
- ATP hydrolysis is a common coupling rxn as its ∆Gº = - 30.5 kJ/mol
- When coupling a rxn, you must add up the free energies of the separate rxns to find the overall free energy
Describe water molecules
- Water is a polar molecule
- The six outer orbital electrons of oxygen atom are distributed in the four sp3 hybrid orbitals
- The two hydrogen atoms are in the two corners of the orbital tetrahedron
- The angle between the two O-H bonds is 104.3º instead of the usual 109.5º because the lone pairs of electrons on water exert greater repulsive forces than the bonded hydrogens.
Describe H bonding
- Hydrogen bonding occurs when an H atom is covalently bonded to an e-neg atom and in close proximity to another e-neg atom (ex. O or N)
- Hydrogen is “shared” between two e-neg atoms
____ is the substance that dissolves a solute, typically present in a greater amount, and is the medium in which the solution occurs (e.g., water in saltwater). _____ is the substance that is dissolved in the solvent, usually present in a smaller amount (e.g., salt in saltwater). Together, they form a solution
Solvent
Solute
Each ion is surrounded by one or more concentric shells of oriented solvent molecules, which essentially spread the ionic charge over a much larger volume. Such ions are said to be ____ or, when water is the solvent, to be _____
solvated
hydrated
Describe hydrophobic interactions
- Non-polar substances tend to aggregate in water. It results from the strong tendency of water to exclude nonpolar groups or molecules
- The introduction of hydrophobic substance causes increased water surfaces and disrupts original water bonds
- The essence of hydrophobic effect is to minimise the cost of disrupting the water hydrogen bonds and increase H2O entropy
- For aggregation process: ∆G<0
Describe the aggregation of hydrophobic substances in water
Water excludes non-polar molecules to avoid disrupting its stable hydrogen bonds. To reduce this disruption, hydrophobic substances aggregate, minimising their exposed surface area to water. This aggregation increases water’s entropy, as fewer ordered water “cages” are needed around the clustered hydrophobic molecules, allowing more water molecules to move freely.
Describe amphiphilicity
- Molecules that are attracted to both polar and nonpolar environments
- All amphiphiles form micelles or bilayers
- The formation of micelles or bilayers are _______ ______
- The size of a micelle and the number of surfactant molecules it contains are influenced by the size of the ____ ____ group and the length of the ____ ____.
thermodynamically favourable
polar head
nonpolar tail
A larger polar head group tends to result in _____ micelles due to increased steric hindrance, while longer nonpolar tails promote ____ micelles by enhancing hydrophobic interactions. This balance between head group size and tail length determines the optimal micelle formation, impacting applications like drug delivery and emulsification
smaller
larger
Describe detergents
- A special group of amphiphile: the molecule which has both polar and nonpolar segments
- Structurally the simplest version of lipids
- A detergent forms micelles in water only when its conc. is above the critical micellar concentration (CMC)
- Depending on the head group, the detergents can be divided into ionic and non-ionic.
- Ionic detergents are more powerful at solubilising proteins, or for washing your clothes
Describe amphipathicity
Molecules that contain both polar and nonpolar groups (i.e fatty acids)
How are amphilicity and amphipathicity different
Amphiphilic refers to a molecule that has both hydrophilic (water-attracting) and hydrophobic (water-repelling) parts and has an affinity for both polar and non-polar environments. It’s typically used in the context of how molecules interact with water and oils, such as surfactants.
Amphipathic also refers to molecules with both hydrophilic and hydrophobic regions, but the term emphasizes the structural division of these regions within the molecule. It’s often used in a biological context, such as describing lipids in membranes.
What are the acid a propos equations?
pH = -log[H+]
pKw = pH + pOH = 14
HA ⇌ H+ + A-
Describe [H+]
This denotes the concentration of hydrogen ions (protons) in a solution. It is a measure of acidity, and higher [H+] concentrations correspond to lower pH values.
Describe [HA]
This represents the concentration of the undissociated weak acid, where “HA” signifies the acid itself (e.g., acetic acid). It is the form of the acid that is not dissociated into ions in solution.
Describe [A-]
This indicates the concentration of the conjugate base of the weak acid, formed when the acid donates a proton. For example, if HA is acetic acid, then [A−] would refer to the acetate ion (CH3COO-).
The pH at the midpoint of a titration provides the ___ value
pKa
Define a weak acid
An acid that IS NOT completely dissociated in the water solt.
Describe buffers
The solt. which contain at least one pair of weak acid and its conjugate base
- The best buffering capacity occurs when there is [HA] = [A-], in other words when pH= pKa
- pKa changes when solt. temp changes
Describe the dissociation constant of a weak acid
Ka = [H+][A-]/[HA]
- A higher Ka value indicates a stronger acid (greater dissociation), while a lower Ka value indicates a weaker acid
- Usually it is products on top and reactants on the bottom
Describe the titration of phosphatic acids
The titration of phosphoric acid (H3PO4), a triprotic acid, involves sequentially adding a strong base, such as sodium hydroxide (NaOH), to determine the pKa values corresponding to its three dissociation steps. Each dissociation yields a conjugate base: H2PO4−, H2PO42- and PO43−, with distinct equivalence points observable in the titration curve. The resulting pH changes at each equivalence point are indicative of the buffer regions, reflecting the acid-base equilibrium of the phosphate species. The titration curve typically displays a characteristic sigmoidal shape, allowing for the precise determination of pKa values and the acid’s overall strength.
