Molecules of Life: Proteins Part 1 from Dundee.ac.uk

Examples of Important Proteins & Peptides

Hormones e.g. Insulin Glucagon Transport /Storage e.g. Haemoglobin Ferritin Growth Factors e.g. TGFß EGF Structural e.g. Actin Collagen Immunoglobulins (antibodies) e.g. IgG IgM IgA Enzymes e.g. DNA Polymerase Trypsin

Hierarchy of Protein Structure

A

• PRIMARY STRUCTURE (A) linear sequence (order) of amino acid residues, joined by peptide bonds
• SECONDARY STRUCTURE (B) the localised conformation of the polypeptide backbone e.g. a helix or ß sheet
• TERTIARY STRUCTURE (C) the 3-dimensional structure of an entire polypeptide, including all its side chains
• QUATERNARY STRUCTURE (D) the spatial arrangement of polypeptide chains in a protein with multiple subunits

H HOHHOH -N-C-C-N-C-C-N- I R 0 B 11 R Z-I N H N H C=0 O R C - R C N- C H N R- H O -N -C R H C 0 0 N-C C H `c/-R N H2N H Fig. 2-10. Primary, secondary, tertiary, and quaternary structures. (A) The primary structure is composed of a linear sequence of amino acid residues of proteins. (B) The secondary structure indicates the local spatial arrangement of polypeptide backbone yielding an extended a-helical or -pleated sheet structure as depicted by the ribbon. Hydrogen bonds between the 'backbone' amide -NH- and -CO- groups stabilize the helix. (C) The tertiary structure illustrates the three-dimensional conformation of a subunit of the protein while the quaternary structure (D) indicates the assembly of multiple polypeptide chains into an intact, tetrameric protein. Copyright 2009, Elsevier Limited. All rights reserved.

Secondary Structure

C 1 R - DSecondary Structure

• a HELIX Examples: · ß SHEETS · Secondary structure refers to the local arrangement of the polypeptide chains . It is determined by hydrogen bonds between the carbonyl oxygen group of one peptide bond and the amide hydrogen 0 B 11 R .- N G-C I N H 1 H O R C O C 11 H C- N R-C H O N -C R H C 0 O N-C C H CR H2N N H

Alpha Helix Structure

CEO N-CHRa Helix

• Described as rod-like . One polypeptide chain, tightly coiled, side chains extend outwards · Mostly right-handed - energetically favourable · Carbonyl oxygen (C=O) of one peptide bond forms a hydrogen bond with the amide hydrogen (N-H) group of another four residues away in the linear sequence Amino terminus H R N H H C N H R o R N C H N C R O H R C N H C N O R H N C R 3.6 residues/turn C H R N N C R C Carboxyl terminus Figure 3-4 Molecular Cell Biology, Sixth Edition @ 2008 W. H. Freeman and Company

Left-handed vs Right-handed Helix

Left-handed helix Right-handed helixß Sheets Polypeptide (pp) backbone almost completely extended Hydrogen bonds between polypeptide chain(s) Compare to the a helix, where the hydrogen bonds are parallel to the direction of the peptide chain Can involve more than one polypeptide Parallel (all strands N->C terminus) Antiparallel (strands running in opposite directions) (a) C N C N (b) N C O C N 2006 Brooks/Cole - Thomson (A) Antiparallel (B) Parallel Molecular Biology of the Cell. 4th edition. Alberts B, Johnson A, Lewis J, et al. New York: Garland Science; 2002.

Phosphoglycerate Kinase Secondary Structure

Phosphoglycerate kinase (an enzyme involved in glycolysis) a helix Different secondary structure elements can occur within one protein Parallel B sheet Phosphoglycerate Kinase domain 2

Tertiary Structures

FIBROUS PROTEINS e.g. collagen, fibroin, keratin GLOBULAR PROTEINS e.g. albumin, myoglobin, haemoglobin Fibrous protein Filament (four right-hand twisted protofilaments) Myoglobin, a globular protein Model of a Globular protein a helices - blue ß sheets - green @ 2006 Brooks/Cole - Thomson · Arrangement of all atoms of a polypeptide or protein in space · Consists of local regions with distinct secondary structure . Either group may have a helix or ß sheets or both

Globular Proteins Characteristics

Globular Proteins Proteins which are folded to a more or less spherical shape . Generally soluble in water and salt solutions . Most of their polar side chains are on the outside and interact with the aqueous environment by hydrogen bonding and ion- dipole interactions . Most of their non-polar side chains are buried inside . Nearly all have substantial sections of a-helix and ß-sheet Examples: MYOGLOBIN HAEMOGLOBIN a helix is the main structural motif

Fibrous Proteins Characteristics

Fibrous Proteins Contain polypeptide chains organised approximately parallel along a single axis · Consist of long fibres or large sheets · Tend to be mechanically strong · Insoluble in water and dilute salt solutions · Play important structural roles in nature Examples: KERATIN - hair, wool, nails, claws COLLAGEN - connective tissue FIBROIN - in silk

