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Structure and Function of Proteins > Lecture 1-7 > Flashcards

Lecture 1-7 Flashcards

1
Q

Define the human proteome?

A

The total number of different proteins, ~>100,000.

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2
Q

What units are used to express the molecular weight of proteins?

A

Daltons (1 Da = 1 amu, absolute molecular weight which is the mass in grams per 1 mole).

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3
Q

How many different types of the post translational modifications are there, and name examples?

A

>

  1. And include phosphorylation, lipid, metal ions, nucleic acids and CHO.
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4
Q

Define amphoteric behaviour?

A

Able to react with acid and base.

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5
Q

What pH does an amino acid form a zwitterion?

A

Neutral.

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6
Q

Which isomer of amino acids is found in proteins L or D?

A

L

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7
Q

Name the two acidic amino acids?

A

Aspartic acid and glutamic acid

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8
Q

Name the three basic amino acids?

A

Arginine, lysine, and histidine.

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9
Q

Is peptide bond formation endothermic or exothermic?

A

Endothermic

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10
Q

What does the resonance stability of the peptide bond affect the bond character?

A

Bond has weak dipole charges and no free rotation as forms a planar six atom structure. Prefers trans configuration over cis, as less repulsion between atoms connected to alpha carbon. Ratio of trans:cis 1000:1.

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11
Q

What is the name of the angle of rotation around the alphaC-C bond.

A

Psi

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12
Q

What is the name of the angle of rotation around the alphaC-N bond?

A

Phi

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13
Q

The final structure of a protein is one that…

A

…is lowest free energy/minimise free energy

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14
Q

Describe the structure of an alpha helix?

A

Single polypeptide chain twisted around itself to form a rigid cylinder. A hydrogen bond forms between every 4th residue, linking the C=O of one amino acid to the N-H of another. every complete turn is 3.6 amino acids. Turn is right-handed and orientates all N-H groups in one direction and all the C=O in the other creating polarity. Peptide backbone is hydrophilic.

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15
Q

Describe the structure of a beta sheet?

A

Neighboring segments of a polypeptide chain that run in the same or opposite direction held together by H bonds, forming a parallel or anti-parallel sheet. Often drawn as an arrow, with the point indicating the N terminus and the blunt end the C.

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16
Q

Describe the structure of a turn?

A

Amino acid n H bonds to amino acid n+3 in a hairpin turn.

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17
Q

Why does proline, unlike other amino acids, prefer the cis confirmation?

A

~30:1 ration because the symmetry between alphaC and sigmaC atoms of proline make cis and trans configurations nearly equal in energy.

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18
Q

DSSP classification is used to describe protein secondary structure using a single letter. What does G, T and E mean?

A 
G = 3-turn helix
T = hydrogen bonded turn 
E = extended strand in a parallel or anti-parallel beta sheet.
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19
Q

Name other secondary structures?

A

Alpha-alpha corner, helix-turn-helix, EF hand, and beta-alpha-beta motif.

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20
Q

Define denaturation and how it is stimulated in proteins?

A

Loss of native structure due to an external stress. Results in the loss of confirmation, loss of solubility. Strong acid/base, conc inorganic salt or organic solvent (e.g. alcohol or chloroform), radiation or heat.

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21
Q

At what frequencies due proteins absorb UV light?

A

280 nm due to aromatic nature of Trp and Tyr or 220 nm due to peptide bonds.

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22
Q

Define size exclusion chromatography?

A

Separation of proteins based on size (or MW). Entropically controlled seperation on the basisi of hydrodynamic molecular volume or size, With proper column calibration or by the use of molecular weigth sensitive detectors, such as light scattering, viscometry or mass spec the MW distribution can be obtained.

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23
Q

What types of ligands are used in affinity chromatography?

A

Substrates, antibodies, lectin, nucleic acid, hormones and metal ions.

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24
Q

In a buffer with a pH greater than the pI the protein of interest will carry a…

A

…net negative charge. Therefore, positively charged anion exchange resins are used to capture protein.

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25
Q

In a buffer with a pH lower than the pI the protein of interest will carry a…

A

…net positive charge. Therefore, a negatively charged cation exchange resin is used to capture protein.

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26
Q

The protein of interest will elute in ion exchange chromatography when…

A

…the pH gradient reaches their pI.

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27
Q

Describe SDS-PAGE gel electrophoresis?

A

SDS and polyacrylamide gel are used to eliminate the influence of protein structure and charge so proteins are solely separated based on chain length. 1.4g of SDS per g of protein. SDS binds at a molar ratio.

28
Q

What is the units used to measure gel mobility?

A

cm^2/sec/v

29
Q

How does varying the percentage of acrylamide in the gel affect SDS-PAGE gel?

A

Creates either low = loose or high = tight mesh. Typcially 1:35 ratio of acrylamide and bisacrylamide.

30
Q

Define 2D electrophoresis?

A

Using first isoelectric focussing (IEF) and then SDS PAGE to separate out proteins.

31
Q

In 2D electrophoresis what does the intensity of the spot of protein correspond to?

A

Protein abundance.

32
Q

What is the resolution of 2D electrophoresis?

A

4000 proteins

33
Q

How are proteins detected on 2D electrophoresis gel?

A

Silver colloid added which binds to cystiene in proteins, sliver darkens on exposure to UV. Or Coomassie Brilliant Blue which complexes with SDS forming a green complex which can be quantified using a Bradford assay.

34
Q

Describe peptide mass fingerprinting?

A

Protein digested using trypsin which cleaves after lysine (K) and arginine (R).
Peptide fragments are ionised and then MS measures the mass to charge ratio of the fragments.
Database is used to identify protein. Can only be performed if protein sequence is already known.