Describe the significance of the x-axis of a titration graph
- Equivalence Points: The inflection points on the sigmoidal curve indicate the volumes of titrant where specific protons from phosphoric acid have been completely neutralised.
- Buffer Regions: Between equivalence points, the curve shows gradual pH changes, reflecting the buffering capacity of the acid-base pairs present.
- Titration Progress: The x-axis illustrates the relationship between titrant volume and pH changes, helping to assess the strength and behaviour of the acid throughout the titration.
Describe zwitterions
Dipolar (containing both + and - charges but overall neutral) amino acids. The predominant form of amino acids in pH neutral conditions. The + group is NH3+ and the - group is COO-. This dual charge allows zwitterions to exhibit unique properties, such as high solubility in water and the ability to act as both acids and bases, influencing their behaviour in biological systems and chemical reactions.
Which amino acids are charged?
Asparagus Glue Lyses Argentinian Hispanics
Asp
Glu
Lys
Arg
His
Which amino acids are hydrophilic?
Ser
Thr
Cys
Asn
Gln
Seriously Thrown Cysts Assassinate Gringos
Which amino acids are hydrophobic
Gly
Ala
Pro
Val
Leu
Ile
Met
Gliding Allow Professionally
Valued Losers Illegitimate Methods
Which amino acids are aromatic?
Phe
Tyr
Trp
Phoenix’s Try Trips
Which amino acids are non-polar?
Gly
Ala
Pro
Val
Leu
Ile
Met
Phe
Tyr
Trp
Gliding Allow Professionally
Valued Losers Illegitimate Methods for Phoenix’s Trying Trips
Which amino acids have uncharged polar side chains?
Ser
Thr
Asn
Gln
Some Asinine Threesomes Golong
Which amino acids contain ionised polar side chains?
Lys
Arg
Asp
Glu
Asparagus Glue Lyses Argentinians
What amino acids contain partially ionised polar side chains?
His
Tyr
Cyst
His Tyred Cyst
Describe the formation of cystine
Describe proline
Why does the ring structure of proline restrict protein geometry?
The cyclic nature of proline results in a rigid structure that restricts the flexibility of the polypeptide chain. Unlike other amino acids, proline’s side chain is part of the backbone, limiting the possible torsion angles around the peptide bond
- All standard amino acids are chiral except for ____
- All L-amino acids are also S-amino acids except for ____
- _____ has four stereoisomers since it contains two different chiral centres
glycine
cystine
Isoleucine
What bioactive molecules are the derivatives of the following amino acids, respectively?:
1. Glu
2. His
3. Tyr
- GABA
- Histamine
- Dopamine or Thyroxine
Describe the dimerisation of glutathione
Two molecules of the tripeptide glutathione (composed of glutamate, cysteine, and glycine) combine to form glutathione disulfide (GSSG) through the oxidation of cysteine’s thiol groups, creating a covalent disulfide bond. This reversible dimerization is key to cellular redox balance. Reduced glutathione (GSH) functions as an antioxidant, while its oxidised form (GSSG) can be converted back to GSH by glutathione reductase, maintaining redox homeostasis during oxidative stress.
The OH groups of Ser, Thr, Tyr are often _____
The NH2 group of Lys is often _____ (H → CH3-C=O)
The ______ is a major amino acid in collagen
The modification of a side chain is called a ______ event
phosphorylated
acetylated
hydroxyproline
posttranslational
Describe how the property of a polypeptide is determined by side chains of amino acids
- By formation of peptide bonds, the ionisable states of -NH2 and COOH are lost and only one -NH2 and one COOH group are left at the end (side chain may also contain the ionisable groups)
- The alpha carbon and the peptide bonds form the backbone of a polypeptide
Describe Hydrophobic side chains
(like those of leucine, isoleucine, and phenylalanine) tend to cluster together in the interior of proteins, helping to stabilise the protein’s three-dimensional structure.
Describe Hydrophilic side chains
(like those of serine, threonine, and glutamine) often participate in hydrogen bonding and are usually found on the protein surface, interacting with water or other polar molecules.
Describe Charged side chains
(like lysine, arginine, and glutamate) play critical roles in forming salt bridges, participating in enzyme catalysis, or interacting with the surrounding environment, depending on their charge at physiological pH.
Describe the Isoelectric point
The isoelectric point (pI) is the pH at which a molecule, such as an amino acid or protein, carries no net electrical charge. At this pH, the concentrations of positively and negatively charged forms of the molecule are equal, resulting in a state of electrical neutrality.
