Announcements TA Office Hours: Brian Eckenroth Monday 3-4 pm Thursday 11 am-12 pm Lecture 7 & 8: PROTEIN ARCHITECTURE IV: Tertiary and Quaternary Structure Margaret Daugherty Fall 2003 Homework II posted on website Primary (1 ) Structure Linear sequence of AAs linked by peptide bonds 1 M R L A F C V L L C A G S L G L C L A F P K E T V R W C T V 31 S S Q E A S K C S S F R H N M K K I L P V E G P H V S C V K 61 R T S Y L E C I R A I L A N E A D A V T I D G G L V F E A G 91 L A P Y N L K P V V A E F Y G S K D D P Q T H Y Y A V A V V Covalent interactions give rise to 1 o structure; Information for folding & function contained in amino acid sequence Structure arises from rotation about the Cα bond Peptide bond characteristics Defined bond lengths and angles Partial double bond character no rotation about C-N! Planar (CO & NH are parallel) Dipolar (δ - on O; δ + on N) Peptide bond consequences Rotation only about Cα ψ angle: rotation about Cα-C(O) bond φ angle: rotation about Cα-N(H) bond Not all ψφ angles are allowed Steric hinderance with R group Steric hinderance with backbone atoms Electrostatic repulsion between like charges (CO-CO or NH-NH) Ramachandran plots Reveal permitted regions of ψφ space Smaller amino acids can occupy more ψφ space Secondary structures assigned to allowed regions.
Secondary structures: local structures stabilized by hydrogen bonds How to tell a left- vs. right-handed a-helix Point your thumb up in the direction of the α-helix (N-->C). Look where the base of the helix spirals are. If they match the knuckles on your right hand it is a righthanded helix. ----------or----------- 3.6 residues/turn; rise = 1.5Å; pitch = 5.4Å left-handed α-helix right-handed α-helix If the helix spirals up in a counterclockwise direction, it is a right-handed helix. Secondary structures: local structures stabilized by hydrogen bonds Human Serum Albumin: Dimensions of a 585 residue polypeptide chain antiparallel β-sheet rise = 3.47 Å ideal hydrogen bonds parallel β-sheets rise = 3.25 Å non-ideal hydrogen bonds
Parallel vs. antiparallel β-sheets need different connectors Turns: provide tight compact means of changing the direction of the polypeptide chain usually α-helix Hydrogen bonding between CO of residue n & NH of residue n+3 Often have proline - forces chain to turn Often have glycine - good residue for a compact structure. Also a turn equivalent to a 3 10 helix (more compact than α-helix) Supersecondary structure Motif= small secondary structure elements; not stable folding units, however are often important parts of the functional sites of proteins Helix loop helix Domain= independently folding structural element; can be a protein in and of itself, oftentime are repeated in proteins Characteristic Compositions Does the AA sidechain (R) really matter in the formation of any of the secondary structures? Troponin-C
Important fact #1: Amino acids are not utilized equally by proteins Important fact #2: How to quickly calculate the MW of a protein Molecular weight is a summation of the masses of the individual AAs; average MW of an AA ~ 110 Daltons (rough estimate of MW = # AA * 110) Units (grams/mole) are expressed as daltons (Da) or kilo-daltons (kda). Tertiary Structure Tertiary structure describes how the secondary structure units associate within a single polypeptide chain to give a threedimensional structure flavodoxin Tertiary Structure: Basic Tenets - the truths 1). All information for folding is contained in the primary sequence. 2). Secondary structure formation is spontaneous - a consequence of the formation of hydrogen bonds. 3). No protein is stable as a single layer - hence secondary structural elements pack together in sheets. 4). Connections between structural elements are short - minimization of degrees of freedom - keeps structures compact. Consequences 1). Secondary structures are arranged in a few common patterns - i.e, resulting in protein families. 2). Proteins fold to form the most stable structure. Stability arises from: formation of large number of intramolecular hydrogen bonds Secondary structure formation reduction in hydrophobic surface area from solvent Association of secondary structure to form tertiary structure
