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 BondRotationTorsion angle defined NH to C  freephi C  to C=Ofreepsi C=O to NHrigid planaromega

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

H bonds between carbonyl O of residue n amide H of residue n+4  - helix

R/V Alpha Helix Woods Hole Oceanographic Institute

Helical parameters – Pitch and Rise

Backbone forms helix Side chains extend outwards  ≈ -57 o  ≈ -47 o 3.6 residues/turn

Helix Types  -helix: C=O H-bonded to NH of residue n+4 (aka helix) 3 10 helix: C=O H-bonded to NH of residue n+3 –(  ≈ -49 o  ≈ -26 o )  -helix: C=O H-bonded to NH of residue n+5 (aka helix) (  ≈ -57 o  ≈ -80 o )

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  -helix) (b) Nbr of atoms in the loop (e.g. 13 for  -helix)

 -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   ≈ -110 o to -140 o   ≈ +110 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

 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 Determined by side chain interactions –Salt links –H-Bonds –Disulfides –Hydrophobic interactions Fibrous Proteins Globular Proteins

Fibrous Proteins Keratin  -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 a-helix Right handed Left handed

Disulfides in the Barber Shop Sodium thioglycolateVarious 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 Fe 2+ to Fe 3+. (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…

British Empire at its Peak A healthy navy is a victorious navy (of course, my ancestors were less than thrilled…)

Protein structure cartoons  -helixAntiparallel  -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

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 – Common patterns found in different proteins Typically have similar function Caused by evolution (gene recombination / duplication)

C-rich Domain of Earthworm Mannose Receptor Fibroblast Growth Factor

Domains can be shared among proteins

Quaternary Structure (Hemoglobin)

Folding Energetics Favoring FoldingFavoring Unfolding -  H from formation of interchain H- bonds and salt links High –  S from going from unfolded  folded state +  S from disulfide formationHigh +  from breaking H-bonds with solvent Enormous +  S from burial of hydrophobic side chains in the interior

Denaturation

Denaturants Heat (increases negative T  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)

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 dithiothreitol From: Isaacson, Weeds, and Fersht (1999) Proc. Nat. Acad. Sci. 96:

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 – 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 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 Scrapie BSE

Spongioform Encephalopathy – your brain on CJD NormalModerateSevere

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 PrPcPrPsc

Vrious Mutations in CJD Prion Proteins CodonAmino acid changeReference 178aspartate to asparagineGoldfarb 1991b 180valine to isoleucineKitamoto 1993a 188threonine to alanineCollins glutamate to lysinePeoc’h **glutamate to lysineGoldgaber valine to isoleucinePeoc’h arginine to histidineMastrianni valine to isoleucinePocchiari glutamate to glutaminePeoc’h methionine to arginineKitamoto 1993a