In electrophoresis, when a protein is subjected to an electric field, it will migrate towards either the ____ (positive electrode) or ____ (negative electrode) depending on its charge relative to the pI. ____ the pI, the protein carries a net positive charge and moves towards the cathode, while _____ the pI, it carries a net negative charge and migrates towards the anode.
anode
cathode
Below
above
How can you tell if an amino acid side chain is ionisable?
- An amino acid side chain is ionisable if it contains functional groups that can gain or lose protons, such as carboxyl (-COOH), amino (-NH₂), sulfhydryl (-SH), or imidazole groups.
- Common ionisable amino acids include aspartate, glutamate, lysine, arginine, histidine, cysteine, and tyrosine. Whether the side chain ionises depends on its pKa value and the surrounding pH
How do you calculate the isoelectric point (pl) for amino acids without ionisable side chains?
- Identify the pKa Values: Most amino acids have two main ionisable groups:
- The carboxyl group (COOH), typically with a pKa around 2.0.
- The amino group (NH3+), typically with a pKa around 9.0. - Use the Formula: The pI is calculated using the average of the pKa values of the groups that are protonated at the pH where the molecule is neutral:
pI = (pKa1 + pKa2)/2
How do you calculate the isoelectric point (pl) for amino acids with ionisable side chains?
- Identify the pKa Values: For amino acids with ionisable side chains (like aspartic acid or lysine), you need to include the pKa of the side chain.
- Determine the Relevant pKa Values: If the pH range of interest spans the pKa of the side chain, use the pKa values for both the terminal amino and carboxyl groups and the side chain.
- Use the Average of the Relevant pKa Values: The pI is calculated by averaging the pKa values of the ionisable groups that flank the neutral species.
So if pKa 1 = 2.0, pKa 2 = 4.0 and pKa 3 = 9.0
pI = (2.0+4.0+9.0)/3 = 7.5
Describe salting out
- A technique for precipitating proteins by increasing salt concentration, usually with ammonium sulphate. As salt concentration rises, water molecules are drawn to the salt ions, reducing the protein’s hydration shell and solubility, causing aggregation and precipitation.
- Used in the initial steps of protein purification to remove unwanted proteins and concentrate the target protein.
Describe dialysis
- A purification method used to remove small molecules, like salts or impurities, from a protein solution by using a semipermeable membrane which allows small molecules to pass through while retaining larger molecules, such as proteins. The protein solution is placed in a dialysis bag or tube, submerged in buffer, where small molecules diffuse into the buffer, leaving the proteins inside.
- This method is commonly applied after salting out to remove excess salts or to exchange buffers after other purification steps
Describe column chromatography
- A purification technique that separates proteins based on their physical or chemical properties as they move through a column filled with a solid matrix. Types include ion exchange, size-exclusion (gel filtration), hydrophobic interaction, and affinity chromatography.
- Proteins are applied to the column and pass through the stationary phase, where separation occurs based on charge, size, hydrophobicity, or affinity to a ligand, depending on the method used.
- It is widely used in protein purification and can be tailored to the protein’s specific characteristics.
Describe Ion exchange chromatography
- Separates proteins based on their net charge at a specific pH.
- In anion exchange, negatively charged proteins bind to a positively charged matrix, while in cation exchange, positively charged proteins bind to a negatively charged matrix.
- Proteins are eluted by increasing salt concentration or adjusting pH to disrupt electrostatic interactions. - It is commonly used to purify proteins with known charge properties, such as separating proteins with slightly different isoelectric points (pI).
Describe Hydrophobic Interaction Chromatography (HIC)
- Separates proteins based on their hydrophobicity. The column matrix is hydrophobic, allowing proteins to bind through hydrophobic interactions, which are enhanced in high salt concentrations.
- When a protein solution with high salt is applied to the column, hydrophobic regions on the protein surface interact with the hydrophobic groups on the matrix. - Proteins are eluted by gradually decreasing the salt concentration, which weakens these hydrophobic interactions.
- This method is used to purify proteins with exposed hydrophobic regions or to remove hydrophobic contaminants
Describe Size-Exclusion Chromatography (Gel Filtration)
- Separates proteins based on size using a stationary phase of porous beads. Larger molecules elute first because they cannot enter the pores, while smaller molecules elute later as they travel through the pores.
- The beads contain specific-sized pores, allowing this separation.
- This technique is commonly used for desalting, buffer exchange, and separating proteins with significant differences in molecular weight, as well as in the final purification steps to remove aggregates or contaminants of varying sizes.
Describe Affinity Chromatography
- A highly specific technique that separates proteins based on their binding affinity to a ligand attached to the matrix.
- The target protein binds to the ligand while other proteins are washed away. The ligand is covalently linked to the matrix in the column, allowing only the target protein to bind when the protein mixture is applied.
- Other proteins are washed away, and the bound protein is eluted by adding a solution with free ligand or by altering conditions (such as pH or salt concentration) to disrupt the interaction.
- This method is often used to purify proteins with specific tags (e.g., His-tagged proteins binding to nickel columns) or proteins with a natural affinity for specific molecules, like enzymes binding to substrates or inhibitors.
Describe SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
- A technique used to separate proteins based on molecular weight. Sodium dodecyl sulfate (SDS) denatures proteins and imparts a uniform negative charge, removing the influence of their native charge and shape.
