Biochemistry - Ch 06 - Lecture 8 Thru 13 - Protein Structure And Folding

Front 1

Define:

Primary Structure

Back 1

Linear sequence of amino acids

Front 2

Define:

Secondary Structure

Back 2

Made up of α-helices and β-sheets.

How polypeptide backbone is folded locally. Held together by hydrogen bonds between the carboxyl groups and the amino groups in the polypeptide backbone.

Front 3

Define:

Tertiary Structure

Back 3

3d shape of the entire protein.

The tertiary structure is held together by bonds between the R groups of the amino acids in the protein, and so depends on what the sequence of amino acids is.

Front 4

What are the 3 types of bonds that bond Tertiary Structures together?

Back 4

Hydrogen Bonds - which are weak.

Ionic Bonds - between R-groups with positive or negative charges, which are quite strong.

Disulfide Bonds - covalent S-S bonds between two cysteine amino acids, which are strong.

Front 5

Define:

Quaternary Structure

Back 5

Spatial arrangement of more than 2 polypeptide chains together.

Front 6

Peptide bonds are formed through _____ reactions.

Back 6

...condensation...

Front 7

How many atoms participate in a peptide group?

Which ones are they?

Back 7

6 atoms.

2 alpha-carbon atoms on either side of the peptide bond (C-N).

1 Double bonded oxygen coming off of C of C-N

1 Hydrogen coming off of N of C-N

2 - The C-N peptide bond itself.

Front 8

A peptide group lies in a _____ _____.

Back 8

...single plane.

Front 9

Peptide groups are usually found in _____ (cis/trans) conformation.

Back 9

...trans...

Front 10

Relative orientation around the peptide groups is specified by what?

Back 10

Φ (phi) and Ψ (psi) angles.

Front 11

Which way does a torsion angle (psi or phi) rotate to increase its angle?

Back 11

Looking down the alpha Carbon toward either N or C, the angle increases by rotating *clockwise*

Front 12

Why are most Φ and Ψ angles not possible?

How can this be represented with a picture?

Back 12

Because of steric hindrance.

This can be represented with a Ramachandran Plot; a plot showing which Φ and Ψ angles are sterically possible.

Front 13

Which amino acid is the most restricted in terms of the Φ and Ψ angles it can take on?

Back 13

Proline.

Front 14

Φ (phi) refers to rotation around which bond?

Back 14

Cα - N

Front 15

Ψ (psi) refers to rotation around which bond?

Back 15

Cα - C

Front 16

Φ and Ψ are called...

Back 16

...torsion angles or dihedral angles.

Front 17

What three things (single letter variables) are helices characterized by?

Back 17

p, n, and d

p ≡ Pitch distance. The distance the helix rises along its axis per turn.

n ≡ # of repeating units per turn.

d ≡ helical rise per repeating unit (p/n)

Front 18

What are 4 characteristics of α-helices?

Back 18

• Stabilized by H-bonding within
helix/strand

• right-handed

• 3.6 residues per turns

• Pitch is 5.4 Å

Front 19

How do you distinguish between a right-handed and a left handed helix?

Back 19

Looking down the middle (axis) of the helix...

...if the helix turns clockwise, it is right-handed.

...if the helix turns counterclockwise, it is left handed.

Front 20

Define:

Helix capping

Back 20

The last 3-4 residues at the end of a helix are not completely hydrogen bonded, so the polypeptide folds back to provide hydrogen donors or acceptors.

Front 21

Define:

β - Sheets

Back 21

Hydrogen bonding occurs between neighboring polypeptide chains.

Front 22

What are the two ways β-Sheets can be oriented?

Describe each setup.

Back 22

Parallel:
Neighboring polypeptides run in the same direction.

Antiparallel:
Neighboring hydrogen bonded polypeptide chains run in opposite directions.

Front 23

4 points that summarize β-Sheets:

Back 23

1. parallel

2. antiparallel

3. most are mixed

4. sheets are pleated and twisted

Front 24

Define:

β-Bilges

Back 24

Amino acid that is not h-bonded to the neighboring strand. Results in a bulge.

Front 25

2 points that summarize the polypeptide backbone:

Back 25

1) Peptide group is planar

2) Conformation of two amine planes around alpha-carbon can be described in phi (Φ) and psi (Ψ) angles.

