The Three-Dimensional Structure of Proteins

Structure Hierarchy Primary structure -- only the sequence of the amino acids and the location of –S-S- bonds Secondary structure -- bond angles (conformations) or shape of the polypeptide backbone in a folded protein (doesn’t include side chains) Tertiary structure -- total 3-dimensional structure of a protein including side chain bond angles Quaternary structure -- in a multi-subunit protein: the arrangement of the subunits through non-covalent interactions (sometimes stabilized by –S-S- bonds)

N H O The Peptide Bond -- revisited Resonance structures Resonance hybrid: 40% double bond character. No rotation; planar; trans; polar; forms H-bonds

(Glycine is the most flexible.) φ ψ φ ψ Phi bond angle Psi bond angle Glycylalanine (a dipeptide) Because of steric clashes, only certain phi and psi angles are allowed, depending on the amino acid. (Glycine is the most flexible.)

Proline Gly-Pro H CH2 O H3N C C N C COO _ + α Reduced double-bond character Cis & trans possible (1:4) Phi angle fixed

Regular Secondary Structure Regions of a protein where: all phi (ϕ) bond angles are equal all psi (ψ) bond angles are equal Some Possibilities: α-helix φ = ─57o ψ = ─47o β-sheet φ = ─119o ψ = +113o (parallel) β-sheet φ = -139o ψ = +135o (antiparallel)

The α-Helix 3.6 amino acids per turn

From the X-ray structure of E.coli aminopeptidase P

The β-Sheet Strands can be parallel (both N- to C-terminal directions the same) or antiparallel (opposite).

“β-pleated sheet”

Tertiary Structure Side chains, all atoms Space-filled

(Orientation of subunits) A tetramer (4 subunits, non-covalent) Quaternary Structure (Orientation of subunits) A tetramer (4 subunits, non-covalent) Colored by subunit

colored by type of amino acid Surface Amino Acids: colored by type of amino acid Red: ─ charged, Asp, Glu Blue: + charged, Lys, Arg Yellow: other polar Grey: hydrophobic

Slice Through the Middle: colored by type of amino acid Red: ─ charged, Asp, Glu Blue: + charged, Lys, Arg Yellow: other polar Grey: hydrophobic

Domains globular; folds independently; spatially separated; may have specific function

Domains are building blocks (modules) A specific domain can be found in many proteins Can carry a specific function Average size: 100 amino acids (<200) A constituent of multidomain proteins Protein C

α/β-domain fold (central β-barrel) Secondary Structure of a Domain called a Fold Fold: specific arrangement of secondary structure elements Lysozyme All α-helix globin fold α/β-domain fold (central β-barrel) α/β-domain fold (twisted β-sheet) All β-domain fold (β-barrel “jelly role”) “Superfolds”

Amino Acid Sequence Determines Protein Folding How? Predicting 3-dimensional structure from amino acid sequence: Improvements in theory and computation Homology modeling: Search X-ray and NMR structure database for a protein that has a similar amino acid sequence (template) Do sequence alignment Use computer programs to “thread” the protein sequence through the X-ray structure

Identity: 33% Similarity: 55%

DNA Mutations Silent: no effect on protein or Effect on protein structure or function folding aggregation enzyme activity protein-protein interaction regulation size (truncation, deletion, insertion) gain of function There are >140,000 disease-associated mutations 80% are in regular secondary structure, tight turns, and bends. They result in decreased stability of the protein structure.

Forces Stabilizing Protein Structures Bond type Bond strength (kcal/mol) Covalent >50 Non-covalent hydrogen bonds 1-7 ionic bonds (+ - interaction) 1-6 (internal) van der Waals <1 hydrophobic interactions 2-3

van der Waals hydrophobic interactions

Forces Stabilizing Protein Structures Bond type Bond strength (kcal/mol) Covalent >50 Non-covalent hydrogen bonds 1-7 ionic bonds (+ - interaction) 1-6 (internal) van der Waals <1 hydrophobic interactions 2-3

Motions in Proteins X-ray structure: average structure over time Proteins move (“jiggle”) over a picosecond time range (10-12 s). Each atom is in constant motion. Much larger conformational changes also occur: Hexokinase + glucose Hexokinase—glucose complex Some proteins or portions of proteins are “unstructured,” and only become structured when they interact with another protein.

A Fibrous Protein--Collagen Most abundant protein in humans (25-30%) Three-chain superhelix (triple helix)

Glycine 33%; Proline 10%; Hydroxyproline (Hyp) 10% Partial Amino Acid Sequence of a Collagen Chain Glycine 33%; Proline 10%; Hydroxyproline (Hyp) 10% “Derived amino acids.” Forms “polyproline type II helix” 3 amino acids per turn Left-handed 2X more extended than α-helix

polyproline helix (G - X - Y)n α - chain Gly Pro Hyp always common common α - chain