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BHCM218 > Module 01 - Section 04 > Flashcards

Module 01 - Section 04 Flashcards

Protein Structure and Function

1
Q

What are the 4 levels of protein structure?

A

Primary, Secondary, Tertiary, Quaternary

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2
Q

What do we mean by “Nested levels” as it pertains to protein structure?

A

The 4 levels of protein structure exist in such a manner that one level higher is comprised entirely of the level below

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3
Q

What is the primary structure of proteins?

A

The linear sequence of amino acids.

It is written and read from the amino terminus to the carboxyl terminus

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4
Q

What are the Peptide backbone constraints?

A

The 4 atoms separating alpha carbon(include carbons) and the 3 covalent bonds lie in the same plane. The peptide bond length is significantly shorter than a normal C-N bond (more like C=N bond), because it is a partial double character due to carboxyl oxygen

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5
Q

Where can the peptide backbone rotate? Where can it not?

A

No rotation around the N-C(alpha)

Free rotation around C(alpha)-C

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6
Q

What are dihedral angles?

A

Torsion angle due to constrained bonds in polypeptide. Phi for N-C(alpha) and Psy for C(alpha)-C

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7
Q

What is a Ramachandran Plot?

A

Way to graphically represent the allowed valued of phi and psi for each amino acid

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8
Q

Why are rotational movements restricted

A

Because of steric hindrance between amino acids side chains

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9
Q

What are the 2 exceptionds to the Ramachandran Plot

A

Glycine, Proline

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10
Q

Why is Glycine an exception to the Ramachandran Plot?

A

Because its side chain is only a H-atom, allowing for a broader range of allowed angles

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11
Q

Why is Proline an exception to the Ramachandran Plot

A

Its structure is greatly restricted because of its cyclic structure covalently bonded to the alph amino group

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12
Q

Which conformations are deemed possible?

A

Conformations that involve little or no interference between atoms. Based on known van der Waals radii and bond angles

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13
Q

How do you read a Ramachandran Plot?

A

Easily allowed conformation are shown in darker shade within the quadrants corresponding to the degree of angle
Lighter = conformations allowed if some flexibilit is permitted in the torsion angles
Unshaded=conformations not allowed or disfavoured

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14
Q

What is the Secondary Structure of Protein

A

Regularly repeating elements within a protein, in which hydrogen bonds form between polar atoms the backbone chain

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15
Q

What are the main secondary structures and residue length?

A

(1) alpha-helix, 10-15 residues long

(2) beta-sheet, 3-10 residues

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16
Q

Which Secondary structure is the most stable?

A

Alpha-helix

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17
Q

What is an alpha-helix?

A

Right-handed helix containing 3.6 amino acids per turn where the H-atom on the amide nitrogen forms a hydrogen bond with the carbonyl oxygen of the 4th residue. The R-group stick out on the outside of the helix

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18
Q

What is the second most stable secondary structure?

A

Beta-sheet

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19
Q

What is a beta-sheet

A

Secondary structure that looks like a pleated sheet. It is made by hydrogen bonding between backbone amide and carbonyl groups of 2 DIFFERENT beta-strands (cant only form one). The R-groups of adjacent amino acids lie on opposite side of the sheet.

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20
Q

What are the beta sheets orientations? (3)

A

(1) antiparallel
(2) Parallel
(3) mix of both

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21
Q

Describe antiparallel beta sheet

A

when beta-strands are oriented in the opposite N- to C-terminal directions

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22
Q

Describe the parallel beta sheet

A

when beta-strands run in the same N- to C- terminal directions

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23
Q

Which beta sheet is more stable and why

A

Antiparallel, because they can form nearly straight hydrogen bonds, which are the most stable of hydrogen bonds

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24
Q

Which amino acids can beta sheet readily accommodate that alpha helix cant?

A 
Aromatic residues (Tyr, Trp, Phe)
and Proline (especially in the edge strands)
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25
Q

What is one function of the beta sheet

A

To form a barrier between greasy and watery environment due to its R groups on opposite sides

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26
Q

Psi and Phi angles that favour Beta sheets

A

Phi: <0 Psi:60 +

1st quadrant ramachandran plot

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27
Q

Psi and Phi angles that favour Right handed alpha helix

A

Phi: <0, Psi: -60

28
Q

Psi and Phi angles that favour left handed alpha helix

A

Phi: 0

29
Q

Describe beta-turns

A

Make a complete reversal of direction using 4 residues

(1) 1st forms a hydrogen bond with amide hydrogen of 4th residue
(2) 2nd and 3rd are not involved in inter-residue bonding
(3) 2nd residue is often proline - readily assume cis conformation which is suitable for tight turns
(4) 3 residue is often glycine - R groups = single hydrogen which can accommodate many conformations

30
Q

Describe gamma turn

A

Complete reversal using 3 residues

1st and 3rd residues form hydrogen bond and 2nd is not involved in inter-residue hydrogen bonding

31
Q

What is the tertiary structure

A

“Packing” or “folding” (3D orientation) of all the different secondary structures, and the turns and lopps that connect them

32
Q

What are the 2 main shape types of proteins

A

(1) Globular

(2) Fibrous

33
Q

Describe fibrous proteins

A

Elongated shape
Play a structural role
ex: collagen and keratin

34
Q

Describe globular proteins

A

Water soluble and spherical in shape

ex: hemoglobin

35
Q

What does the tertiary structure involve?

