Lecture 4

Question 1

Big Picture Items

Answer 1

• Amino acids are the building blocks of proteins
• Stretches of proteins often fold into “α-helices” and “β-strands”
• α-helices and β-strands are building blocks of “domains”
• Domains are building blocks of proteins
• Fibrous and globular proteins differ in architecture
• Membrane proteins are often all-α helix or all-β strand
• Hydrophobic interactions play a key role in protein folding
• All proteins undergo continuous thermal motion
• Many proteins undergo functional conformational changes

Question 2

Primary Structure

Answer 2

Linking amino acids by forming peptide units.

The order of the amino acids is called the “Primary Structure” of a protein

Question 3

Secondary Structure

Answer 3

helix

Question 4

tertiary structure

Answer 4

one complete protein chain (b chain of hemoglobin)

Question 5

quarternary structure

Answer 5

the four separate chains of hemoglobin assembled into an oligomeric protein

Question 6

Linking amino acids together via peptide bonds

Answer 6

An extended chain of a polypeptide

Emphasizing the planar peptide units (blue-greenish)

Question 7

Two main chain torsional angles per residue:

phi (Φ) and psi (Ψ)

Answer 7

If one peptide unit is kept fixed, Φ and Ψ define the orientation of the second peptide unit.

So, in a first approximation, the course of a polypeptide chain is defined by a pair of dihedral angles (Φ and Ψ) per amino acid residue.

Each peptide unit contains six atoms: Cα, C, O, N, H and the next Cα.

Question 8

The definition of phi (Φ)

Answer 8

If one peptide unit is kept fixed, Φ and Ψ define the orientation of the second peptide unit.

So, in a first approximation, the course of a polypeptide chain is defined by a pair of dihedral angles (Φ and Ψ) per amino acid residue.

Question 9

The definition of psi (Ψ)

Answer 9

If one peptide unit is kept fixed, Φ and Ψ define the orientation of the second peptide unit.

So, in a first approximation, the course of a polypeptide chain is defined by a pair of dihedral angles (Φ and Ψ) per amino acid residue.

Question 10

Secondary Structure Elements

Answer 10

• Proteins fold in complex ways with variations in Φ, Ψ angles per residue.
• In many instances, however, there are stretches of amino acids in a protein with a more regular structure
• These stretches have “secondary structure”
• In globular proteins, the major secondary structure elements
are:
• The α-helix
• The β-strand

Question 11

The α-helix

Answer 11

VITAL CHARACTERISTICS:
• 1.5 Å rise per residue
• 3.6 residues per turn
• 100 degrees RIGHT-HANDED rotation from residue to residue viewed along the axis
• For all residues the same main chain dihedrals:
Φ ≈ −570 and Ψ ≈ −470
• Hydrogen bonds between:
• main chain C=O of residue n
• the main chain NH of residue n+4

Question 12

Right-handed and left-handed helices

Answer 12

A (discontinuous) helix is defined by:

1. An object (e.g. a step from a staircase)
2. The helix axis
3. A rotation of the object about the helix axis
4. A translation of the object parallel to the helix axis

Question 13

Myoglobin

Answer 13

The predicted α-helix was confirmed when this protein structure was determined.
Myoglobin is an “all-α” soluble, globular protein. It contains eight α-helices.

Myoglobin also contains a “heme group” which contains an Fe(II)ion.

Question 14

β-sheets: Antiparallel and parallel β-strands

Answer 14

NH to C=O hydrogen bonds between peptide units from different parts of the chain

Note the different pattern of hydrogen bonds in parallel versus antiparallel

Question 15

β-sheets form a “pleated sheet”

Answer 15

In both parallel and anti-parallel β-sheets:
The side chains point alternatingly in opposite directions

There are also many MIXED ß-sheets, with some strands parallel and others antiparallel

Question 16

β-sheets in globular proteins are often twisted

Answer 16

Carboxypeptidase A consists of a central MIXED β-sheet surrounded by α-helices.

The twist of the sheet is LEFT-handed when viewed in the plane of the sheet perpendicular to the β-strands.

This is due to the fact that when viewed along the β-strand, each strand has a RIGHT-handed twist.

This left-handed twist of the β-sheet is similar in parallel, anti-parallel and mixed β-sheets.

Question 17

FIBROUS PROTEINS: Keratin and coiled coil α-helices

Answer 17

α-keratin is the principal protein of mammalian hair, nails, skin.

Question 18

Keratin and coiled coil α-helices

Answer 18

The central 310-residue portion of α-keratin has a pseudo-repeat sequence a-b-c-d-e-f-g with nonpolar residues at a and d.

Note left-handed twist of theright-handed (!) α-helices.

In the coiled-coil structure the a:d’ and d:a’ contacts make a hydrophobic strip of contacts.

Question 19

Fibrous Protein: The Collagen Triple Helix

Answer 19

Collagen: the most common vertebrate protein
A single collagen molecule consists of three
chains

Question 20

Type I Collagen:

Answer 20

• Two α1 and one α2 chains
• Each chain about 1000 residues long
• Width: 14 Ǻ, length: ~ 3000 Ǻ
• About 30% of residues are Gly, 15-30% are 4 hydroxyPro
• Repeat of Gly-X-Y, often Gly-Pro-Hyp
• Three of these helices are wound around each other with a gentle, RIGHT-handed twist
• A Gly at every third position in the chain makes it possible to bring the chains very close together in the center of the triple helix.

Question 21

The collagen triple helix

Answer 21

Interchain hydrogen-bonding pattern viewed along the collagen helix axis.

• Three consecutive residues from each chain shown in ball-and-stick
• Dashed lines are hydrogen bonds from Gly NH to Pro O in adjacent chains
• Dots show van der Waals surfaces.
• Note the close packing near the helix axis made possible by glycine residues at every third position.