Front 26

Define:

Regular Secondary Structures

Back 26

Composed of sequences of residues with repeating Φ and Ψ values.

Front 27

Describe the structure of fibrous proteins.

Back 27

Dominated by a single type of secondary structure.

Front 28

What are the 3 types of fibrous proteins we talked about?

Back 28

1) Keratin

2) Fibroin

3) Collagen

Front 29

What role do fibrous proteins play in the body?

Back 29

Protective
Connective
Supportive

Front 30

What are some examples structures with α-keratin in them?

Back 30

Skin, hair horns, feathers

Front 31

Name 7 physical characteristics of:

α-keratin

Back 31

• Two α-helices (right-handed)

• Parallel

• Left handed coiled-coil ( for both helices together)

• The pitch is 5.1 Å (instead of 5.4 Å)

• Stabilized by van der Waals forces

• 7 residues pseudo-repeat (a-b-c-d-e-f-g)

• a and d are hydrophobic

Front 32

Where are hydrophobic and hydrophilic residues located in α-keratin?

Back 32

Hydrophobic residues are on the inside of the coil.

Hydrophilic residues are on the outside

Front 33

Name the 4 structures of α-keratin, from smallest to largest.

Back 33

α-helix --->

Dimer --->

Protofilament --->

Microfibril

Front 34

In α-keratin, dimers are able to cross-link to each other via...

Back 34

...disulfide bonds.

Front 35

The two helices in dimers in α-keratin are kept together via...

Back 35

...hydrophobic interactions.

Front 36

Name the 3 forces that help stabilize α-keratin.

Back 36

1) Individual α-helices are stabilized by hydrogen bonds between backbone residues

2) vdW forces between monomers
7 residue pseudo repeat
H-phobic strip along the side of the monomers

3) disulfide bonds between dimers

Front 37

Define:

Fibroin

Back 37

A stack of β-Sheets

Front 38

What is the most abundant vertebrate protein?

Back 38

Collagen

Front 39

What bodily structures does collagen help provide strength to?

Back 39

Bone
Cartilage
Tendons
Skin
Blood vessels

Front 40

How many α-helix coils make up collagen?

Back 40

3

Front 41

What 3 amino acids largely make up the structure of collagen?

Back 41

Glycine (35%)

Proline (20%)

Alanine (10%)

Hint 41

GPA

Front 42

What are the 5 characteristics of the Quaternary structure of Collagen?

Back 42

1. 3 helices run parallel

2. Wind around each other
(right handed helix)

3. Glycine in the center,

4. Proline on the outside

5. Molecule is stabilized by H-bonds

Gly N-H to Pro C=O

Front 43

Collagen:

Prolyl is converted to _____ by what enzyme?

When does this reaction take place?

What molecule does the enzyme require to maintain its activity?

Back 43

...hydroxyprolyl..., by prolyl hydroxylase

Reaction takes place after proline is incorporated into the growing polypeptide.



Vitamin C

Front 44

Vitamin C deficiency could lead to what disease in humans?

Back 44

Scurrrveee (arr!)

Front 45

Collagen:

Lysyl is converted to...

What do these residues function as attachment sites for?

Back 45

...hydroxylysyl

Sugars

Front 46

Describe the first 2 steps in the process of cross-linking allysine residues.

Back 46

pg. 139

(1) Lysyl oxidase deanimates (gets rid of amine) Lysine to form Allysine.
*Step (1) happens to two Lysine AA's.

The two Allysine react together via. an aldol condensation reaction (releases water) to form allysine aldol.

This reaction creates a double bond between the two allysine molecules.

Front 47

True or False:

In native (folded) proteins, non-repetitive structures are less ordered than helices and β-Sheets.

Back 47

False.

They are simply irregular and hence more difficult to describe.

Front 48

What are 2 methods that can be used to determine protein structure?

Back 48

1) X-Ray Crystallography (XRC)

2) Nuclear Magnetic Resonance (NMR)

Front 49

What are the 5 steps to viewing a protein via. X-Ray Crystallography?

Back 49

1) *Purify* protein

2) Produce a *crystal*

3) Expose crystal to a beam of x-rays to get a *diffraction pattern*

4) Translate diffraction pattern data into an *electron density map*.

