CH339K

Proteins: Higher Order Structure

Higher Levels of Protein Structure

Repetitive background: -N-C-C-N-C-C-

Side chains hang off the backbone

The shape of the peptide chain can be defined by the three consecutive bond torsional angles

Bond Rotation Torsion angle definedNH to C free phiC to C=O free psiC=O to NH rigid planar omega

Since is constrained, only and can vary

There are steric restrictions on what values they can assume

Permissable Angles(Ramachandran Plot)

Secondary Structures• Represent interactions among

backbone atoms

• Examples -helices Other helices -sheets - and -turns

These structures have characteristic and angles

Pauling, Corey, and Branson (1951)

H bonds between

• carbonyl O of residue n

• amide H of residue n+4

Each amino acid is rotated 100o from the previous one.

3.6 amino acids per turn

-helix

R/V Alpha Helix

Woods Hole Oceanographic Institute 1966-2011

Helical parameters – Pitch and Rise

Backbone forms helix

Side chains extend outwards ≈ -57o

≈ -47o

3.6 residues/turn

Helix Types

-helix: C=O H-bonded to NH of residue n+4 (aka 3.613 helix)

310 helix: C=O H-bonded to NH of residue n+3 – ( ≈ -49o ≈ -26o)

-helix: C=O H-bonded to NH of residue n+5 (aka 4.116 helix)

( ≈ -57o ≈ -80o)

Helix terminologyH-bond makes a closed loop from amide H through backbone through carbonyl ODefine helix by (a) Nbr of residues per turn (e.g. 3.6 for -helix)(b) Nbr of atoms in the loop (e.g. 13 for -helix)

NH CH C

R

O

N CH C

R

O

N CH C

R

O

N CH C

R

O

N CH C

R

O

N CH C

R

OH H H H H H

etc

3103.613

4.116 or

Idealized Helices

-Sheets

• Can be thought of as helix with two residues per helix

• Backbone atoms run in a plane

• Side chains extend up and down from plane

≈ -110o to -140o

≈ +110o to +135o

C=O of residue n with N-H of residue n+3

Gamma Turns:

C=O of residue n with N-H of residue n+2

Angles for Secondary Structures

NOTE: Left-handed -helix has = +57, = +47

Ramachandran Plot: Blue areas are permitted and angles

Ramachandran plot for pyruvate kinase

Tertiary Structures

• Three dimensional folding

• Determined by side chain interactions– Salt links– H-Bonds– Disulfides– Hydrophobic interactions

• Fibrous Proteins

• Globular Proteins

Fibrous ProteinsKeratin

-keratin: hair, horns, and hoofs of mammals

-keratin: scales, claws and shells of reptiles, beaks and claws of birds, porcupine quills

-keratin• Lots of Ala, Gly, Cys• All -helix (well, almost)

Right handed

Left handed

Disulfides in the Barber Shop

Sodium thioglycolate Various peroxides

Fibrous Proteins - Fibroin

75-80% Ala/Gly

15% Ser

Within a fiber: crystalline regions are separated by amorphous regions.

Fibrous Proteins - Collagen

Left handed helix of tropocollagen forms right handed triple helix of collagen.

Hydroxyproline participates in H-bonding between tropocollagen chains

In the absence of vitamin C, reaction 2 oxidizes Fe2+ to Fe3+.

(1)

(2)

Lack of hydroxyls causes serious destabilization of the triple helix

Scurvy• Weakness• Paleness• Sunken eyes• Tender gums and/or

tooth loss• Muscular pain• Reopening of old

wounds or sores• Internal bleeding• Loss of appetite• Bruising easily• Weight loss; inability

to gain weight• Diarrhea• Increased heart rate• Fever• Irritability• Aching and swelling in

joints• Shortness of breath• Fatigue

Arrrrr…

The Brits Found the Link Between Fruits and Veggies and Healthy Sailors

Walk wide o' the Widow at Windsor, For 'alf o' Creation she owns: We 'ave bought 'er the same with the sword an' the flame, An' we've salted it down with our bones. (Poor beggars! -- it's blue with our bones!)

The Widow at Windsor – Kipling

We broke a King and we built a road --A court-house stands where the reg'ment goed.And the river's clean where the raw blood flowedWhen the Widow give the party.(Bugle: Ta--rara--ra-ra-rara!)

