Biochem Ch. 4 Pt 1

What are the 4 tiers/levels of protein structure?

Primary, Secondary, Tertiary, Quaternary

What defines primary structure?

Amino acid sequence & location of S-S (disulfide) & amide bonds, held by covalent bonds

What defines secondary structure?

Interaction of amino acids close to each other forming specific patterns (eg alpha helix, beta sheet), stabilized by H-bonds

What defines tertiary structure?

3D structure of a single polypeptide (folded shape), stabilized by mostly hydrophobic effect (drivwn by entropy of water molecules)

What defines quaternary structure?

Arrangement of multiple folded protein subunits in a multi-subunit protein, stabilized by only hydrophobic effect (no covalent/disulfide bonds - compared to tertiary where we also do)

What % double bond (DB) character does the peptide bond have?

40% DB character (shown by Pauling & Corey using X-ray)

Why does the peptide bond have partial double bond character?

Resonance between the C=O and C-N forms (lone pair on N delocalizes into C=O)

What does the 40% DB character of the peptide bond cause?

It makes the peptide bond planar, so there is no rotation around it/ partial positive on N & neg on O-

Because the peptide bond is planar with no rotation, what isomerism can occur?

Cis/trans isomerism

Which peptide bond configuration is far more common, cis or trans?

Trans (nearly all peptide bonds are trans - more stable)

What % of peptide bonds preceding a proline may be cis?

About 10%

Since the peptide bond itself cannot rotate, where does rotation occur in the backbone?

Around the alpha carbon (Cα)

What is the name of the dihedral angle at Cα-N?

Phi (φ)

What is the name of the dihedral angle at Cα-C?

Psi (ψ)

What are phi and psi values for a fully extended polypeptide chain?

Both +180° or -180°

What limits rotation around phi and psi angles?

Steric hindrance (nonbonded atom clashes)

What is a Ramachandran plot?

A calculated plot of all sterically allowed psi & phi angles for a polypeptide chain

Who created the Ramachandran plot and when?

G.N. Ramachandran, 1963

What does the Ramachandran plot show about possible conformations?

Only a limited amount of permissible configurations exist - lots of empty space; (shown as shaded areas on the graph)

What is protein secondary structure defined as?

The general 3D form of local segments of biopolymers giving regular, recurring local conformations

Give examples of protein secondary structure elements

Alpha helix, beta strand, beta bend, collagen triple helix, loops & turns

What atoms form H-bonds in secondary structure?

Backbone amide NH of one amino acid & backbone carbonyl C=O of another

Who proposed the alpha helix and when?

Linus Pauling (with Robert Corey), through model building in 1951

Between which residues do H-bonds occur in an alpha helix?

Between residue n and residue n+4 (they form between adjacent AA’s to give curved polypeptide helix).

( n —> n+4 = alpha helix)

What Nobel Prize did Linus Pauling win and for what?

Nobel Prize in Chemistry, 1954, for research into the nature of the chemical bond and its application to elucidating the structure of complex substances

Who are considered the founders of Structural Biology?

Linus Pauling and Robert Corey

In the alpha helix, how do R groups point relative to the helix axis?

Outward, ~perpendicular to the axis

In the alpha helix, how are H-bonds oriented relative to the helix axis?

~Parallel to the helix axis

What is the pitch of an alpha helix? ****!

5.4 Å (0.54 nm) (the height of one complete 360 turn)

How many residues per turn does an alpha helix have? ****!

3.6 residues/turn

What is the rise per amino acid in an alpha helix? **!

1.5 Å/AA (5.4/3.6) - (distance of one amino acid residue)

What handedness are most alpha helices?

Right-handed (left-handed alpha helices, αL, are rare)

Does the alpha helix have a dipole?

Yes—all C=O groups point toward the C-terminus, giving the whole helix a dipole with (+) N-terminus and (-) C-terminus

What charge is typically found at the C-terminus of a stable alpha helix and why?

A positively charged amino acid, to neutralize the helix dipole moment (C=Oδ-)

Name a protein that is predominantly alpha helices (all-alpha)

Myoglobin - only secondary structure

Why does proline destabilize an alpha helix?