Tertiary Structures Note that some parts of a protein structure are not regular (i.e., helicallike or sheet-like). These are often referred to as disordered or random coil regions. However a better nomenclature All betais (retinol natively binding random. protein) All alpha (human growth hormone) Alpha-beta barrel (triose isomerase) Fibrous proteins: Filamentous; play a major structural role in cells & tissues insoluble in H 2 O Tertiary Structures Globular proteins: compact structures; different folds for different functions Hydrophobes inside, hydrophiles outside Membrane Proteins: found associated with various membrane systems inside out insoluble in H 2 0 soluble in detergents Fibrous Proteins Share properties that give strength &/or flexibility to the structures in which they occur; Fundamental unit is a simple repeating element of secondary structure; Insoluble in water; large percentage of hydrophobic amino acids; Usually the hydrophobic surfaces are hidden in the elaborate supramolecular complexes; mechanically strong ; perform important structural functions Strength is enhanced by cross-links (disulfide bonds). Secondary Structures and Properties of Fibrous Proteins Structure α-helix, Cross-linked by disulfide bonds Characteristics Tough, insoluble protective structures of varying hardness and flexibility Examples of occurrence α Keratin of hair, feathers and nails β-conformation Soft, flexible filaments Silk fibroin Collagen triple helix High tensile strength, without stretch Collagen of tendons, bone matrix
FIBROUS PROTEINS: α-keratin What: Part of the intermediate filament proteins which have major structural roles in nuclei, cytoplasm and cell surfaces Where: Found in hair, fingernails, claws, horns, animal skin Composition: Long stretches of α-helices (> 300 residues) monomer: α-keratin type I or II coiled-coil: α-keratin type I + II; parallel protofilament: coiled-coil pair Coiled-Coils Interactions are stabilized by hydrophobic interactions between the α- helices; Heptad repeat ( a-b-c-d-e-f-g) n where a & d are nonpolar & lie in the center of the coiled coil; Evolved for strength; helical nature confers flexibility Coiled-coil is a super twist left-handed helix Distortion of helix to 3.5 residues/turn Hydrophobic faces interacting in a close interlocking pattern filament: 4 protofilaments α-keratin: A Human Hair Contact side chains (red balls) interlock
The Keratin Beauty Parlor Disulfides between molecules make the overall structure more rigid; The numbers & positions of these disulfides determine the amount of curling; Getting a permanent involves reducing these disulfides, reorganizing the hair & allowing the disulfides to reform; Blow drying just rearranges H-bonding. Why is rhino horn so strong? Strength of Keratin Results from the number of disulfides! In rhino horn, 18% of the AAs are Cys and involved in disulfides! Stephen Everse 2002 Stephen Everse 2002 FIBROUS PROTEINS: β-keratin COLLAGEN What: Part of the fibroin proteins Where: silk, bird feathers Composition: stacked anti-parallel β-sheets; strength Sequence: Alternating Gly-Ala/Ser Gly face interacts with another gly face Ala/Ser faces interact with one another What: Greek for glue; defined as that constituent of connective tissue which yields gelatin on boiling Where: Principal component of mammalian tissue; constitutes ~25% of a mammals protein content; more than 30 varieties Composition: Triple helix Sequence: Gly-X-Y; X usually Pro, Y usually Pro/HyPro > 3000Å long; 15Å in diameter
Collagen Primary Structure Approx 1000 AA/chain Repeats of Gly-X-Y where X is often Pro and Y is often hydroxyproline or proline MODIFIED AMINO ACIDS post-translational modifications add functionality to amino acid Composition G ~ 35% A ~ 11% P/HP ~ 30% Hyp: stabilizes tropocollagen via intrachain H-bonds Hyl: stabilizes fibrils via its ability to crosslink; attachment of CHO groups Consequences of Collagen Primary Structure Consequences of Collagen Primary Structure Distortion of backbone due to high content of glycines and prolines Can t form normal secondary structures Forms triple helix Every third residue faces inside Interior is compact; hence interior residue is glycine For comparison α-helix Fit occurs because Gly strand1 lies adjacent to X strand2 and Y strand3 Stabilization from hydrogen bonds Gly strand1 N-H to X strand2 C=O hydrogen bond Hydroxyproline forms hydrogen bonds