EEP Flashcards

Question

How was the sequence of redox complexes in the ETC discovered?

Answer 1

Redox groups are ‘coloured’- they have distinct spectra in the visible light, which differ in the oxidized and reduced states. By measuring these spectra we can follow the change in redox state of each component.

Answer 2

Electron transfer from reduced ubiquinone (QH₂, carrying 2e-) to cytochrome c (carrying 1e-) requires a repetitive cycle in which a radical intermediate (semiquinone, Q*-) is held inside the enzyme.

Answer 3

Purple bacteria, Cyanobacteria, mosses, ferns, algae (phytoplankton to seaweeds), and higher plants.

Answer 4

Differences: size (chloroplasts are bigger) and sub-compartmental structures, inner membrane of chloroplasts completely detached (thylakoid membranes) Similarities: mechanisms and their orientation with respect to stroma/matrix; electron transport chain, chemiosmosis

Answer 5

Photosynthesis uses light energy to transform CO₂ into organic carbon molecules. Two major steps: 1. Energy capture (production of ATP, NADPH) 2. Build up of organic carbon molecules from CO₂ (consumption of ATP and NADPH

Answer 6

Antenna complex: collects energy and tunnels it to the reaction centre Reaction centre: produces high energy electron and passes it to quinone

Answer 7

1. Decay by giving off light and heat 2. Decay by resonance energy transfer 3. Decay by successive electron transfers

Answer 8

In the antenna complex, decay by resonance energy transfer. In the reaction centre, decay by successive electron transfers.

Answer 9

Rings. Magnesium (Mg) at centre. Chlorophyll a has a hydrophobic tail region.

Answer 10

P900 (Purple bacteria) P680 (PSII) P700(PSI) Main differences are structure, and donor of low-energy electrons (cytochrome, water, and plastocyanin respectively)

Answer 11

- Structural proteins - Scaffold proteins - Enzymes - Membrane transport proteins - Regulatory proteins - Motor proteins Non-exhaustive list. Proteins can belong to more than one functional class.

Answer 12

They determine cell shape and contribute to the extracellular environment. Include actin and tubulin- filaments that control movement within cells, movement of cells, and define cell shape. They can form molecular machines by self-assembly.

Answer 13

They bring proteins together into an ordered complex; allows increased efficiency than if the proteins were not assembled together.

Answer 14

Biological catalysts.

Answer 15

They are responsible for moving material from one side of the membrane to the other. This can be solutes, ions, proteins, or signals.

Answer 16

Signals, sensors, and switches that control cell function by altering the function of other proteins. These include receptors and signalling proteins like kinases.

Answer 17

They move other proteins, organelles, cells, or even whole organisms (think actin/myosin in muscles). Proteins do ‘work’- they use chemical energy to beat entropy.

Answer 18

Large complexes of proteins that assemble to accomplish their actions efficiently.

Answer 19

- Catalysis - They bind (to other proteins, to DNA, to substrates) - Fold into specific shapes- confirmations

Answer 20

Molecules that exhibit molecular complementarity can form multiple non-covalent interactions at close range. When such molecules collide, these multiple interactions cause them to stick together-bind.

Answer 21

Because the non-covalent interactions that might hold them together are weak and are transient under physiological conditions (pH, salt, temperature, etc).

Answer 22

- Salt bridge (ionic interactions) - Hydrogen bond - Hydrophobic interactions - van der Waals forces

Answer 23

The geometry of the two binding partners, where no other arrangement of atoms will produce as energetically favourable complex.

Answer 24

The higher the affinity of two molecules, the better the ‘molecular fit’ between them, the more non-covalent interactions can form and the more tightly they (usually) bind. The greater the number of interactions, the better the specificity.

Answer 25

The individual components of the protein bind one another by a particular surface of the protein (no change in shape necessary).

Answer 26

Some enzymes permit some (small) latitude in the structure and shape of a substrate, leading to the ‘induced-fit’ concept.

