Announcements. Primary (1 ) Structure. Lecture 7 & 8: PROTEIN ARCHITECTURE IV: Tertiary and Quaternary Structure

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1 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.

2 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 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

3 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

4 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

5 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

6 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

7 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

8 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