- Mechanism:
1. SDS Binding: SDS coats proteins with negative charges proportional to their length, causing them to unfold.
2. Gel Matrix: Proteins are loaded onto a polyacrylamide gel, where an electric current causes the negatively charged proteins to migrate towards the positive electrode.
3. Separation by Size: The gel acts as a sieve, with larger proteins moving more slowly than smaller ones.
4. Staining: After electrophoresis, proteins are visualised using dyes like Coomassie Brilliant Blue or silver stain. - Application: SDS-PAGE is widely used to estimate protein molecular weight, assess purity, and monitor protein expression in research and diagnostics.
Describe Native Gel Electrophoresis
- Proteins are separated based on size and charge without denaturing agents like SDS, allowing them to retain their native structure. Migration depends on their overall charge at the buffer’s pH, with positively charged proteins moving towards the negative electrode (cathode) and negatively charged proteins towards the positive electrode (anode).
- This method is useful for studying protein-protein interactions and enzyme activities, as the proteins maintain their functional form.
Describe Isoelectric Focusing (IEF)
- Separates proteins based on their isoelectric point (pI). Proteins are applied to a gel with a pH gradient, and under an electric field, they migrate to the region where the pH matches their pI, stopping their migration.
- This technique provides high-resolution separation of proteins with small pI differences and is often used in two-dimensional gel electrophoresis (2D-PAGE) for detailed protein separation by both charge (IEF) and size (SDS-PAGE)
How do you calculate the sedimentation coefficient
Describe the sedimentation coefficient
- Measured in Svedbergs (S), indicates how fast a particle sediments in response to centrifugal force. It’s calculated by dividing sedimentation velocity by the applied centrifugal force. Larger, denser, and more spherical particles typically have higher sedimentation coefficients, meaning they sediment more quickly, while smaller, less dense, or elongated particles sediment more slowly.
How do you interpret the sedimentation coefficient
Mass: Heavier particles have higher sedimentation coefficients and move faster during centrifugation.
Shape: Spherical particles experience less frictional resistance and sediment faster than elongated or irregularly shaped particles of the same mass, leading to a higher s-value.
Density: Denser particles sink more quickly, increasing their sedimentation coefficient
What are four factors that affect the sedimentation coefficient?
- Particle Mass : Larger mass increases sedimentation rate.
- Particle Shape : Spherical shapes sediment faster than elongated ones.
- Particle Density : Denser particles settle more quickly.
- Medium Properties : Denser or more viscous media slow down sedimentation.
- The sedimentation coefficient is essential in ultracentrifugation for determining the size, shape, and interactions of biomolecules like proteins, ribosomes, and macromolecular complexes
Describe protein sequencing
The process of determining the amino acid sequence of a polypeptide or protein. This process typically involves a series of chemical and enzymatic steps to break down the protein into smaller fragments, identify these fragments, and then reconstruct the sequence
What are the five steps of protein sequencing?
Describe Edman degradation
- The process involves treating the peptide with phenylisothiocyanate (PITC), which reacts with the free N-terminal amino group to form a phenylthiocarbamoyl (PTC) derivative, which is then cleaved into a cyclic phenylthiohydantoin (PTH) for identification.
- This iterative process removes one amino acid at a time from the N-terminus, allowing for sequential determination of the peptide sequence.
- Efficient for peptides of up to approximately 50 amino acids; for longer sequences, the protein must be fragmented and the fragments sequenced individually.
How do you determine PTH-amino acids from edman degradation?
Can be effectively achieved through chromatography, particularly High-Performance Liquid Chromatography (HPLC), which is the most common method, and Thin-Layer Chromatography (TLC).
Ignore
IGNORE
____ is preferred for its high sensitivity and resolution, while ____ provides a simpler, though less precise, alternative. Both methods rely on the UV absorbance of the PTH group for identification and quantification of the amino acids.
HPLC
TLC
Describe Tandem Mass Spectrometry (MS/MS)
An advanced analytical technique used to identify and characterise biomolecules, particularly proteins and peptides, based on their mass-to-charge ratio (m/z). Here’s how it works:
1. Ionisation
* This process converts molecules in the sample into charged ions, which can be manipulated in the mass spectrometer.
2. First Mass Analysis (MS1)
* The generated ions are then introduced into the first mass spectrometer (MS1), where they are separated based on their m/z ratios.
3. Fragmentation
* Selected precursor ions are then fragmented in a collision cell, where they collide with neutral gas molecules. This collision causes the ions to break apart into smaller fragments, which provides structural information about the original molecules.
4. Second Mass Analysis (MS2)
* The resulting fragment ions are then analysed in a second mass spectrometer (MS2). This stage measures the m/z ratios of the fragment ions, allowing for the determination of their structure and the reconstruction of the original molecule’s sequence.