The Widow’s Party - Kipling

British Empire at its Peak

• A healthy navy is a victorious navy (of course, my ancestors were less than thrilled…)

Protein structure cartoons

-helix Antiparallel -sheet

Globular Proteins (examples)

Structural Motifs – “supersecondary structures”

common stable folding patternsFormed from consecutive sequencesFound in proteins w/ different functionsresult from the physics and chemistry of the structure

Greek Key Motif (antiparallel -sheets)

a) Schematic of motifb) Staphylococcus nuclease protein

More motifs

Ricin B chain

• Two domains

• Each domain is a trefoil

• 3 repeats of a sheet-loop structure

• i.e. 6 repeats of a primitive fold

Domains – • Stable, independently folded, globular units• Common patterns found in different proteins• Typically have similar function• Caused by evolution (gene recombination / duplication)• Frequently (not always!) correspond to exons in genes

C-rich Domain of Earthworm Mannose Receptor

Fibroblast Growth Factor

Domains can be shared among proteins

Quaternary Structure (Hemoglobin)

Folding EnergeticsFavoring Folding Favoring Unfolding

-H from formation of intrachain H-bonds and salt links

High +S from going from folded unfolded state

+S from disulfide formation High -from making H-bonds with solvent

Enormous +S from burial of hydrophobic side chains in the interior

Denaturation

Denaturants

• Heat (increases negative TS contribution)

• Cold (H2O becomes less disordered)

• Pressure

• High and low pH (electrostatic effects)

• Low-polarity and non-polar solvents (e.g. EtOH)

• Chaotropes (urea, guanidinium chloride)

• Milliseconds to seconds

• Rapid nucleation and hydrophobic collapse to “molten globule”

• Slower compaction into the native state

• Disulfides lessen negative S

• Larger proteins often have multiple structural domains

• Each domain folds by mechanisms similar to those above.

• Once folded, domains reshuffle to form the final native structure.

Protein Folding

Effects of disulfides on folding

Denaturation of gelsolin with (open circles) and without (solid circles) 1 mM dithiothreitolFrom:Isaacson, Weeds, and Fersht (1999) Proc. Nat. Acad. Sci. 96: 11247-11252.

Rapid 2o structure formation

Collapse to molten globule

Reshuffle to final state

Heat Shock Proteins• Nucleotide binding domain – binds ATP and hydrolyzes it to ADP.

• Protein binding domain – contains a groove with an affinity for neutral, hydrophobic amino acid residues. The groove can interact with peptides up to seven residues in length.

• C-terminal domain –acts as a 'lid' for the substrate binding domain.

When an Hsp70 protein is ATP bound, the lid is open and peptides bind and release relatively rapidly.

When Hsp70 proteins are ADP bound, the lid is closed, and peptides are tightly bound to the protein binding domain.

Chaperonins - GroEL

Simpler Picture of GroEL Action

A Problem in FoldingCreutzfeldt-Jakob Disease,

Mad Cows, and the Laughing Disease of the New Guinea Cannibals

Initially, persons may have difficulty sleeping, experience depression, problems with muscular coordination, impaired vision, and personality and behavioral changes such as impaired memory, judgment, and thinking. As the disease progresses, mental impairment becomes severe and involuntary muscle jerks (myoclonus) often occur along with blindness. Eventually, the ability to move or speak is lost and the person enters a coma until death occurs. (100% fatal)

Prion Diseases• Human Prion Diseases

• Creutzfeldt-Jakob Disease (CJD)

• Variant Creutzfeldt-Jakob Disease (vCJD)

• Gerstmann-Straussler-Scheinker Syndrome

• Fatal Familial Insomnia

• Kuru

• Animal Prion Diseases• Bovine Spongiform Encephalopathy (BSE)

• Chronic Wasting Disease (CWD)

• Scrapie

• Transmissible mink encephalopathy

• Feline spongiform encephalopathy

• Ungulate spongiform encephalopathy

• Kuru• Scrapie

• BSE

Spongioform Encephalopathy – your brain on CJD

Normal Moderate Severe

Brain atrophy in CJD – you’re usually dead before it reaches this stage

Prion Proteins

PrPc

Normal cellular prion protein (PrPc) – mostly -helical C-terminal domain

Prion Proteins – C terminal region

PrPc PrPsc

Infectious Proteins

The presence of one misfolded PrPsc causes adjacent PrPc to toggle into the misfolded state.

Various Mutations in CJD Prion Proteins

Codon Amino acid change Reference

178 aspartate to asparagine Goldfarb 1991b

180 valine to isoleucine Kitamoto 1993a

188 threonine to alanine Collins 2000

196 glutamate to lysine Peoc’h 2000

200** glutamate to lysine Goldgaber 1989

203 valine to isoleucine Peoc’h 2000

208 arginine to histidine Mastrianni 1996

210 valine to isoleucine Pocchiari 1993

211 glutamate to glutamine Peoc’h 2000

232 methionine to arginine Kitamoto 1993a