Lecture 4

Question 1

How many levels of protein structure are there?

Answer 1

There are four different levels of protein structure.

Question 2

What is a peptide?

Answer 2

Peptide is the term for the shortest proteins, which are typically less than 50 residues in length (in the primary structure)

Question 3

What are polypeptides?

Answer 3

Primary structure with a sequence longer than 50 residues that fold and is part of a quaternary structure.

Question 4

What are proteins?

Answer 4

Proteins are usually defined as polypeptide chains longer than ~50 residues in length

Question 5

Primary structures determine?

Answer 5

Primary structure determines all higher order structure (conformation)

Question 6

Primary structure?

Answer 6

Sequence of amino acid residues linked by covalent peptide bonds.

Question 7

Secondary structure?

Answer 7

Localized folding mediated by H bonds

Question 8

Tertiary structure?

Answer 8

3D packing of entire protein, involving multiple non-covalent and hydrophobic effect

Question 9

Quaternary structure?

Answer 9

Spatial arrangement of polypeptide chains in a multi-subunit protein.
- Each subunit is a polypeptide

Question 10

Peptide bonds limit..?

Answer 10

Peptide bonds limit conformational flexibility. Peptide bonds are planar - they do not rotate.
- There is rotation around the N - alpha C bond and the alpha C-C bond in the peptide backbone. These bond angles are called phi and psi respectively.

Question 11

Why are peptide bonds planar?

Answer 11

Peptide bonds are planar because they are amide bonds, which
have partial double bond character due to resonance. They can move between forms, having partial double bond character

Question 12

What is resonance?

Answer 12

Resonance refers to the delocalization of electrons such that
two arrangements are possible. Note that the second form
(right) is a double bond (which cannot rotate).

Question 13

Dihedral angle preferability?

Answer 13

Some dihedral angles (φ,ψ) are preferred. Rotation about the dihedral angles φ and ψ is constrained in the peptide chains of stable proteins. Some rotational angle combinations are incompatible with certain structures (or never occur at all).

• The φ and ψ dihedral angles define the direction/shape of the peptide chain.

Question 14

Secondary structure - alpha helix?

Answer 14

This structure relies on an H bond between the C=O oxygen of one residue (i) and the H-N hydrogen of
the residue four places forward (toward the C-terminus) at each position of the sequence. (See each
H bond as “—-“ in diagram.)

Question 15

Right handed alpha helix - how many residues per turn?

Answer 15

Right-handed α-helix (3.6 residues or 5.4 Å per turn, Hydrogen bonding is key for stability)

Question 16

R groups of alpha helices?

Answer 16

The R-groups (grey) point outward. Some R-groups destabilize the helix. The R groups of the amino acids determine the surface properties of the α-helix.

Question 17

Proline is an imino acid, where the sidechain is covalently
bonded to the backbone amine. How may this influence
the ability of proline to adopt an α-helical structure?

Answer 17

Once Pro forms a peptide bond, as shown here in Ala-Pro, there is no N-H available to hydrogen bond with a C=O four positions back. So, this residue is considered to be a helix breaker.
- Therefore, they are not normally observed in helices, as they break them.

Reasoning: N is already bound and cannot carry the H as it normally would. That Nitrogen a therefore not hydrogen bond with the carboxyl group or the residues behind it - disrupts H bond that stabilizes the helix and breaking it.

Question 18

Secondary structure - beta sheets?

Answer 18

The beta sheet occurs between two adjacent strands; the two strands can be paired to the same polypeptide/protein chain that looped around and came back, or they can be side by side.

They are an extended sort of zigzag conformation. If you looked at one chain of the protein, it would just be a zigzag; altogether, they form a surface/sheet almost like a sheet of paper.

Question 19

Beta sheets - Hydrogen bonds?

Answer 19

The structure relies on H bonds between the C=O and N-H of residues in adjacent strands of the protein. These bonds hold strands together, forming a sheet.

Question 20

Beta sheets - R groups?