Describe primary protein structures
- Its linear sequence of amino acids linked by peptide bonds
- The backbone conformation of a polypeptide chain is defined by the torsion angles Φ (phi) and Ψ (psi), representing rotations around the N-Cα and Cα-C bonds
- These angles are limited by steric interference in the peptide backbone, constraining the protein’s possible conformations
- Can be visualised on a Ramachandran plot that maps the allowed Φ and Ψ values, indicating regions where alpha helices and beta sheets are likely to form.
Describe secondary protein structures
The secondary structure arises from hydrogen bonding patterns between the carbonyl oxygen (C=O) and the amide hydrogen (N-H) of the polypeptide backbone. The main types of secondary structures are the alpha helix, beta strands/sheets, and PII helices.
Describe alpha helices
A right-handed helical structure where hydrogen bonds form between the carbonyl oxygen of one residue and the amide hydrogen of another residue four positions ahead (n+4 pattern). The structure stabilises through these internal hydrogen bonds. The amino acid composition of an alpha helix is critical, with small, non-polar residues (like alanine) often promoting helix formation.
Describe Beta strands/sheets
- Extended strands that align next to each other, forming beta sheets.
- There are two types of beta sheets: parallel (where strands run in the same direction) and antiparallel (where strands run in opposite directions).
- In both, hydrogen bonds form between neighbouring strands. Beta turns, common in antiparallel sheets, allow the polypeptide chain to reverse direction. Type I and II beta turns are stabilised by a hydrogen bond between the first and fourth residues, with glycine (flexible) and proline (rigid) often found in these turns.
What is the difference between a type I and type II beta turn?
- Type I beta turn, the carbonyl oxygen of the second residue points inward, while in a type II beta turn, the carbonyl oxygen points outward. Additionally, type II turns often feature glycine as the third residue to avoid steric clashes.
- Only Type I β-turns are stabilized by hydrogen bonds, not Type II β-turns.
Describe the tertiary structure of proteins
Refers to the three-dimensional (3D) folding of a single polypeptide chain, stabilised by various interactions like hydrophobic effects, hydrogen bonding, ionic interactions, and disulphide bonds. The folding process ensures that secondary structures (like alpha helices and beta sheets) arrange themselves to create a compact, functional protein.
Describe the PII helix
A left-handed helical structure, prominent in proteins like collagen, where it forms a triple helix. The PII helix is stabilised by specific hydrogen bonds, and collagen’s composition—rich in glycine, proline, and hydroxyproline—ensures the formation of this unique structure.
Describe haemoglobins 3D structure
- Haemoglobin is a good example of tertiary and quaternary structure. Its tertiary structure shows how helices and beta sheets fold to form functional domains. In haemoglobin, each subunit contains alpha helices that come together in a compact structure to bind oxygen.
Describe the quaternary structure of proteins
- The assembly of multiple polypeptide chains (subunits) into a functional complex. In proteins like haemoglobin, four subunits (two alpha and two beta chains) come together to form the functional oxygen-carrying protein.
- Quaternary structure is stabilised by the same types of interactions as tertiary structure but between different polypeptide chains.
What are the pros and cons of quaternary structures?
Pros:
1. Easy to fix the defect of the final product
2. Synthesis and assembly can be done at different locations, easy for translocation
3. Easy to maintain the genetic information
4. Easy to provide a regulatory mechanism, especially for heterocomplexes
5. Bring linked functional components into close proximity
Cons:
1. The expression of different subunits has to be coordinated - both spatially and temporally
In some _____ enzymes, the active site is formed by amino acid residues from different polypeptide chains. For example, histidine (His) and cysteine (Cys) may be located in the same subunit, while aspartate (Asp) comes from a neighbouring subunit.
oligomeric
Subunit symmetry in oligomeric proteins refers to the organised arrangement of _____ (the individual subunits) in a symmetrical manner, which is crucial for the protein’s structural integrity and functionality
protomers
Describe protomers
These are the individual polypeptide chains or subunits that constitute an oligomeric protein. Protomers can be identical (homooligomers) or different (heterooligomers), and their arrangement is fundamental to the protein’s quaternary structure.
As an example hemoglobin consists of four protomers: two alpha subunits and two beta subunits
Describe oligomers
An oligomer is a protein complex composed of multiple protomers. The interactions between these subunits can result in diverse structural motifs and functional properties, enabling the protein to perform specific biological roles.
Describe mirror synthesis
This symmetry type occurs when a protein can be divided into two identical halves, akin to a mirror image. It reflects the spatial arrangement of protomers such that one side is a mirror image of the other, contributing to the stability and function of the protein.
Describe rotational symmetry
In proteins exhibiting rotational symmetry, the arrangement of protomers allows for a rotation around a central axis, resulting in an identical appearance after a certain angle of rotation. This symmetry is characterised by an n-fold rotational axis, where “n” denotes the number of protomers involved.
Describe helical symmetry
Helical symmetry is a specific arrangement where protomers are arranged in a helical pattern around a central axis. This results in a twisted structure that can accommodate varying lengths of the oligomer while maintaining a consistent structural motif, often observed in fibrous proteins.
Describe Cyclic Symmetry:
Cyclic symmetry refers to a circular arrangement of protomers around a central axis, allowing for rotational symmetry. For example, a protein with a 6-fold cyclic symmetry has six identical subunits arranged in a ring, contributing to the formation of structures like viral capsids or symmetric enzymes.