Its cyclic side chain sterically destabilizes the helix, so proline is not likely found in alpha helices

What other factors destabilize an alpha helix besides proline?

Steric/charge repulsion from adjacent bulky or like-charged amino acids; R-groups too large (tryptophan, tyrosine); R-groups too small (glycine)

• be able to apply rules - eg if you see one of these groups - not likely to form alpha helix

What is a helical wheel diagram used for?

Visualizing the arrangement/distribution of side chains around an alpha helix

What is an amphipathic alpha helix?

A helix with hydrophobic residues on one side and hydrophilic residues on the opposite side

What is alpha-keratin?

A coiled coil motif of 2 amphipathic alpha helices (a supersecondary structure)

What structures does alpha-keratin make up in the body?

Hair, skin, and nails (also horns, covering a core of live bone)

What is the basic assembly hierarchy of keratin in hair?

Dimer → Protofilament → Microfibril → Macrofibril

Why is alpha-keratin rich in cysteine important?

Cysteine residues form -S-S- (disulfide) crosslinks that stabilize keratin structure

What happens chemically during a hair perm?

Disulfide bonds are reduced to 2 RSH (eg. with ammonium thioglycolate), hair is curled, then re-oxidized (eg. with H2O2) to reform -S-S- bonds in the new shape

Can the perm process also be used to straighten curly hair?

Yes, the same reduction/re-oxidation process works for relaxing (straightening) curly hair

What can alpha-keratin (hair) be used to detect?

Heavy metal poisoning - love to bind sulfur; (eg. thallium, mercury, lead, arsenic bind to S-S sites in hair)

What is a beta strand?

A polypeptide chain that is almost fully extended

Who discovered Beta Sheets

Pauling & Corey

What is a beta sheet?

Multiple beta strands arranged side-by-side

Can beta strands in a sheet come from different parts of the same polypeptide or different polypeptides?

Yes—from the same polypeptide, different sections of a polypeptide, or even different polypeptides

In a beta sheet, what plane are the strands in?

The X plane

In a beta sheet, where do H-bonds occur relative to strand direction?

Between amino acids on adjacent strands, perpendicular to strand direction (Y axis)

In a beta sheet, how do side chains (R groups) point?

They alternate pointing above and below the plane, perpendicular to both H-bonds and strands (Z axis)

*be able to apply - if you see alternating r groups — most likely a beta sheet

Why can beta sheets easily accommodate bulky or like-charged residues?

Because side chains alternate above/below the plane, avoiding direct clashes seen in alpha helices

What is the difference between antiparallel and parallel beta sheets?

Antiparallel strands run in opposite N-to-C directions; parallel strands run in the same N-to-C direction

What position are H-bonds stronger

when they are linear - antiparallel Beta sheets

Describe the overall conformation shape of a beta sheet

Extended zig-zag (ruffled) conformation (arrow points towards the C terminus)

How many residues are typically involved per strand in a beta sheet?

Usually 6-12 residues

What is the repeating distance per 2 amino acid residues in a beta sheet, and per single AA?

7 Å per 2 residues (3.5 Å per AA)

Can beta sheets be amphipathic?

Yes—one surface can have hydrophobic side chains while the other surface has hydrophilic ones

What structural feature do beta strands typically show besides being extended?

They twist

Name a protein that is predominantly beta sheets (all-beta)

Concanavalin A

What secondary structure motif is silk fibroin made of?

A beta pleated sheet (extended layers of antiparallel beta sheets)

What is the repeating amino acid sequence motif in silk fibroin?

(-Gly-Ser-Gly-Ala-Gly-Ala-)n — roughly alanine alternating with glycine

How does silk fibroin achieve its strength?

Extended layers of antiparallel Beta sheets

Alanine from one sheet interdigitates (locks) with alanine from another sheet

How does silk fibroin's strength compare to steel?

It is about 5x stronger than steel (in spider web)

What is collagen and where is it found?

The main structural protein in the extracellular matrix (ECM) of connective tissues; found in bone matrix, tendons, cartilage, blood vessels, skin, etc.

How abundant is collagen in mammals?

It is the most abundant protein in mammals, making up ~1/3 of total protein mass in large animals

What is the basic unit of collagen called?