A

Compromises between tendency of peptide backbones to form the regular helices and sheets and the tendency of the side chains to twist the backbone into less regular configurations

36
Q

When do proteins fold?

A

spontaneously as theyr as biosynthesized

37
Q

Which structure level is more conserved across species and within protein families?

A

tertiary

38
Q

What is the Quaternary Structure of proteins

A

the connections between two or more polypeptide chains

39
Q

What is a dimer?

A

A protein consisting of 2 polypeptides subunits

40
Q

Why is a multi-subunit protein favorable energy wise?

A

If 1 domain of a single large protein did not fold properly the entire protein would lack function and its wasted energy. if 1 subunit misfolds it just wont be included in the oligomer, and the waster energy is much smaller

41
Q

Protein composed of multiple polypeptide is called?

A

Oligomer or multimer

42
Q

Individual polypeptide chains in a multimer are called&

A

subunits of protomers

43
Q

Prefix used when all subunits are identical?

A

Homo-

44
Q

Prefix used when subunits are not all identical?

A

Hetero-

45
Q

What is a domain?

A

A separate folding unit

46
Q

Domain basic facts (3)

A

(1) A protein with 2 or more domain may perform a single function or may have 1 function per domain
(2) Although independent folding, they often interact
(3) you can find the boundaries between domains by assessing the protein structure or by limited proteolysis

47
Q

How can you predict protein folding from the primary structure?

A

you cant yet

48
Q

What are the 4 main steps in the protein purification process?

A

Cell lysis, centrifugation, fractionation, protein detection

49
Q

What is the main goal of cell lysis?

A

Open the cells to get access to the proteins

50
Q

What are the 4 most common methods of cell lysis?

A

Detergent, shear force, low ionic salt, changes in pressure

51
Q

Describe the use of detergent in cell lysis

A

Detergents compromise the intergrity of cell membranes, thereby facilitating lysis of cell and extraction of soluble protein

52
Q

Describe the use of shear force in cell lysis

A

Applying high frequency sonic waves to agitate and break the cell membrane, or by rapidly shaking the sample

53
Q

Describe the use of low ionic salt in cell lysis

A

Treatment of cell cultures with low ionic salt concentrations causes cells to osmotically absorb water and burst easily

54
Q

Describe the use of pressure changes in cell lysis

A

High amounts of pressure can cause the cell to break

55
Q

describe Centrifugation of tissues (4 steps)

A

(1) low-speed, pellets contain whole cells, nucei, cytoskeletong, plasma membranes
(2) Medium-speed, Pellets contain mitochondria, lysosomes and peroxisomes
(3) high-speed, pellets contain microsomes and small vesicles
(4) very high-speed, Pellets contain ribosome and large macromolecules

56
Q

What are the 2 phases of Column Chromatography?

A

(1) Mobile phase; fluid in which the mixture is dissolved

(2) Stationary phase; structure containing another material through which the mobile phase is passed

57
Q

What process is used for fractionation ?

A

Column chromatography

58
Q

Describe the process of column chromatography

A

(1) protein mixture is applied to a column containing resin or matrix that interacts differently with various proteins
(2) Buffer is passed through the column to thoroughly was away any protein that do not bind to matrix
(3) Elution: Another buffer is applied that causes bound proteins to dissociate from the matrix and carry them out in the buffer flow
(4) Eluted proteins are collected in a fraction collector

59
Q

What are the 3 types of column chromatography I should know?

A

(1) Ion exclusion chromatography
(2) Size-exclusion chromatography
(3) Affinity chromatography

60
Q

describe ion-exchange chromatography

A

Proteins are separated by charge

  • Anion exchange resin (coated with cations), which is positively charged and binds cations
  • Cation exchange resin (coated with anions), is negatively charged and binds . cations
61
Q

Describe binding and elution in ion-exchange chromatography

A

Binding:
Use this property to chose the resin;
When pHpI, protein has an increased anionic character (Net -)
Elution:
Add counter-ions (NaCl) to the bound proteins on the column, as they compete for the ionic interactions and lead to elution

62
Q

Describe size exclusion chromatography

A

Column matrix contain beads that have size specific pores. Proteins that can enter the pores will take longer to migrate through the column while the larger proteins will more around the pore and elute earlier

63
Q

Describe affinity chromatography

A

Takes advantage of the fact that many proteins specifically bind other molecules or ligands as part of their functions

64
Q

What is the acronym of sodium dodecyl sulfate-polyacrylamide gel electrophoresis?

A

SDS-PAGE

65
Q

What is SDS-PAGE

A

Protein detection:

Uses an electrical current to separate proteins according to their size within a polyacrylamide matrix

66
Q

Describe the SDS-PAGE process

A

(1) Treated samples are loaded into the wells at the top of the gel, electric field is applied to the gel
(2) Proteins migrate through the gel at different rates according to their molecular mass (smaller = faster)
(3) When proteins have been separated, gel is removed and soaked in acidic buffers to “fix” the proteins to prevent diffusing out of the gel.
(4) gel is treated with a dye that selectively binds to proteins

BHCM218 (24 decks)

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