Answer 27

Proteins can bind small ‘ligands’ (like cAMP) and proteins can bind to other proteins. Binding of a ligand (or another protein) can induce a change in protein confirmation. This can be associated with a change in activity. Binding can be quantified and measured. E.g. cAMP-dependent protein kinase A. There are two Regulatory subunits (R) that have binding sites for cAMP, and two Catalytic subunits (C) that contain kinase activity. R and C associate tightly into an R2C2 complex with no kinase activity. When cAMP levels rise, cAMP is tightly bound by the R subunits resulting in a conformational change that weakens the association between R2 and C subunits, so the C subunits fall off and their catalytic activity is turned on.

Answer 28

The binding association constant K_d. P + L ⇌ PL K_d = [P] • [L]/[PL] The dissociation constant is calculated from the concentrations of the three components when they are at equilibrium. Alternatively, K_d = k_off/k_on, where k_on is the association of the ligand with the protein (→) , and k_off is the disassociation of the ligand with the protein ( ←).

Answer 29

The lower the K_d, the lower the concentration of L is needed to bind half of P. Hence, the lower the K_d, the tighter the binding and hence the higher the affinity. A large bio molecule like a protein can have multiple binding surfaces and exhibit very tight binding.

Answer 30

Using an equilibrium experiment, or a kinetic experiment.

Answer 31

One determines the extent of the reaction as a function of the concentration of one of the reactants. Analysis of these data gives the equilibrium constant.

Answer 32

One determines the rate of the forward and reverse reactions as a function of the concentration of one of the reactants. Analysis of these data gives the rate constants for the forward and reverse reactions. The ratio of these rate constants gives the equilibrium constant.

Answer 33

E.g. a labelled ligand e.g cAMP binding to purified Regulatory subunit of cAMP-dependent protein kinase A. A dialysis chamber is used. There are two sides of the chamber separated by a semi-permeable membrane(the membrane has holes that allow free diffusion of small molecules like cAMP. cAMP is added to one side of the chamber at a known concentration. After a few hours, when equilibrium is reached, the [cAMP] on each side of the semi-permeable membrane will be equal. Replicate the experiment, adding R subunits to one side of the semi-permeable membrane. They are too big to pass through, but cAMP still can, unless it’s bound to R. The free [cAMP] reached at equilibrium will reflect the fact that some of the cAMP has bound the protein. How much cAMP bound at any given concentration of cAMP reflects the affinity of the R subunits for cAMP. Vary [cAMP] and determine [Bound] and [Free] at constant protein concentration.

Answer 34

Primary structure- the sequence of the amino acids Secondary structure- the core elements of protein structure α-helix, β-sheet, β-turn due to Hydrogen bonding of the protein backbone Tertiary structure- three-dimensional folding pattern of the polypeptide chain due to side chain interactions Quaternary structure- protein consisting of more than one amino acid chain

Answer 35

They have resonance structures. They are a hybrid of the two resonance structures (1: Peptide bond is shown as a single C-N bond (C=O double bond); 2: Peptide bond is shown as a double C=N bond (C-O single bond)) in which electrons are delocalised over the carbonyl oxygen, the carbonyl carbon, and the amide nitrogen. Rotation around the C-N bond is restricted due to the double bond nature of the resonance hybrid form. Note: Peptide bonds are polar. The oxygen and nitrogen atoms have partial negative and positive charges respectively.

Answer 36

A common structural motif. Hydrogen bonds between every N-h group and the C=O group in the turn of the helix, four amino acids down the chain. - 3.6 residues (amino acids)/turn. - Always right handed. - R-groups point outwards. - Proline residues are helix-breakers.

Answer 37

Unlike the α-helix, the β sheet is formed by hydrogen bonds between protein strands, rather than within a strand. The amino acids are more extended than in an α-helix. The R-groups alternate above and below the plane of the sheet. Hydrogen bonds are formed across the strands. Sheets may be parallel or anti-parallel.

Answer 38

These allow polypeptide chains to tun abruptly and go in the opposite direction. Allows proteins to attain a compact (globular) chain. Pro and Gly are commonly found in such turns, proline because of its cyclic structure and glycine by virtue of its small size making it flexible.

