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biochem > topic 03 protein structure & analysis > Flashcards

topic 03 protein structure & analysis Flashcards

1
Q

when and between what is the peptide bond formed?

A

between alpha-amino group of one amino acid and the alpha-carboxyl group of another in a condensation reaction

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2
Q

how are peptides written?

A

N-terminal first.

Gly-ser does not equal Ser-gly

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3
Q

what is the repeating pattern of a peptide?

A

NCC

NCC-NCC-NCC

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4
Q

even though peptide bond is a single bond, why does it shares characteristics with a double bond? what is steric interference?

A

due to the resonance between the C-N and C-O bonds

the atoms are coplanar and there is no free rotation around the C-N axis which constrains flexibility and could prevent some folding patterns. this is steric interference

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5
Q

define primary, secondary, tertiary, and quaternary structure in general terms

A

primary: sequence of amino acid residues

secondary: local folding pattern of polypeptide backbone

tertiary: 3D structure of entire polypeptide chain including side chains

quaternary: arrangement of polypeptide chains in a protein with multiple subunits

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6
Q

what are the bonds involved in the primary structure?

A

covalent bonds between the residues

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7
Q

what are the bonds involved in the secondary structure?

A

hydrogen bonds between the backbone NH and CO groups

in an alpha-helix, a carbonyl is hydrogen bonded to an amine, 4 residues away

in a beta-sheet, bonds formed between neighbouring beta strands

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8
Q

between a parallel and non-parallel beta-strand, which one is stronger? why? why is the other not as strong?

A

the non-parallel beta-strand:
more stable bc of the straight hydrogen bonding

the parallel beta-strand is not as strong due to the bends and kinks

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9
Q

what are the bonds involved in the tertiary structure?

A

all 4 forces: ionic, hydrogen, disulfide, and van der waal

between side chains or side chain and backbone (distant in the sequence)

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10
Q

what are the bonds involved in the quaternary structure?

A

all 4 forces: ionic, hydrogen, disulfide, and van der waal

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11
Q

what are the two major secondary structures?

A

alpha-helix and beta-strand

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12
Q

which amino acids cause destabilizing kinks in the alpha-helix? why?

A

proline
it can’t fit into into the backbone’s pattern and it has an imidazole ring that constrains bond angles

glycine
too flexible. can disrupt secondary structures

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13
Q

how are side chains position in an alpha-helix? beta-sheet?

A

alpha-helix: protrude out

beta-sheet: up and down

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14
Q

what does the folding of a secondary structure depend on?

A

interaction between side chains

steric interference from large side chains

charge repulsion between side chains

presence of proline/glycine

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15
Q

what is the most important force in the folding of tertiary structures?

A

entropy & the hydrophobic effect

entropy:
when folding, hydrophobic area comes together in protein’s interior, expelling water. even though the peptide’s entropy decreases, as it goes from unfolded to folded, overall entropy increases because of the water’s entropy. when unfolded, the water in the protein shields the hydrophobic parts from the rest of the water. when folded, water is released into the solution and returned to high entropy state

the hydrophobic effect:
proteins are most stable when hydrophobic residues are in the core and hydrophilic on the surface

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16
Q

describe the disulfide bond. where can it be found? why?

A

covalent bond between cysteine residues (their thiol groups)

only present in (oxidized environments) non-cytoplasmic proteins bc in cytoplasmic proteins, there exists enzyme systems which remove the disulfide bonds

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17
Q

give two examples of quaternary protein structures

A

collagen: triple helix made from 3 polypeptides

elastin: made of individual elastin peptides cross linked

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18
Q

why is a folded protein considered its native state?

A

said to be the conformation with the least free energy

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19
Q

what denatures a protein?

A

treatments with solvents that weaken bonds

extreme pH

high temperatures

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20
Q

describe a denatured protein

A

has a random, flexible conformation

usually lacks biological activity

may aggregate or precipitate due to exposed hydrophobic groups

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21
Q

if the denaturing condition is removed, some proteins will refold and regain activity. what does this renaturation tell us?

A

that all the info for folding is within the primary structure

22
Q

describe molecular chaperones

A

proteins that help others fold and renature

they mask hydrophobic areas to prevent aggregation during the process - play important role in newly synthesized proteins

they can also act as a chamber to provide the protein with the right environment

23
Q

define conformation changes

A

movement of domains connected by flexible linkers

24
Q

describe a domain

A

independently folded part of the domain that folds into stable structures

a protein could have one or more domain

a domain is in between secondary and tertiary structure

have structural and functional purposes

25
Q

what are domains derived from?

A

primordial proteins that existed on their own, that’s how they can fold independently

26
Q

what separates domains?