Tropocollagen — a triple helix of 3 polypeptide chains, ~1000 amino acids long each

What is the general repeating unit in each collagen strand?

Gly-Pro-Pro/Hyp (thus called a "Proline Helix")

Why must glycine be invariant at every third position in collagen?

Glycine (the smallest amino acid) must be located along the central axis of the triple helix — no other residue can fit there

Are collagen H-bonds interchain or intrachain?

Interchain (between amide NH of one helix and carbonyl oxygens of another); no intrachain H-bonds occur

What amino acid derivative also forms interchain H-bonds to stabilize collagen?

HydroxyProline (Hyp)

How many strands form the collagen triple helical coil, and what is it compared to?

3 strands; described as strong, like a rope twist or metal cable

What is osteogenesis imperfecta (brittle bone disease) caused by?

A mutation in a collagen gene causing substitution of glycine for another amino acid (eg. serine), preventing normal mature collagen production

What genetic connective tissue disorder is associated with the aorta/heart and tall stature (FYI)?

Marfan syndrome

What condition is associated with hyperextensible/stretchy skin and joint hypermobility (FYI)?

Ehlers-Danlos syndrome

What two things are required to form hydroxyproline and hydroxylysine in collagen?

O2 and ascorbic acid (vitamin C)

What enzymes catalyze formation of hydroxyproline and hydroxylysine?

Prolyl hydroxylase (for hydroxyproline) and lysyl hydroxylase (for hydroxylysine)

What disease results from vitamin C deficiency and why?

Scurvy — lack of hydroxyproline leads to insufficient interchain H-bonding, causing weak collagen (skin lesions, fragile blood vessels, bleeding gums)

What is denatured collagen commonly known as?

Gelatin (Jello!)

Besides H-bonds, what covalent crosslinks strengthen collagen fibrils?

Intrachain lysine-lysine crosslinks and interchain hydroxypyridinium crosslinks

What happens to collagen crosslinks over time, and what is the effect?

They accumulate over time; increased crosslinks = less elastic collagen = more brittle bones/tendons, contributing to signs of old age

What is the function of loops and turns in protein structure?

They allow chain reversal (non-regular secondary structure)

Where are loops and turns normally located in a globular protein?

On the surface (they are hydrophilic)

What is an example of a larger loop structure named for its shape?

The Omega (Ω) loop

What does a beta turn (beta bend/tight turn) allow?

The polypeptide in an antiparallel beta sheet to reverse direction

Between which residues does the H-bond form in a beta turn?

Between residues 1 and 4 (carbonyl O of residue 1 H-bonded to amide H of residue 4)

What are the two types of beta turns, and what residue is often found at position 2 in both?

Type I and Type II; Proline is often the 2nd residue in both types: Type 2 has a glycine

Where do Beta sheets show up on ramachandran plot

Top left

What does tertiary (3°) structure result from?

The folding of a polypeptide chain into a closely-packed 3D structure, mainly due to hydrophobic interactions/effect

Can amino acids far apart in primary sequence come together in tertiary structure?

Yes, they may be brought together during folding

In globular (cytosolic) proteins, how are hydrophilic and hydrophobic residues typically arranged?

Hydrophilic residues on the outside, hydrophobic residues packed on the inside

What does the term "tertiary structure" essentially mean?

The 3D structure of the protein (can be shown many different ways

What technique is used to determine 3D protein structure in atomic detail from crystals?

X-ray crystallography

What is required to perform X-ray crystallography on a protein?

Protein crystals (eg. myoglobin forms crystals in 3M ammonium sulfate)

Describe the basic X-ray diffraction setup

X-rays from a source hit a protein crystal, producing diffracted beams that are captured by a detector (eg. film)

What mathematical process converts a diffraction pattern into an electron density map?

Fourier transform

How is protein resolution measured, and which value is better?

Measured in Ångströms (Å = 10⁻¹⁰ m); smaller numbers indicate better resolution (eg. 1.1 Å is better than 5 Å)

What limitation does X-ray crystallography have?

It's difficult to get crystals for many proteins, and it doesn't reveal structure of proteins in solution