Answer 20

These may stabilize or destabilize the sheet structure, depending on the residues.

Question 21

Beta sheet alignment?

Answer 21

Beta sheets can be parallel or anti-parallel.

Parallel: called beta loops, the strands pointing the same way with their N and C terminus’ pointing the same direction. Bonds are diagonal.

Antiparallel: called beta turns, the strands pointing the opposite ways, N and C terminus’ pointing the opposite ways. bonds are straight

Question 22

Peptide backbone?

Answer 22

Many diagrams that show protein structures show only the “backbone”.
* The backbone is the path through the central C and N atoms that follows the chain of the peptide bonds.
* Three-dimensional diagrams of proteins often only show the path of the peptide backbone.
* Essentially, anything that is in the protein other than the R groups

Question 23

4 types of visualizations of proteins??

Answer 23

Backbone
Wire Frame
Ribbon
Space Fill

Question 24

Backbone?

Answer 24

Basic structure with no R groups

Question 25

Wire Frame?

Answer 25

Same basic info with added structure of R groups

Question 26

Ribbon?

Answer 26

Backbone, formed by secondary structures. Ribbons = helixes Arrows = sheets Lines = beta turns

Question 27

Space fill?

Answer 27

Helps with the surface appearance of the protein, determining if it's charged, or if there's a cavity in the protein. Might be coloured to show charges, hydrophobicity, polarity, particular residues, etc.

Question 28

"Supersecondary" structure?

Answer 28

Motifs and domains

Question 29

Protein motifs?

Answer 29

Small regions with defined sequence or structure that often serve a common function in different proteins

Question 30

Protein domains?

Answer 30

Sub-regions of single polypeptide chains that can fold and function independently (sometimes correlated with exons)

Question 31

Pyruvate?

Answer 31

Pyruvate kinase has 3 domains

Question 32

"EF hand"?

Answer 32

Ca binding motif. Found in a lot of intracellular proteins. It is a helix and a loop with beta sheets and in the middle is a coordinate calcium residue. Occurs in many calcium regulated proteins. - Mix of secondary structures but doesn't have a hydrophobic core or complex folding of tertiary structures.

Question 33

A solution contains a typical protein containing many acidic and basic amino acid residues. If the pH of the solution is less than the pI of the protein: A. The protein will tend to be deprotonated, causing it to have a net positive charge B. The protein will tend to be protonated, causing it to have a net positive charge C. The protein will tend to be deprotonated, causing it to have a net negative charge D. The protein will tend to be protonated, causing it to have a net negative charge E. The protein will have a net charge of 0.

Answer 33

The answer is B. The protein will tend to be protonated, causing it to have a net positive charge. When the protein is at its pI what is its charge? Zero! When the protein is in a more acidic environment (more H+ available), it will tend to acquire some of those protons from the solution. Then, it will be protonated and have a net + charge.

Question 34

What forces stabilize the protein's tertiary structure?

Answer 34

Tertiary structre of a protein is usually an assembly of secondary structures in unstructured bits, like loops, that come together to form a more complex fold that has a core that you really wouldn't be able to see. Below, we have a zigzag protein with a helical structure, beta sheets, metal ion coordination, hydrophobic effects, disulfides & hydrogen bonding and electrostatic/ionic interactions.

Question 35

Tertiary structure bonds?

Answer 35

Other than disulfide bonds, all bonds in the tertiary structure are non covalent. The hydrophobic effecr is not a bond, but is also present and allows a protein to come together around a hydrophobic core, shielding it from the aqueous solvent, making it more energetically favourable.

Question 36

Types of forces stabilizing tertiary structure?

Answer 36

Helical structure Sheet structure Metal ion coordination hydrophobic interactions Disulfide bond Side chain hydrogen bonding Electrostatic attraction - All of these interactions cause the protein to fold into its inactive statewhere the hydrophobic residues are buried.

Question 37

Favourable vs unfavourable folding?