Question 22

Handedness summary

The α-helix:

Answer 22

The α-helix:

• RIGHT-handed, when viewed along the helix axis.

Question 23

Handedness summary

In ß-sheets:

Answer 23

In ß-sheets:
• The individual ß-strand is slightly RIGHT-handed twisted, when viewed ALONG the length of the strand.
• In a ß-sheet, the strands are arranged LEFT-handed with respect to each other, when viewed IN the plane of the sheet PERPENDICULAR to the strands.

Question 24

Handedness summary

In α-keratin:

Answer 24

In α-keratin:
• Each individual α-helix is RIGHT-handed
• The two helices are twisted around each other in a LEFT-handed manner.

Question 25

Handedness summary

In collagen:

Answer 25

In collagen:

• The three strands are twisted around each other in a RIGHT-handed manner

Question 26

Domains

Answer 26

Combinations of many secondary structure elements by turns and loops, and form a compact unit.

Question 27

Triose Phosphate Isomerase (TIM)

Answer 27

A domain which occurs in a many proteins.

Note the “β-barrel” in the center surrounded by α-helices

Note the 8-fold repeated β-α motif

Question 28

Membrane proteins are often either all-α or all-β

Answer 28

The protein avoids placing main chain C=O and NH groups in the hydrophobic bilayer)

Bacteriorhodopsin: α-HELICES crossing the membrane

OmpF Porin: β-BARREL crossing the membrane

Question 29

Multi-domain proteins are very common

Answer 29

Combining domains is a way to to bring different functions of different domains together

N-terminal domain (gold) and C-terminal domain (blue) of the enzyme:
“Glyceraldehyde-3-phosphate dehydrogenase” (GAPDH)
(Actually only one subunit from the tetrameric protein GAPDH is shown)

C-terminal domain:
Substrate binding

N-terminal domain:
Cofactor binding

Question 30

Proteins are only marginally stable:

Answer 30

A 100 residue protein is typically stable by only about 10 kcal (40 kJ) per mole.

Proteins have not evolved to be optimally stable, they have evolved to be optimally functional.

(Note: proteins are often modular, i.e. folded into multiple domains.
Therefore larger proteins are usually not more stable than smaller proteins)

Question 31

Electrostatic effects:

Answer 31

Salt bridges between opposite charges relatively weak due to electrostatic screening by water

Question 32

Disulfide bonds:

Answer 32

SS-bridges are an important stabilizing factor for extracellular proteins;

intracellular proteins very rarely have SS-bridges.

Question 33

Hydrogen bonds:

Answer 33

Important to make H-bonds in native state since they are made with water in the unfolded state;
Native proteins almost never have unpaired donors/acceptors in the core!

Question 34

Van der Waals

Answer 34

interactions:

Important to make these in the native state since they are made with water in the unfolded state

Question 35

Conformational Entropy:

Answer 35

The protein has a much greater entropy in the unfolded than in the folded state!

Question 36

Hydrophobic interactions:

Answer 36

Nonpolar sidechains come together in the folded protein to minimize the contact with water.
A major determinant of protein stability is the entropy gain of bulk water!

Question 37

Protein denaturation (in vitro)

Answer 37

Loss of secondary, tertiary & quaternary structure without peptide bond hydrolysis.

Popular denaturants:
• Heat
• Guanidinium chloride (6M)
• Urea (8M)
Breaking SS bridges is not required for denaturation, but is required to arrive at a true random coil.
β-Mercaptoethanol (BME) is often used to reduce SS bridges.

Denaturation is often reversible!

Question 38

Sequence determines the structure of a protein

Answer 38

The conclusion of Anfinson on protein denaturation-renaturation was that the primary sequence dictates the folded structure.

Hence: the secondary and tertiary structure is determined by the protein’s sequence.

Question 39

Ribonuclease A

Answer 39

After denaturation, refolding needs careful attention regarding oxidation agents in order
to obtain correct disulphide bridges.

Question 40

Myoglobin:

Answer 40

After denaturation and dialysis, it was found necessary to provide the heme moiety to
obtain native protein.

Question 41

Muscle aldolase:

Answer 41

This tetrameric protein could be recovered in high yield after acid denaturation, showing
that assembly of the quaternary structure also follows from the protein’s sequence.

Question 42

Folding pathways and energy landscapes in protein folding

Answer 42

Folding pathway
(hypothetical, yet
capturing current
thinking):

Proteins fold in a
hierarchical manner.
First, small local
elements of secondary
structure form.

Then, these coalesce
to yield larger
supersecondary
structure units.

These units coalesce
with other units to
form larger elements:
domains and the
complete folded chain.

Question 43

Atoms are closely packed in the interior of a protein

Answer 43

Proteins are usually packed as tightly as organic crystals

However, there are two types of motion which are critical:

1. Thermal motion around equilibrium positions of all protein atoms;
2. Functional motions (“conformational change”) in response to
   - encounters with other molecules
   - changes in pH

Question 44

Conformational Change: Calmodulin

Answer 44

Protein structure is important.

Yet, without functional conformational changes of proteins, life would be pretty miserable.

Question 45

Atoms are Dynamic

Answer 45

Thermal motions around the equilibrium positions of atoms

Molecular Dynamics of Myoglobin

Calculated “snapshots” of myoglobin at intervals of 5 x 10-12 seconds superimposed

The protein is only represented by a Cα trace – blue lines

The heme group is yellow; a key histidine residue is gold.

Question 46

Proteins are Dynamic

Answer 46

Thermal motions around the equilibrium positions of atoms can be surprisingly large as seen in a movie produced by a special purpose molecular dynamics computer called “Anton”.