A

separated by loosely folded regions and may create clefts between them

27
Q

describe a protein family

A

proteins related by evolution

they have similar primary sequence, functions, structure, and domains

28
Q

give examples of protein families and describe them

A

lipoproteins: combined with lipids

metalloproteins: combined with proteins

glycoproteins: modified by attachment of carbohydrates

hemoproteins: attached heme-group

29
Q

describe a conserved residue

A

amino acid residue that’s necessary for function and could be found in domains

30
Q

describe membrane proteins

A

inserted or tightly bounded into the membrane

31
Q

define proteases

A

enzymes that break down proteins

32
Q

define homologous proteins

A

have the same protein family

33
Q

what are the purification steps of a protein

A
  1. cell breakage
  2. centrifugation for initial fractionation
  3. column chromatography to separate proteins from one another
  • small samples of the preparations are monitored at different stages of by SDS (sodium dodecylsulfate) polyacrylamide gel electrophoresis (SDS-PAGE)
34
Q

describe SDS-PAGE

A

sodium dodecylsulfate polyacrylamide gel electrophoresis, the most common analytical separation technique

separates by the polypeptide chain size

charged molecule migrates in electric field

gel acts as a molecule sieve: smaller molecules go faster

the gel can be a single concentration or a gradient. gradients are used to examine a range of different protein sizes

a low % gel is used for high molecular weighed proteins

35
Q

what are the different types of liquid chromatography and what differences do they exploit to separate them?

A

gel filtration: size

ion exchange chromatography: net charge

affinity chromatography: presence of binding affinity for a specific ligand

36
Q

what techniques can be used to determine 3D structures?

A

x-ray (crystallography) diffraction or NMR (nuclear magnetic resonance)

cyro-EM (cyro-electron microscopy) can be used to solve structures of large proteins and protein complexes that are difficult to obtain in large amounts and to crystallize

37
Q

describe column chromatography

A

contains insoluble beads with different characteristics

applies proteins in solution where they will pass through and around - some will emerge from the bottom where they’re collected and separated due to the time it takes to go through. a “fraction” is collected every few minutes

38
Q

what is effluent?

A

discharged liquid collected in fractions - every few minutes

39
Q

how does one tell which collected fraction (effluent) contains proteins?

A

using the absorbance at 200-280 nm

aromatic amino acids absorb at 280nm

other amino acids at 200nm due to carboxyl group

40
Q

describe gel filtration chromatography

A

the beads contain holes

if a protein gets into the hole, it is slowed down - otherwise, it will come out early. this is an example of “size exclusion”.

smaller proteins fit into the bead more easily, therefore, larger proteins are more likely to elute faster

41
Q

describe ion exchange chromatography

A

relies on attraction of opposite charges

to remove proteins that are stuck on sides, increase salt concentration or change the pH

42
Q

define isoelectric point (PI)

A

pH at which a specific molecule carries no net electrical charge

if pH < PI, there’s a positive charge

43
Q

describe affinity chromatography

A

relies on interaction between protein and a specific ligand

if the ligand is attached to a column matrix, certain proteins will bind to it and unbound proteins are washed away by the buffer

most powerful chromatography method but needs the knowledge of what ligand to use

protein of interest is eluted by adding competing free ligand, or by changing the pH which changes the ionization of the group responsible for binding

44
Q

what is polyacrylamide?

A

long chain of hydrophilic polymer

the gel in SDS-PAGE is polyacrylamide

45
Q

what does the SDS in SDS-PAGE do?

A

denatures proteins and give a negative charge

this must be done before electrophoresis

beta-mercaptoethanol added to break disulfide bonds

46
Q

differentiate between old and new technological methods of analysis

A

old tech
- purify proteins, then sequence (chemical method)
- identify genes, then deduce amino acid sequence

new tech
- use mass spectrometry to characterize proteins in complex mixture (proteomes)
- can provide info on identity (sequence), abundance, and various modifications

47
Q

what can mass spectrometry measure?

A

molecule size

mass/charge ratio

whether it’s protonated or not

48
Q

what are the steps of mass spectrometry?

A

now, steps 1-3 can be forgone

  1. extract protein
  2. run on SDS gel
  3. cut out desired band
  4. resolved by liquid chromatography. before this step, sample is treated with an enzyme to fragment the proteins into manageable bits

LC-MS
5. sample run in mass spectrometer 2
6. mass determination

LC-MS/MS
5. mass spectrometer
6. collision cell
7. mass spectrometer 2
8. amino acid sequence identification

49
Q

describe x-ray crystallography

A

obtain a highly pure protein, then get it to form a crystal by concentrating protein solution so much - then blast it with x-rays. a pattern will be caught on the detector due to the diffraction

diffraction pattern of crystal = diffraction pattern of molecules

the diffraction pattern yields an electron density map

50
Q

describe NMR (nuclear magnetic resonance)

A

can determine the structure of the solution directly (in the solution, the protein has more conformational freedom)

the environment of the nucleus affects the magnetic resonance

generates a 2D NMR for 2 different nuclei in protein (H & a heavy isotope)

atm, NMR can only be used for small proteins

51
Q

describe and list steps of cryo-EM

A

cryo-electron microscopy. used for larger proteins

  1. vitrify solution: preserve proteins in native solution state
  2. electron microscopy: imaging electron density to build model of structure - image one single molecule at a time

biochem (15 decks)

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