Answer 39

Hydrogen bonding, ionic bonding, hydrophobic interactions, and van der Waals forces.

Answer 40

They are covalent linkages between the sulphur-containing side chains of Cys; much stronger than the other types of bonds that contribute to tertiary structure (and sometimes quaternary).

Answer 41

Amino acids with nonpolar, hydrophobic R groups cluster together on the inside of the protein, leaving hydrophilic amino acids on the outside to interact with surrounding water molecules.

Answer 42

Proteins fold to the lowest free energy conformation. Linear chains of amino acids have multiple possible confirmations so high entropy. As folding proceeds, both free energy and the entropy decrease to a minimum. Proteins can independently refold following denaturation, meaning all information required to fold is contained in the amino acid sequence.

Answer 43

Hydrophobic and hydrophilic. Most proteins begin to told during translation.

Answer 44

A partially folded protein state: conserves a native-like secondary structure content but without the tightly packed protein interior. Van der Waals forces help the atoms in a protein pack together.

Answer 45

Native⇌Molten Globule = slow Molten Globule ⇌ Denatured = fast

Answer 46

Natural selection has strongly favoured protein sequences that have a single confirmation that forms easily and seldom makes mistakes (think α-helix, β-sheet).

Answer 47

The observed tendency of non-polar molecules to aggregate in water. It is an indirect effect resulting from a peculiarity of water structure. Water molecules exchange hydrogen bonds with neighbours at a rate of about 10¹¹s^-1. At the interface between water and a non H-holding group such as a methyl group, CH₃, water molecules have fewer opportunities for H-bond exchange leading to longer than usual lifetime of H-bonds, an ice-like state at the interface, and consequently decrease in entropy. Thus water at the interface is rotationally and translationally constrained.

Answer 48

Each H₂O molecule is rapidly forming H bonds with ~7-8 other H₂O molecules.

Answer 49

Minimising entropy loss. Clustering of hydrophobic residues within the core of a protein is an important force in protein folding.

Answer 50

Domains are a conserved part of a protein structure that can evolve, function, and exist independently. They are an independently folding unit of a polypeptide chain that usually carries specific function. Vary from 25-500 amino acids in length. Many proteins consist of different domains linked together. Because they fold independently, t is ‘easy’ to move these between different proteins either by evolution or genetic engineering. In molecular evolution, such domains may have been utilised as building blacks, and may have been recombined in different arrangements to modulate protein function through evolution.

Answer 51

Src contains 3 domains that are shared with other proteins. SH3 binds polyproline motifs. SH2 binds peptides phosphorylated on Tyr. Kinase phosphorylates other proteins.

Answer 52

>110 proteins in humans contain an SH2 domain. They are structurally conserved and can autonomously fold. They also exhibit regions of primary sequence conservation.

Answer 53

A motif is usually a sequence of amino acids which is predictive of belonging to a particular group. For proteins, this means we can use these motifs to predict which proteins belong to a particular protein family. The kinase motifs are an example of this.

Answer 54

There is significant correlation between the borders of exons and domains for both invertebrates and vertebrates. Extensive exon shuffling events during evolution significantly contributed to the shaping of eukaryotic proteomes.

Answer 55

Larger proteins e.g. 1000 aa’s are unlikely to ever fold by themselves into the final structure. Sub dividing the structure into domains which fold autonomously means that each domain reaches its energy minima (stable final confirmation) sometimes while the rest of the protein is still being synthesised on the ribosome. ‘Linker’ regions between domains are often unstructured. Act like a flexible hinge.

Answer 56

Basic Local Alignment Search Tool is an algorithm for comparing primary illogical sequence information, such as the amino-acid sequences of proteins.

Answer 57

An MSA is a sequence alignment of three or more protein sequences. From the resulting MSA, sequence similarity can be inferred and phylogenetic analysis can be conducted to assess the sequences’ shared evolutionary origins. MSA is often used to assess sequence conservation of protein domains, tertiary and secondary structures, and even individual amino acids.

