CH339K

Proteins: Higher Order Structure

Higher Levels of Protein Structure

Side chains hang off the backbone Repetitive background: -N-C-C-N-C-C-

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

bond torsional angles Bond

NH to C a C a to C=O C=O to NH

Rotation

free free

rigid planar Torsion angle defined

phi psi omega

Since constrained, only f w and is y can vary There are steric restrictions on what values they can assume

Permissable

F-Y

Angles (Ramachandran Plot)

Secondary Structures

• Represent interactions among

backbone

atoms • Examples  a -helices     Other helices b -sheets b - and g -turns These structures have characteristic f angles and y

a -

helix

• •

H bonds between carbonyl O of residue n amide H of residue n+4

R/V Alpha Helix Woods Hole Oceanographic Institute 1966-2011

Helical parameters – Pitch and Rise

Backbone forms helix Side chains extend outwards

f

≈ -57 o

y

≈ -47 o 3.6 residues/turn

Helix Types

a

-helix:

C=O H-bonded to NH of residue n+4 (aka 3.6

13

helix)

3 10 helix:

C=O H-bonded to NH of residue n+3

– ( f ≈ -49 o y ≈ -26 o ) p

-helix:

C=O H-bonded to NH of residue n+5 (aka 4.1

16 ( f ≈ -57 o y ≈ -80 o )

helix)

Helix terminology

H-bond makes a closed loop from amide H through backbone through carbonyl O Define helix by (a) Nbr of residues per turn (e.g. 3.6 for a -helix) (b) Nbr of atoms in the loop (e.g. 13 for a -helix) H H N H C O C H N R H C O C H N R H C O C R H

3 10

N H C O C H N

3.6

13

H C O C H N

4.1

16 or

 H C O C R R R etc

b

-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   f y ≈ -110 o ≈ +110 o to -140 o to +135 o

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

F-y

Angles for Secondary Structures

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

Ramachandran Plot

: Blue areas are permitted F angles and Y

Ramachandran plot for pyruvate kinase

Tertiary Structures

• Determined by

side chain

– Salt links – H-Bonds – Disulfides – Hydrophobic interactions

interactions • Fibrous Proteins • Globular Proteins

Fibrous Proteins

Keratin a

-keratin:

hair, horns, and hoofs of mammals b

-keratin:

scales, claws and shells of reptiles, beaks and claws of birds, porcupine quills

a

-keratin

• Lots of Ala, Gly, Cys • All a-helix

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

(1) (2) In the

absence

of vitamin C, reaction 2 oxidizes Fe 2+ to Fe 3+ .

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…

British Empire at its Peak

• A healthy navy is a victorious navy (of course,

my

ancestors were less than thrilled…)

Protein structure cartoons

a

-helix Antiparallel

b

-sheet

Globular Proteins (examples)

Motifs

– common stable folding patterns Found in proteins w/ different functions result from the

physics and chemistry

of the structure

More motifs

Domains

•Common patterns found in different proteins •Typically have similar function •Caused by –

evolution

(gene recombination / duplication)

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

C-rich Domain of Earthworm Mannose Receptor Fibroblast Growth Factor

Domains can be shared among proteins

Quaternary Structure

(Hemoglobin)

Folding Energetics

Favoring Folding

D H from formation of interchain H bonds and salt links + D S from disulfide formation

Enormous

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

Favoring Unfolding

High  – D S from going from unfolded folded state High + DH from breaking H-bonds with solvent

Denaturation

Denaturants

• Heat (increases negative T D S contribution) • Cold (H 2 O becomes less disordered) • Pressure • High and low pH (electrostatic effects) • Low-polarity and non-polar solvents (e.g. EtOH) • Chaotropes (urea, guanidinium chloride)

Protein Folding • Milliseconds to seconds • Rapid nucleation and hydrophobic collapse to “molten globule” • Slower compaction into the native state • Disulfides lessen negative D 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.

Effects of disulfides on folding Denaturation of gelsolin with (open circles) and without (solid circles) 1 mM dithiothreitol From: Isaacson, Weeds, and Fersht (1999) Proc. Nat. Acad. Sci.

96

: 11247-11252.

Rapid 2 o 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

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

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 Folding

Creutzfeldt-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)

• Kuru • BSE • Scrapie

Spongioform Encephalopathy – your brain on CJD Normal Moderate Severe

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

Prion Proteins

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

Prion Proteins – C terminal region PrPc PrPsc

178 180 188 196 200** 203 208 210 211 232

Vrious Mutations in CJD Prion Proteins

Codon Amino acid change

aspartate to asparagine valine to isoleucine threonine to alanine glutamate to lysine glutamate to lysine valine to isoleucine arginine to histidine valine to isoleucine glutamate to glutamine methionine to arginine

Reference

Goldfarb 1991b Kitamoto 1993a Collins 2000 Peoc’h 2000 Goldgaber 1989 Peoc’h 2000 Mastrianni 1996 Pocchiari 1993 Peoc’h 2000 Kitamoto 1993a