Answer 37

Unfavourable to have any hydrophobic regions sticking out - favourable to have all the hydrophobic regions in together surrounded by hydrophilic regions./

Question 38

Folding in a nutshell?

Answer 38

The hydrophobic effect and favourable interactions cause proteins to fold.

Question 39

The Anfinsen experiment?

Answer 39

Native protein structure is encoded by its sequence. A lot of proteins won't fold on their own in solution (test tube) outside the body. RNAse-A however, is likely to gold up all on its own and doesn't need ANY accessory factors.

Question 40

Process of folding?

Answer 40

1. Native RNAse A catalytically active with proper disulfide bonds 2. Reduce (mercaptoethanol) 3. Denature/unfold (urea) 4. Oxidize first then renature (misfiled protein aggregate due to improper disulfide bond pattern) 4. Renature (remove urea via dialysis) 5. Oxidize (then restart)

Question 41

Protein refolding?

Answer 41

Some (but not all) proteins can refold on their own

Question 42

Molecular folding in action?

Answer 42

*Proteins fold much faster than random chance would allow (ms vs. 10^27 years) * Initial secondary structure elements guide/restrict protein folding (allows less variation in folding and the native form is favoured) * Some intermediates may promote misfolding or aggregation

Question 43

Protein funneling?

Answer 43

Protein folding as free energy "funnel" Check slide 24

Question 44

Protein folding and misfolding?

Answer 44

Folding: 1. Newly synthesized polypeptide 2. folding 3. native struture Misfolding: 1. Newly synthesized polypeptide 2. misfolding 3. misfiled structure 4. aggregation (BAD - clumps of misfolded proteins) 4. degradation (taken apart and reused/metabolized and used for other parts of the cell) 4. refolding (sometimes mistakes are noticed and proteins are refolded right away)

Question 45

Guaranteed protein folding?

Answer 45

Protein folding is not always guaranteed and misfolding can be irreversible with serious physiological consequences

Question 46

Conformational diseases?

Answer 46

Proteins that have a job if folded properly, but if folded improperly they can cause illness or contribute to illness. Ex. Alzheimer's disease is caused by one shift in the human amyloid peptide 1-40, causing a very stable beta sheet stack to form and cause the illness.

Question 47

Mad cow disease cause?

Answer 47

Prions (Mad cow disease) is involved with scrap disease (sheep), mad cow disease (bovines) and several diseases in humans. - Folded tertiary structure is native structure but they refold into amloid conformations thjen they form a new conformation that's bad.

Question 48

Mad Cow disease outbreak?

Answer 48

An outbreak in 1993 (~120,000 cattle) * Abnormal posture, weight loss * Incurable, always fatal, months to decades long incubation period * Can infect humans (variant Creutzfeldt-Jakub disease) – Still a few cases per year * Due to a protein, nothing more (no virus or bacteria)

Question 49

Protein stability?

Answer 49

Native state (N) is the functional state of a protein. Stabilized by: hydrophobic effect enthalpic interactions

Question 50

Protein denaturation?

Answer 50

Denatured state (D) is a non-functional state of protein. Stabilized by: high entropy of the protein

Question 51

What can denature a protein?

Answer 51

Heat, pH extremes, chemicals and mutations can denature proteins

Question 52

Which of these statements about protein folding is false? A. The native state is stabilized by the high entropy of water (hydrophobic effect) B. The native state is destabilized by the low entropy of protein C. Certain residue orientations (phi/psi angles) are more favourable than others D. When folding, a protein samples all possible conformations to find the native state E. The native state is stabilized by favourable enthalpic interactions, such as hydrogen bonds

Answer 52

B and D are false statements!

Question 53

Quaternary structure - hemoglobin A?

Answer 53

Protein complexes contain protein subunits. Can be dimer, trimer, etc. Can be homo (same), or hetero (different). Hemoglobin is a heterotetramer with two alpha subunits and two beta subunits

Question 54

Benefits of hemoglobin structure?

Answer 54

* Small subunits can fold more easily * Subunits can be used in different protein complexes * Quaternary structure can be regulated