35
Q

Describe tandem mass spectroscopy?

A

Protein digested using trypsin which cleaves after lysine (K) and arginine (R).
Peptide fragments are ionised and then MS measures the mass to charge ratio of the fragments. Generating peptide fingerprint.
Individual peptide fragments are selected and enter a region of the MS called the collision cell where further fragmentation occurs. Producing fragments that differ by an amino acid.

36
Q

In peptide tandem mass spec, N terminal charged fragments ions are classed as:

A

a, b, and c

37
Q

In peptide tandem mass spec, C terminal charged fragment ions are classed as:

A

x, y, and z.

38
Q

State and describe methods of peptide fragmentation?

A

Low energy collision induced dissociation: b and y ions main product.
High energy collision induced dissociation: all types of fragments produced.
Electron transfer dissociation and electron capture dissociation: predominant ions c, y, z+1, z+2 and sometimes w.
Post source decay: in MALDI a, b, y ions common.

39
Q

State the limitations of the transition state theory?

A

Assumes that each intermediate is long-lived enough to reach Boltzmann distribution of energies before continuing to the next step. When intermediate is short lived TST fails.
Assumes that the atomic nuclei behaves according to classical mechanics. It assumes unless atoms collide with enough energy to form the TS the reaction doesnt occur.

40
Q

Define the rate constant?

A

K describes what proportion of A will react per unit of time. Rate constant is proportional to the conc of the transition state.

41
Q

Why are enzymes required even if the reaction is thermodynamically favouable?

A

Enzymes are required for the reaction system to reach the required activation energy/transition state.

42
Q

Define the activation energy?

A

The difference in energy between the reactant and the transition state is the activation energy of a reaction. Once the transition state is reached a 51% chance of product formation.

43
Q

How many folds does a catalyst typically increase a reaction rate?

A

10^5 to 10^7 fold.

44
Q

How do enzymes speed up reaction rates?

A

Reducing the activation energy by stabilizing the TS.

45
Q

State the two catalytic mechanisms used by enzymes?

A

Changing the change in enthalpy = general acid base or electrostatic catalysis.
Changing the change in entropy = proximity and orientation effects and covalent catalysis.

46
Q

State the five ways enzymes catalyse reactions?

A

Bond strain, proximity and orientation, proton donor and acceptors, electrostatic catalysis and covalent catalysis.

47
Q

What evidence is there of the formation of an enzyme substrate complex?

A

At a constant conc of enzyme the reaction rate increases with increasing substrate conc until a maximal velocity is reached. In contrast uncatalysed reactions do not show this saturated effect.
The spectrscopic characteristics of many enzymes and substrates change on the formation of an ES complex.
X ray crystallography has provided high resolution images of substrates and substrate analogs bound to the active site.

48
Q

Define binding energy?

A

The energy released by the formation of a large number of weak interactions between a complementary enzyme and substrate. Binding energy lowers the activation energy. The interaction of the E and S is fleeting, with molecular movements resulting in the optimal alignment of functional groups at the active site so maximal bonding can occur.

49
Q

What are the units of the rate constant of a first order reaction?

A

s^-1

50
Q

What are the units of the rate constant of a second order reaction?

A

M^-1 s^-1

51
Q

Define Vmax?

A

The maximum velocity catalysed by a given conc of enzyme, rate at enzyme saturation.

52
Q

Define Km?

A

Equals the substrate conc at which the reaction rate is half the Vmax, therefore the units are conc. Give info about the affinity of an enzyme for a substrate. High Km - relatively weak substrate binding in mM. Low Km - relatively strong substrate binding in microM. Km is not the same as binding affinity.

53
Q

Define Kcat?

A

Is a measure of molecules of substrate transformed per unit of time, usually per sec.

54
Q

What assumption are made by steady state kinetics?

A

When [E]

55
Q

What is the specificity constant?

A

Kcat/Km units M^-1 s^-1. Allows for the comparison of different enzymes acting on different substrates. Max speed is ~10^8 to 10^9 M^-1 s^-1.

56
Q

What is the difference between reversible and irreversible inhibitors?

A

Reversible do not react covalently with enzyme. Irreversible changes the enzyme chamically by covalent modification of a key amino acid residue need for enzymatic activity.

57
Q

Describe a competitive inhibitor?

A

Resembles enzyme substrate and binds to active site. increases Km and no effect on Vmax.

58
Q

Examples of competitive inhibitors?

A

Sulfanilamide, methotrexate, folic acid, cyanide and sidenafil.

59
Q

Describe a non-competitive inhibitor?

A

Inhibitor binds at the allosteric site. No effect on Km and decreases Vmax. Equal affinity for E and ES.

60
Q

Examples of non-competitive inhibitors?

A

Strychnine, Penicillin, and glucose-6-phosphate.

61
Q

Describe a uncompetitive inhibitor?

A

The uncompetitive inhibitor binding site is only created when E forms a ES complex.

62
Q

Describe a mixed inhibitor?

A

Hinders the binding of the substrate and decreases the turnover number of the enzyme.

63
Q

Define a partially competitive inhibitor?

A

EIS complex either increases or decreases catalytic activity.

64
Q

Describe substrate/product inhibition?

A

Substrate or product inhibits enzyme. Product via negative feedback. Or two substrate binding inhibits acitivty.

65
Q

Describe slow-tight inhibition?

A

Occurs when initial EI complex undergoes isomerisation to a second more tightly bound EI complex. But inhibition is still reversible. Goes against M-M kinetics.

66
Q

Examples of irreversible inhibitors?

A

Dilsopropylfluorophosphate (DEP) and alpha-difluoromethyltornithine.

Structure and Function of Proteins (3 decks)

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