Describe Dihedral symmetry
- Combines both rotational and mirror symmetry. It describes arrangements where protomers are organized in such a way that there are symmetrical relationships between pairs of protomers, often resulting in more complex quaternary structures.
Describe n-fold Axis:
This term denotes an axis of symmetry around which a protein can be rotated by a specific angle (360°/n) to produce an identical arrangement. For instance, a 5-fold axis indicates that the structure can be rotated by 72° to achieve a congruent configuration, allowing for diverse oligomeric arrangements.
Describe Two-fold Axis:
This specific type of n-fold axis indicates that a protein can be divided into two symmetrical halves by a 180° rotation around the axis. This symmetry is commonly found in dimeric proteins and contributes to their stability and interaction with ligands or substrates.
Describe Tetrahedral Symmetry:
Tetrahedral symmetry describes a three-dimensional arrangement where protomers are positioned at the corners of a tetrahedron. This symmetry is characteristic of certain viral capsids and multi-subunit proteins, facilitating a stable structure that can withstand environmental stresses
Describe zinc finger motifs
- A type of domain motif functional bending
- A beta sheet’s cystines bind with the histidines of an alpha helix via a zinc bridge to form a finger looking bend between the two
- This domain motif then binds to the major groove of DNA and helps regulate transcription
Describe ion pairs in myoglobin
Ionic pairs in myoglobin form between oppositely charged amino acids, like lysine and glutamate/aspartate, contributing to its structural stability. These electrostatic interactions help maintain the protein’s tertiary structure, particularly near the heme group or on the surface, though they are not as prevalent as in other proteins. The charged residues are well-positioned to support the formation of ionic pairs in myoglobin
Define hydropathy
- The study of the hydrophobic and hydrophilic properties of molecules
- Quantified using a hydropathy index, which measures the relative hydrophobicity or hydrophilicity of amino acids; positive values indicate hydrophobicity, while negative values indicate hydrophilicity
- Helps predict membrane-spanning regions of proteins
Describe the dichotomy between protein structure and function
- Some proteins share similar structural features but carry out different functions
- Proteins with different structures can carry out similar functions
- Switching two neighbour amino acids could significantly affect its 3D-structure
Describe chaotropic agents
Substances that denature proteins into random-coils by allowing water molecules to solvate nonpolar groups in the interior of proteins
Describe the experiments of Christian Anfinsen on RNase A
- Described principles of denaturation and renaturation
- RNase A was denatured by exposing it to a chaotropic agent, such as urea, which unfolded the protein and caused it to become denatured and “scrambled.” The scrambled form of RNase A retained some structural characteristics but lacked its original enzymatic activity
What are the steps of protein de- and re-naturation
Describe protein renaturation
- A denatured protein regains its native structure and function upon removal of the denaturing agent. Anfinsen found that when the chaotropic agent was removed, RNase A could refold into its native state and regain its activity, provided the conditions were suitable.
The speed of protein _____ varies widely and is influenced by several factors, including the protein’s size, complexity, and the folding pathways it can take. Proteins often follow specific pathways to reach their native state, which can be represented as a ___ of ___ ____. That shape illustrates how the protein transitions from higher-energy, denatured states to lower-energy, native states during the folding process.
renaturation
funnel of free energies
How do you calculate the favorability of a folding process?
∆Gfolding=∆Hprotein-T∆Sprotein+∆Hsolvent-T∆Ssolvent
A _____ ΔGfolding indicates that the folding reaction is thermodynamically favourable and is likely to occur spontaneously.
negative
The protein _____ _____ determines the tertiary structure
primary structure
What is the presumed order of protein folding?
Random coil → Form 2º structure → Stabilise 2º structure → Domain formation → Native form
The free energy difference between the ____ (folded) and _____ (unfolded) must be favourable (G < 0).
native
denatured
Define molecular chaperones
A diverse group of proteins that assist in the correct folding, assembly, and maintenance of other proteins in vivo, ensuring they achieve their functional conformations. They play a crucial role in preventing misfolding and aggregation, particularly during stressful conditions that can lead to protein denaturation.
Describe assembly chaperones
These chaperones facilitate the assembly of multi-subunit protein complexes, ensuring that individual subunits are correctly incorporated into the final structure.
Describe Folding Chaperones:
These proteins aid in the proper folding of nascent polypeptides, preventing aggregation and promoting correct conformational pathways.
Describe Heat Shock Proteins (HSPs):
A family of highly conserved molecular chaperones that play a crucial role in maintaining protein homeostasis, especially under conditions of cellular stress such as heat, oxidative damage, or toxic compounds.
What are the seven types of heat shock proteins?