Answer 58

X-ray crystallography enables us to visualise protein structures at the atomic level and enhances our understanding of protein function. X-ray crystallography can be considered a form of microscopy. The amount of detail or the resolution of any microscope is limited by the wavelength of the EM radiation used. X-ray wavelength is around 0.1nm (or 1Å).

Answer 59

- The protein in question must crystallise in an ordered fashion (not all proteins do). - Many only crystallise at non-physiological pH or [salt]- hence relevance may be debatable. - You see a single static image of a protein, and get no indication of its dynamics.

Answer 60

NMR spectroscopy- information on structure under more physiological conditions (in aqueous buffer). Electron microscopy- overall shape of the molecule.

Answer 61

This method is based on the observation that two proteins belonging to the same family (and sharing similar aa sequences) will have similar 3D structures. In reality, the degree of conservation of protein three-dimensional structures within a family is much higher than the conservation of the sequence.

Answer 62

Modelling a protein’s 3D structure using a known experimental structure of a homologous protein (the template). The ‘low-resolution’ structure provided by homology modelling contains sufficient information about the spatial arrangement of important residues in the protein. In the pharmaceutical industry homology modelling is valuable in structure-based drug discovery and drug design.

Answer 63

The analysis of a complex mixture of proteins (e.g. in a cell).

Answer 64

A useful simple approach to begin to fractionate complex mixtures. Works best on water soluble proteins. Easy and cheap. Higher molecular weight proteins are eluded first.

Answer 65

The pH at which a protein has no net charge. At a pH above the isoelectric point a protein carries a net negative charge. At a pH below the isoelectric point a protein carries a net positive charge.

Answer 66

Positively charged resin/beads, bings negatively charged proteins. Positively charged proteins flow through.

Answer 67

Negatively charged resin/beads, binds positively charged proteins, negatively charged proteins flow through.

Answer 68

Protein mixture is added to column containing a polymer-bound ligand specific for protein of interest. Unwanted proteins are washed through column. Protein of interest is eluded by ligand solution.

Answer 69

A substance that increases the rate of a chemical reaction without itself undergoing any permanent chemical change.

Answer 70

By reducing activation energy, which is the energy that must be supplied in order for molecules to react with one another. This is achieved by stabilising a transition state - *ES *- they do not affect the reaction equilibrium. They have very high catalytic efficiencies. E + S → *ES * → E +P

Answer 71

Rate constant x concentrations of reactants. Rate = k[E][S]

Answer 72

In classes by the type of reaction they catalyse (hydrolyses, oxidoreductases, lyases, transferases, ligases, and isomerases).

Answer 73

Oxidation-reduction reactions where electrons or hydrogens are transferred between molecules.

Answer 74

Transfer of functional groups (e.g. methyl, amino, glycosyl, nucleotidyl) from one molecule to another.

Answer 75

Hydrolysis reactions- breaking bonds by adding water.

Answer 76

Additional of groups to double bonds, or removal of groups to form double bonds, without hydrolysis or oxidation.

Answer 77

Structural rearrangements within a molecule (isomerisation).

Answer 78

Joining f two molecules, usually coupled with ATP hydrolysis.

Answer 79

The location on an enzyme where the substrate specifically binds and catalysis occurs: stabilises the transition state. It contains groups that bind the substrates in a defined orientation and groups that take part in catalysis. It is not a linear sequence of amino acids. Active sites form 3D clefts or crevices where water is excluded unless it is a reactant. Substrates bind to enzymes by multiple weak interactions (H bonds, van der Waals forces, electrostatic and hydrophobic interactions)

Answer 80

The active site of an enzyme is complementary in shape to the substrate; no structural change occurs in the enzyme upon substrate binding. It explains enzyme specificity, but not flexibility or catalytic efficiency. It doesn’t account for the dynamic nature of proteins, and is less accurate for enzymes that undergo conformational changes.

