Molecular Biology and BioChem 1 Flashcards
What is the difference between tertiary and secondary structure?
Tertiary structure is the coming together of all the elements of secondary structure to form a complete native folded protein
Secondary structure is α helix and β sheet, the 3-D structure of local segments
What forces drive the formation of tertiary structure?
The same forces that drive secondary structure formation For example: Hydrogen bonding Salt bridges Hydrophobic effect Disulphide bridges
What is the advantage of haemoglobin being able to bind 4 haem groups?
It has a very fine control over the binding of oxygen. The binding of oxygen onto the first haem group affects the binding of oxygen onto the other haem groups.
Quaternary structure can be crucial to the function of proteins
What are the main methods of determining protein structure?
X-ray Crystallography- Reveals atomic structure- not all proteins can be crystallised.
NMR Spectroscopy- Similar level of detail as X-ray Crystallography but you don’t need crystals- can only solve small proteins.
Cryo-electron microscopy- You don’t need crystals and can be used for large structures- not that good at small proteins.
For membrane-bound proteins where does the hydrophobic section of the protein sit?
The hydrophobic section of the protein will sit within the membrane.
What are polar residues within the transmembrane α helices indicative of?
A channel
What are the key characteristics of membrane-spanning α helices?
Very hydrophobic
About 20 amino acids long
Not many polar residues
What is the difference between integral membrane proteins and peripheral membrane proteins?
Integral membrane proteins span the membrane, and so tend to be rich in α helices.
Peripheral membrane proteins associate with head groups of the membrane or integral membrane proteins or associated with fatty acid groups in the membrane.
Describe how proteins fold
Progressive stabilisations of small regions. The most stable secondary structures fold first and other structures fold against. Fold in an energetically favourable way to maximise non-covalent bond formation.
Describe the findings of the Anfinsen Experiment.
All the information about the tertiary structure is found in the primary structure
When you remove the denaturing agents the protein can refold.
Discuss what cofactors and what prosthetic groups are and what the difference is between them
Cofactors are small organic compounds or metals that help to enhance the diversity of the functional groups in proteins
Prosthetic groups are permanently associated cofactors
Give an example of a cofactor and describe what it does
Haem- in haemoglobin - metal (iron)- helps with the carrying of oxygen
NAD+- accepts OH- groups and then donates it later- acts as a transient carrier of important functional group
What do Zinc fingers bind to? Where do they insert?
They bind to DNA - they insert into the major groove
What are the different types of post-transcriptional modification and how to they change to properties of the protein?
Glycosylation- addition of carbohydrate group- makes proteins more hydrophilic and more soluble
Hydroxylation- addition of a OH group- increases H-bonding potential- makes the protein more rigid, can form stronger structure e.g. collagen
Phosphorylation-addition of phosphate group from ATP- adds to negative charge- changes charge distribution- can change the structure of a protein
Acetylation- addition of acetyl group- neutralises positive charge from lysine- causes DNA to be less densely packed around histones and be more accessible
What is specific activity in terms of protein purification?
Number of enzyme units per milligram of total protein.
What are some key factors to consider when planning an experiment and deciding on how to purify a protein?`
Yield vs purity (specific activity)
The source of the protein
Solubility, size, charge, surface hydrophobicity, binding for a ligand (of target protein)
Describe the stages of liquid chromatography
Pass protein mixture through solid matrix of beads in a column. Choose beads depending on the property you want to exploit (charge/ size). Use UV light to detect protein peaks as fractions pass through the column
Proteins with a higher affinity for the matrix- bind with higher affinity- elute later
Ion-exchange chromatography- separate by charge
Size-exclusion chromatography- separate by size
What is activation energy?
Activation Energy is the minimum amount of energy that is required to activate atoms or molecules to a condition in which they can undergo a chemical transformation.
In transition-state theory, the energy required to reach transition state.
What is reaction rate?
Reaction rate is the rate at which reactants are converted to products.
Reaction rate can be described as the rate constant * concentration of reactants.
If X -> Y,
Rate = k[X]
(Square brackets = Concentration)
What is the transition state?
In any chemical reaction, the reactants go through a transition state.
The Transition state the highest energy configuration of molecules throughout the process of the reaction.
What are the best ways to speed up a reaction?
By changing heat and pressure.
This isn’t really possible under physiological conditions…
Instead, we use enzymes. Enzymes lower the activation energy under physiological conditions, by stabilising the transition state.
What is Vmax defined as?
The maximum rate that a given concentration of an enzyme can achieve
What is Km defined as?
Km is the Michaelis constant – substrate concentration at Vmax/2
What are enzymes? How do they affect the rate of a reaction? How do they alter activation energy?
Enzymes are biological catalysts, they reduce the rate of reaction but are unchanged themselves. They reduce the activation energy by lowering the energy of the transition state.
What is the Michaelis- Menten equation?
V = Vmax X [S] / Km + [S]
What are the 3 assumptions made for the MM equation?