Hsp40
Hsp60
Hsp70
Hsp90
Hsp100
Hsp110
Small HSPs
Describe the function of Hsp60
Hsp60 (Chaperonins):
Hsp60 serves as a molecular folding machine, providing a protected chamber-like structure where proteins can fold correctly without interference from the surrounding environment. It operates as a large multimeric complex, such as GroEL-GroES in bacteria or mitochondrial Hsp60 in eukaryotic cells. Hsp60 is particularly important for the folding of newly synthesised mitochondrial proteins and for maintaining the stability of cytosolic proteins. Its mechanism involves encapsulating misfolded or unfolded proteins in its cavity and utilising ATP-dependent conformational changes to facilitate proper folding.
Describe the function of Hsp70:
It binds to hydrophobic regions of unfolded or nascent polypeptides, preventing their aggregation and assisting in their proper folding. Hsp70 is also crucial for transporting proteins across membranes, such as into mitochondria or the endoplasmic reticulum, where further folding occurs. In addition to its role in folding, Hsp70 recognises damaged proteins during stress and either refolds them or directs them to degradation pathways, such as the proteasome or autophagy. Its activity is tightly regulated by co-chaperones like Hsp40 and nucleotide exchange factors, ensuring efficient energy utilisation during the chaperoning process.
Describe the function of Hsp90:
Hsp90 protects proteins from misfolding and helps maintain their functional integrity, even under stress conditions. Its role extends to critical cellular processes such as signal transduction, cell cycle control, and immune responses. Clinically, Hsp90 is significant because it is often overexpressed in cancers, where it stabilises oncogenic proteins.
Describe the function of Hsp40:
It enhances the ATPase activity of Hsp70, which is essential for the binding and release of substrate proteins during the chaperoning cycle. Hsp40 recognises and binds unfolded or misfolded proteins, delivering them to Hsp70 for further processing and repair. In addition to its co-chaperone role, some Hsp40 proteins directly participate in disaggregation and refolding of damaged proteins. This makes Hsp40 essential during acute stress conditions, where it identifies destabilised proteins and ensures their stabilisation or repair
Describe the function of Hsp110:
Hsp110 is a large chaperone protein that primarily acts as a nucleotide exchange factor (NEF) for Hsp70. By facilitating the exchange of ADP for ATP, Hsp110 ensures the regeneration of the ATP-bound active form of Hsp70, enabling it to continue its chaperoning cycle. In addition to its role as a co-chaperone, Hsp110 also assists in protein stabilisation and refolding, especially during severe stress.
Describe the function of Small HSPs:
Small heat shock proteins (sHSPs) function as a first line of defence against protein misfolding and aggregation. These proteins bind to unfolded or misfolded proteins, preventing them from aggregating, and serve as reservoirs for damaged proteins, which can later be refolded by larger chaperones like Hsp70 and Hsp100. They also play a role in down modulating apoptosis by interacting with apoptotic signalling pathways, often promoting cell survival under stress conditions.
Describe the function of Hsp100 (Clp):
Hsp100 (Clp), found in prokaryotes and lower eukaryotes, is part of the Clp protease system, which specialises in protein degradation. It uses ATPase activity to unfold damaged or misfolded proteins and translocates them to a proteolytic core like ClpP for degradation. Unlike eukaryotic Hsp110, which focuses on protein folding, Hsp110 (Clp) ensures proteostasis by eliminating irreparably damaged proteins, with a structure tailored for degradation rather than refolding.
Match letters to numbers
A) Hsp40
B) Hsp60
C) Hsp70
D) Hsp90
E) Hsp100
F) Hsp110
- Stabilises signalling proteins, kinases, and hormone receptors.
- Nucleotide exchange factor for Hsp70, severe stress protection.
- Protein folding in isolated environments (chaperonin).
- Co-chaperone, delivers substrates to Hsp70.
- Protein disaggregation and recovery.
- Protein folding, translocation, aggregation prevention.
A-4
B-3
C-6
D-1
E-5
F-2
Describe Clamp-type Chaperone Proteins:
Clamp-like chaperone proteins are molecular chaperones that bind to unfolded or misfolded proteins via specific regions, typically hydrophobic patches, in a manner akin to a “clamp.” This binding prevents protein aggregation and assists in proper folding or transport. These chaperones are characterised by their ability to undergo conformational changes, driven by ATP binding and hydrolysis, which regulate substrate binding and release
E.g: Hsp40, Hsp90, Hsp70
Describe Chamber-type Chaperone Proteins:
Such as Hsp60, these proteins provide a protective environment (often in the form of a barrel-like structure) where polypeptides can fold without interference from other cellular components.
Describe the Chaperone-independent Folding pathway
the spontaneous process where polypeptides fold into their native conformations without assistance from molecular chaperones. This typically occurs in proteins with simple structures that have a strong intrinsic ability to adopt their functional forms.
Describe the Hsp70-assisted Protein Folding pathway
Hsp70-assisted protein folding involves Hsp70 chaperones binding to nascent or partially folded polypeptides. By stabilising exposed hydrophobic regions, Hsp70 prevents premature folding and aggregation, facilitating correct conformational development in an ATP-dependent manner.