5) Fit polypeptide sequence to the map.

Front 50

Crystals are made up primarily of what?

Back 50

40-60% water

Front 51

What are 3 characteristics of NMR?

Back 51

1) Protein is in solution

2) Relatively small proteins ( < 40 kDa)

3) NMR gives information about the *distance between atoms*.

Front 52

What are the 5 stages of NMR?

Back 52

1. Get nuclei with magnetic moment (^1H, ^13C, ^15N)

2. Align in a magnetic field

3. Excite with radio frequency pulses

4. Reversion to low energy state and
emission of radio frequency signal

5. Frequency gives information about the
molecular environment

Front 53

When constructing a picture of the protein, NMR is limited by what kind of restraint?

Back 53

Distance restraint (between the two nuclei)

Front 54

What are the 3 main differences for X-Ray Crystallography and NMR?

Back 54

XRC:
1) Crystal
2) Electron density map
3) No size limitation

NMR
1) In solution
2) Distance restraints
3) 40 kDa size limit

Front 55

What 3 models/methods are used to structurally visualize proteins?

What are some of their advantages/disadvantages?

(This is not X-Ray Cryst. and NMR)

Back 55

1) Ball and Stick - Gives details, but is cluttered.

2) Space fitting - Only shows the outside

3) Artistic rendition - Gives overview, but is imprecise.

Front 56

What 3 main things have we learned from the 60,000 protein structures we've solved?

Back 56

1) Side chain location varies with polarity

2) Helices and β-strands/sheets combine in a variety of ways

3) Large proteins contain domains.

Front 57

AA Side chain location in globular proteins varies with _____.

Back 57

...polarity.

Front 58

AA Side chain location in globular proteins:

Which 5 AA's are mainly found in the center?

What type of polarity do they possess?

Back 58

{M, I, L, F, V}

Methionine
Isoleucine
Leucine
Phenylalanine
Valine

**NON-POLAR RESIDUES**

Hint 58

IV MLP

Front 59

AA Side chain location in globular proteins:

Which 5 AA's are mainly found on the surface?

What type of polarity do they possess?

Back 59

{R, H, K, D, E}

Arginine
Histidine
Lysine
Aspartic Acid
Glutamic Acid

**CHARGED POLAR RESIDUES**

Front 60

AA Side chain location in globular proteins:

Which 5 AA's are mainly found on the outside?

What polarity do they possess?

Back 60

{S, T, N, Q, Y}

Serine
Threonine
Asparagine
Glutamine
Tyrosine

**UNCHARGED POLAR RESIDUES**

Hint 60

3 of the 5 are AA's with -OH groups

Front 61

Globular proteins are built from...

Back 61

...combinations of secondary structural elements

Front 62

Define:

Supersecondary structures (motifs)

Back 62

Groupings of secondary structural elements in globular proteins.

Front 63

What are the 4 types of supersecondary structures (motifs) we looked at?

Back 63

βαβ motif - alpha helix connects two beta sheets

β hairpins - antiparallel strands connected by relatively tight turns

αα motif - two successive antiparallel alpha helices pack against each other with their axes inclined.

β barrels - β-sheets rolled up.

Front 64

Define:

Domains

Back 64

The separate globular clusters of large proteins.

Front 65

What are 5 characteristics of Domains?

Back 65

1) Two or more layers of secondary structures.

2) Function independently

3) Fold independently

4) Connected by a single polypeptide strand

5) Binding sites for small molecules at the interface between two domains

Front 66

Define:

Kinase

Back 66

An enzyme that transfers a phosphoryl group between ATP and another molecule.

Front 67

Domains assume their normal 3d structure when...

Back 67

...isolated from the rest of the protein.

Front 68

What has the greatest influence on protein stability?

Back 68

The Hydrophobic Effect

Front 69

Define:

Hydrophobic Effect

Leads to what?

Back 69

Tendency of water to minimize its contacts with nonpolar substances.

Induces substances to aggregate.

Front 70

Define:

Ion Pairs (i.e. Salt Bridges)

Back 70

Association of two ionic protein groups of opposite charge.

EXA: Lysine (+'ve) and Glutamate (-'ve)

Front 71

Ion pairs are located mostly on the _____ of proteins

Back 71

...surface...