Answer 81

The active site complements the transition state and is only fully formed after the substrate is bound to the enzyme. Substrate binding induces confirmational changes in the enzyme aligning catalytic residues and enhancing reaction efficiency. It better explains enzyme structural flexibility and transition state stabilisation, more realistic for most enzymes, and accounts for dynamic interactions and regulatory mechanisms.

Answer 82

Enzymes from evolutionary distinct organisms with low overall protein sequence similarities may have similar mechanisms of action due to arrangements of residues in their active sites e.g. serine proteases from mammals and bacteria catalyse peptide bonds with a similar mechanisms using a conserved catalytic triad (Ser/His/Asp) on their active site.

Answer 83

Many enzymes require a non protein cofactor to assist them in their reaction. The protein portion of the enzyme, apoenzyme, combines with that cofactor to form the whole enzyme, holoenzyme. Cofactors can be metals/ions (such as Zn⁺², Mg⁺², and Mn⁺²), or small organic molecules (frequently vitamins or derived from vitamins such as biotin or Coenzyme A).

Answer 84

Enzyme activity strongly depends on temperature, pH, substrate and enzyme concentrations. These factors influence protein structure, ionisation, and reaction kinetics.

Answer 85

The kinetic energy of the system increases with the increase in temperature → higher chance for the reactants to collide with enough energy to form the [ES] complex. Temperatures higher than the optimal for catalysis lead to breakage of hydrogen bonds and protein denaturation. Optimal temperature is organism/enzyme dependent.

Answer 86

pH contributes to maintaining the 3D shape of enzymes but maintaining the required ionic bonds in the protein. Optimal pH is also dependent on the type of enzyme.

Answer 87

Assuming a non-limiting [S], increasing [E} will increase the reaction rate. At constant [E] and low [S] the substrate will be a limiting factor for the enzymatic reaction. As the [S] increases, the reaction rate will increase to the point where all the active sites are occupied and the reaction cannot increase any more.

Answer 88

Experimentally, we can measure the accumulation of product under specific conditions and generate progress curves of the reaction. Generally, we can measure by spectrophotometric methods: product absorbs light at specific wavelength.

Answer 89

V₀: initial reaction rate when [P] is virtually zero. V₀: linearly proportional to [S] if this is very low, but independent from [S] if this is in excess. V_max: plateau at equilibrium, no net change in [S] or [P].

Answer 90

Initially linear, reaching a plateau with time.

Answer 91

Initial rate is directly proportional to enzyme concentration, hence initial rate gives a measure of enzyme activity.

Answer 92

The rate of an enzyme catalysed reaction where S → P increases with substrate concentration to a plateau.

Answer 93

v = v_max[S]/K_m + [S] v = the reaction rate v_max = maximum reaction velocity (directly proportional to [E]) K_m = Michaelis-Menten constant ([S] required to give half of maximum reaction rate; it is independent of [E]) [S] = substrate concentration

Answer 94

Ability of an enzyme to selectively bind a substrate or group of substrates to catalyse a specific reaction; determined by the active sites of an enzyme.

Answer 95

Absolute specificity: one substrate only Group specificity: substrates with similar functional groups Stereospecificty: distinguishes between stereoisomers

Answer 96

E + S ⇌ ES → E + P k₁ = → of ⇌ k_-1 = ← of ⇌ k₂ = → Rate of formation of ES= k₁[E][S] Rate of breakdown of ES = (k_-1 + k₂)[ES] K_m = (k_-1 + k₂)/k ₁

Answer 97

How effectively an enzyme converts substrate into product

Answer 98

(= V_max) the number of substrate molecules converted into product per molecule of enzyme per unit of time. This equals the kinetic constant k₂, also called k_cat

Answer 99

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Answer 109

The potential energy between two separated opposite charges trying to move towards each other due to the electric force of attraction. Measured in volts or milivolts.

Answer 110

Ions of opposite sign on either side of a membrane. All cells under resting conditions have a negative membrane potential.

Answer 111

When the driving force of concentration = driving force of charge. Influx = efflux. Net flux = 0.

Answer 112

If there is a concentration gradient for X⁺ and the membrane is selectively permeable to X⁺.

EEP Flashcards

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