Describe the Folding Assisted by Hsp70 and Chaperonin Complexes pathway
This process involves both Hsp70 chaperones and chaperonins (like Hsp60). Hsp70 initially binds to nascent polypeptides to prevent aggregation and then transfers them to chaperonins, which provide a protected environment for proper folding, ensuring that proteins achieve their correct structures
Describe Hsp70 in the context of E. coli
Hsp70 proteins, such as DnaK in Escherichia coli, play a crucial role in the folding of nascent polypeptides during their synthesis on ribosomes. These chaperones specifically recognise exposed, hydrophobic regions of polypeptides, preventing nonproductive associations and ensuring the polypeptide remains unfolded until productive folding interactions can occur. Hsp70 consists of two main domains: a 44-kDa N-terminal ATP-binding domain that regulates its activity and an 18-kDa central domain that binds to hydrophobic residues. By stabilising nascent chains, Hsp70 facilitates correct folding processes critical for protein functionality, especially under stress conditions that can induce misfolding.
The GroES-GroEL complex in E. coli is an example of _____ chaperones, also known as _____, which assist proteins in completing their folding after release from ribosomes. These large, cylindrical protein complexes are formed from two stacked rings of subunits, creating an enclosed space referred to as an “_____ ___.”
Hsp60
chaperonins
Anfinsen cage
Describe what happens within the Anfinsen cage
Within this cage, substrate polypeptides undergo forced unfolding, exposing buried hydrophobic residues, followed by folding processes that promote the correct formation of their native structure. The functioning of the GroES-GroEL complex is ATP-driven, ensuring energy is supplied for the folding process.
The GroES-GroEL Complex has rings, where the ____ ring initially binds the substrate, and the ____ ring is involved in subsequent folding steps. This intricate process is essential for ensuring proteins achieve their functional conformations.
cis
trans
What are the four steps in the functional cycle of GroEL/ES
Describe Ubiquitin-dependent protein degradation
Describe protein triage
- Definition: Cellular process for assessing and managing newly synthesised proteins to ensure proper folding and functionality.
- Folding on Ribosomes: Proteins begin to fold on ribosomes; some may misfold or remain partially folded.
- Role of Molecular Chaperones: Chaperones like Hsp70 and chaperonins (e.g., GroEL/GroES) bind to nascent or misfolded proteins, preventing aggregation and assisting in proper folding.
- Quality Control Mechanisms: Detect misfolded proteins, which may be targeted for degradation via the ubiquitin-proteasome system or redirected for refolding attempts.
- Outcome of Protein Triage: Determines whether proteins achieve native conformations, are refolded, or are degraded, ensuring cellular proteostasis and function, especially under stress conditions
Describe alzheimer’s as a transmissible neurodegenerative disease
Prions are misfolded proteins that can induce other proteins to misfold, leading to the spread of misfolding through the brain.
The primary protein involved in Alzheimer’s disease is amyloid-beta. In Alzheimer’s, amyloid-beta aggregates into plaques that are toxic to brain cells. Similarly, tau proteins also misfold and form tangles within neurons.
Research has suggested that in rare cases, misfolded tau and amyloid-beta proteins may spread between individuals in specific contexts, potentially through medical procedures or other routes.
Describe Transmissible Spongiform Encephalopathies (TSE)
represent another class of neurodegenerative diseases caused by prions, which are infectious agents composed solely of misfolded proteins. These diseases include conditions such as Creutzfeldt-Jakob disease and bovine spongiform encephalopathy (mad cow disease).
The abnormal prion protein, denoted as ____ (scrapie form), induces misfolding of the normal cellular prion protein (___). This conversion results in the accumulation of insoluble protein aggregates known as ___ ____, leading to spongiform changes in the brain tissue.
PrPsc
PrPc
prion rods
Describe how protein disulphide isomerase catalyses disulfide interchange
Protein disulfide isomerase (PDI) plays a crucial role in ensuring proper protein folding by catalysing the formation and rearrangement of disulfide bonds between cysteine residues. PDI’s thiol groups form mixed disulfide bonds with substrate proteins, allowing incorrect disulfide bonds to be swapped for correct ones through a process called disulfide interchange. This activity is essential for stabilising protein structure, particularly for secretory and extracellular proteins. PDI also aids in protein quality control, ensuring that misfolded proteins are corrected before they are either exported or degraded, preventing cellular dysfunction and diseases linked to misfolding
Describe Intrinsically Disordered Proteins (IDRs)
Intrinsically Disordered Proteins (IDPs), or Intrinsically Disordered Regions (IDRs), are protein regions that lack a stable, well-defined three-dimensional structure under physiological conditions.
IDRs exhibit flexibility and randomness, adopting multiple conformations instead of folding into a single stable structure.
These regions play crucial roles in cellular processes such as signal transduction, transcription regulation, and molecular recognition.
IDPs are typically rich in polar, charged, and unstructured amino acids, contributing to their disordered nature.
Describe CREB (cAMP Response Element-Binding Protein)
A prominent example of an IDR. As a transcription factor, it regulates gene expression in response to various cellular signals, primarily through cyclic AMP (cAMP). The flexible disordered regions of CREB facilitate interactions with multiple protein partners, allowing it to dynamically modulate transcriptional activity, which is essential for processes such as neuronal plasticity, learning, and stress responses.