Front 72

How can metal ions influence protein structure?

What is an example?

Back 72

Metal ions may function internally to cross-link proteins.

EXA = Zinc finger

Front 73

True or False:

The aggregate of all the forces that hold proteins together makes them rigid structures.

Back 73

False.

Proteins are very dynamic structures.

Front 74

Define:

Breathing (in terms of proteins)

Back 74

Conformational flexibility

Allows for structural displacement of up to 2 angstroms, and for small molecules to diffuse in and out of the interior of some proteins.

Front 75

What are 4 things that can lead to protein denaturization?

Back 75

1) Increase in temperature

2) pH charge distribution

3) Detergens
Interact with non-polar side chains

4) Chaotropic agents
Increase the solubility of nonpolar sidechains

Front 76

How do the functions of Detergens and Chaotropic agents lead to protein denaturization?

Back 76

They interfere with hydrophobic interactions (i.e. the main force that maintains protein stability)

Front 77

What are two examples of chaotropic agents?

Back 77

Guanidinium

Urea

Front 78

What are 2 reasons that protein folding in-vivo is different than in-vitro?

Back 78

1) Proteins start folding during synthesis in-vivo.

2) Proteins are present in-vivo that help with folding

Front 79

What are the 3 steps in the protein folding pathway?

Back 79

1) Elements of secondary structure fold. (5 ms)

2) Molten globule (collapsed state from step (1)) (5 - 1000 ms)
- Further stabalization of secondary structures.
- Formation of super-secondary (motifs) structures or subdomains.

3) Protein 'wobbles' into its final position over the next few seconds.

Front 80

What does the trough on a "Folding Funnel" graph represent?

Back 80

The native structure of the protein.

Front 81

What 2 functions does PDI (Protein Disulfide Isomerase) perform?

Back 81

1 - Rearranges disulfide bonds

2 - Catalyzes initial disulfide bond formation

Front 82

Why is PDI needed to rearrange disulfide bonds?

Back 82

Because improperly folded proteins expose non-polar regions on their surfaces. This would promote aggregation of proteins due to the hydrophobic effect.

Front 83

Define:

Chaperones

Back 83

Help proteins fold properly


Detailed (pg. 165)
Essential proteins that bind to unfolded and partially folded polypeptide chains to prevent the improper association of exposed hydrophobic segments that might lead to non-native folding as well as polypeptide aggregation and precipitation.

Front 84

Describe the GroES/GroEL reaction cycle

Back 84

pg. 166 - 168

Improperly folded protein = *

Big barrel binds * via. it's hydrophobic parts.

1 - GroES caps off GroEL, resulting in a conformational change.
In this change, the upper chamber expands, hydrophobic portions turn into hydrophilic (allowing release of *) while helping to pull * apart allowing for it to fold back into its native shape.

2 - Hydrolysis of 7 ATP's releases 7 phosphates. This weakens GroES/GroEL connection.

3 - 7 ATP bind to bottom of barrel, along with another *.

4 - Binding from (3) releases GroES, 7 ADP, and the now properly folded protein. GroEL turns 180 degrees and the process can start over.

Front 85

What are 4 reasons for proteins to make multiple subunits?

Back 85

1 - There can be different sites for subunit and end-product assembly.

2 - Defective subunits can be replaced.

3 - Less genetic information.

4 - Additional properties for protein (cooperation)

Front 86

Define:

Oligomer

Back 86

A protein containing multiple subunits

Front 87

Define:

Protomer

Back 87

The identical (repeating) subunits of a protein.

EXA:
In hemoglobin, there are 2 α parts and 2 β parts.
1 protomer is one αβ complex.

*Note: Protomer is not the two repeated α's or β's as a group.

Front 88

What are 3 properties of multi-subunit proteins?

Back 88

1 - The have identical or non-identical subunits

2 - They're usually non-covalently associated

3 - Interfaces often contain tightly packed non-polar residues.

Front 89

Proteins only have _____ symmetry.

Back 89

...rotational...

Front 90

Define:

Cyclic symmetry

Back 90

Protomers are related by a single rotational axis.

Front 91

In Dihedral symmetry, how do you find 'n' in:

D_n

Back 91

(total # of subunits) / 2

Front 92

Which dihedral symmetry is the most common?

Back 92

D_2