- ) Progress from the activated enzyme-substrate complex to the formation of products is irreversible
- ) Substrate is available in very large amounts compared to enzyme
- ) The enzyme-substrate complex is at steady state (unchanging over time)
Briefly explain the meaning of dynamic equilibrium and “steady state approximation”.
Dynamic equilibrium: The rate of the forwards and backwards reactions are equal- hence the relative concentrations of reactants and products are unchanging.
Steady state approximation: The concentration of the ES is low and constant due to a.) the slow complex formation and b.) the rapid consumption (once formed it is converted into products rapidly)
True or false, “Km can be used as a measure of binding affinity?
True
What does a large Km value suggest?
Large Km = weak binding (low affinity) and small Km = strong binding (high affinity)
Large Km = more substrate needed to achieve ½ Vmax = enzyme and substrate have low binding affinity for each other. Graph will look less steep.
What is meant by the saturation of the enzyme? Where does it occur?
Occurs at Vmax. More substrates than active sites of enzyme.
“Maximum theoretical rate”
What is Kcat? What are its units and how is it used?
Kcat: number of moles of product generated per mole of enzyme per second = rate of turnover = Vmax / E tot
Enzyme efficiency = Kcat / Km
Why do you need catalytic efficiency to compare? Why not use simply Km?
Km only measures how much substrate is required to get enzyme activity. Varies widely. Different qualities required in different enzymes: substrate affinity, turnover rate, substrate specificity.
Enzyme efficiency = Kcat / Km. increase enzyme efficiency by increasing Kcat or reducing Km
Describe the two models of enzyme binding.
What are the main differences?
Lock and key binding or induced fit.
In induced fit, the binding of a substrate induces a conformational change in the active site- the strain causes the substrate into a conformation similar to the TS.
What is the benefit of transition state stabilisation?
The transition state is the highest energy state of the reaction and so the most unstable. Stabilising it will reduce the activation energy, so allow the reaction to occur at an increased rate. Note: Vmax is larger where TS energy is lower- the reaction goes faster with a stabilised TS.
What are the two forms of Enzyme inhibition?
Competitive inhibition
Non-Competitive inhibition
How do competitive and non-competitive inhibition differ?
Competitive inhibition is regulation by substrate analogues.
Non-competitive inhibition is regulation by binding at a distinct site called an allosteric site.
What are the catalytic sites of ATCase?
The enzyme is made of catalytic trimer (catalytic) and regulatory dimer (binds inhibitor)
The Quaternary structure reorganises when there’s no ligand bound (no active site). Catalytic site lies between C subunits.
What inhibits ATCase and how? What is the biological reason for this inhibition?
Regulated by CTP - end product in pathway to ensure correct amount of CTP is made in cell
T-state = poor substrate binding/activity, stabilised by inhibitor binding, prevents transition to R-state
R-state = highly active, binds substrate over inhibitor
R and T exist in equilibrium, CTP shifts equilibrium towards less active T-state, and ATP shifts the equilibrium towards the more active R state (this is in order to maintain the ratio between purines and pyrimidines).
Substrate binding at one active site causes other attached enzymes to bind substrate too.
Describe what is meant by allosteric inhibition
Binding of an inhibitor at one site on a protein/enzyme which brings about an effect at a distance is allosteric inhibition
Which ion is needed for DNA Polymerase activity?
Mg 2+
During DNA replication, where are new nucleotides added?
3’ -OH of the previous nucleotide’s sugar group
Which of these histone proteins does NOT form part of the octamer?
- H1
- H2A
- H3
- H4
H1 - It’s a linker protein
Which of these protein structure determination methods can resolve larger proteins, but not smaller ones? -X-Ray Crystallography -NMR -Cryo-Electron microscopy
Cryo-Electron microscopy
Which form of ATCase favours substrate binding?
- T Form
- S Form
- Q Form
- R Form
R Form
Difference between Nucleoside and Nucleotide?
Nucleoside = base + sugar Nucleotide = base + sugar + phosphate
Which base pairing is the most stable?
C-G
This is because the pairing has 3 hydrogen bonds
What forces are involved in base stacking?
Hydrophobic interactions
Base stacking reduces exposure of the hydrophobic bases to the aqueous environment
Why is DNA better as an information storage substrate than RNA?
The O-H bond in the ribose of RNA makes the molecule more reactive, compared to DNA. RNA is unstable under alkaline conditions, and it is more susceptible to enzyme attack. RNA is degraded quickly.
What are the forces contributing to the stability of DNA?
The stability of the DNA double helix depends on a fine balance of interactions including hydrogen bonds between bases, hydrogen bonds between bases and surrounding water molecules, and base-stacking interactions between adjacent bases.
How does the proofreading subunit of DNA polymerase detect errors in DNA
replication?
By reading the minor groove of DNA and checking for structural deformities in double helix caused by DNA damage (e.g. wrongly incorporated bases)
What is a nucleosome? Describe the structure of a nucleosome
The basic unit of DNA packaging - consists of octameric histone core around which DNA is wound (~146 bases wrapped around core) with additional linker DNA between nucleosomes.