Secondary Structures and Properties of Fibrous Proteins

Secondary Structures and Properties of Fibrous Proteins STRUCTURE CHARACTERISTICS EXAMPLES a Helix, crosslinked by disulphide bonds Tough, insoluble structures of varying hardness and flexibility a-Keratin of hair, nails and feathers ß Conformation Soft, flexible filaments Fibroin of silk Collagen Triple Helix High tensile strength without stretch Collagen of tendons, bone matrix Cross-linked Elastin Chains Two-way stretch with elasticity Elastin of ligaments

Forces Stabilising Tertiary Structures

Forces Stabilising Tertiary Structures: Tertiary structure is the 3D, folded and biologically active conformation of a protein. It is determined by interactions between amino acid side chain (R) functional groups. These interactions include: 1. Disulphide bonds (covalent) 2. Hydrogen bonds 3. Salt bridges (ion pairs) 4. Hydrophobic interactions 2 Cys (- S - S + Cys Ser- O -H . O =C- Gln 3 1 NH2 O Glu - C-0 + NH3 - Lys vvv- Valv CH3 / H3C CH3 Phe- 4. Hydrophobic interactions CH3 HC ICH3 CH2 Copyright 2009, Elsevier Limited. All rights reserved.

Types of Bonds in Tertiary Structure

1. Disulfide bonds 2. Hydrogen bonds 3. Salt bridgesDisulphide Bonds

• A covalent bond between 2 cysteine side chains i.e. - SH -SH -> -S-S- · Can be between Cysteine residues in the same protein, or different polypeptides · A significant bond in the stabilisation of tertiary structures cysteine cysteine S H + C H S oxidation reduction cystine S S disulfide bond A 100 40 Q S 50 Figure 2.51 Biochemistry, Seventh Edition 2012 W. H. Freeman and Company Bovine ribonuclease http://img.docstoccdn.com/thumb/orig/119813108.png 10 EROHM D 1 20 +H_N-K S 2 30 80 KM MON c S R S G (N S 90 120 G L K 124SADFHV NG N) T E K) K P 110 N SQ 4 60 C C 70 G K A N) S SAAST S · Involves an oxidation reaction

Tertiary Structure Example: Myoglobin

Tertiary Structure - e.g. Myoglobin CD C FG D -Heme group (Fe) F B F AB H COO- NHỜ GH 2006 Brooks/Cole - Thomson TOOC COO- 1 1 HỌC CH2 1 HỌC CH9 1 H 1 C C C = HỌC-C C-CH3 N N C C = HC Fe (II) CH C C N N C-C C-CH3 H C C C C H H3C C= CH2 H Heme (Fe-protoporphyrin IX) 2006 Brooks/Cole - Thomson · A globular protein · Contains 8 a helical regions (no ß pleated sheet) • Contains a Haem group: contains iron, Fe(II) called a prosthetic group · Haem group = iron + protoporphyrin · Binds one oxygen molecule per myoglobin protein · Stores oxygen in muscle · Myoglobin minus haem = apoprotein C C HỌC G EF

Quaternary Structure of Proteins

Quaternary Structure Proteins which contain more than one polypeptide chain i.e. between 2 and 12+ identical or different subunits Generic name = oligomer Dimer = 2 subunits Trimer = 3 subunits Tetramer = 4 subunits Generally held together by: Electrostatic interactions Hydrogen bonds Hydrophobic interactions

Quaternary Structure Example: Haemoglobin

Quaternary Structure e.g. Haemoglobin Haemoglobin: • transports oxygen in the blood 4 subunits (tetramer) • 2a and 2ß chains • (not to be confused with a helix and @ sheet) . each contains a Haem group · each subunit can bind one O2 Positive Cooperativity: Binding of one O, molecule increases the affinity of the other subunits to O2 Heme group (Fe) a a B B @ 2006 Brooks/Cole - Thomson

Amino Acid Substitutions and Protein Structure

Amino Acid Substitutions and Protein Structure SICKLE CELL ANAEMIA: . In the coding sequence of the ß-subunit of haemoglobin, a single nucleotide is changed (GAG -> GTG) • During translation, this causes a Glutamic acid residue to be substituted by Valine • Glutamic acid ionises at physiological pH and is negatively charged (now called glutamate) and can form ionic bonds or hydrogen bonds with water or other amino acid side chains . However, Valine is hydrophobic and interacts with other hydrophobic amino acids CONSEQUENCES: • The haemoglobin behaves normally until exposed to low oxygen tensions where it forms large fibrous aggregates distorting the erythrocytes to the sickle shape. . This alters the flow properties of the cells, which may block blood flow in the vessels and capillaries. . These red cells are fragile leading to haemolysis & anaemia.

Folding Polypeptide Chains

Folding Polypeptide Chains . The primary structure of a protein contains all the information needed for its 3-dimensional shape · Proteins may fold spontaneously into their correct shape . This can be a slow and erroneous process - protein may begin to fold incorrectly before it is completely synthesised - it may associate with other proteins before it is folded properly e.g. Alzheimer's, Parkinson's, Creutzfeldt-Jakob disease (CJD) . Sometimes the folding process is aided by other specialised proteins called chaperones

Protein Folding - Prion Disease

Protein Folding -Prion Disease e.g. Bovine spongiform encephalopathy (BSE), Scrapie, Human spongiform encephalopathy · Prion proteins are normal glycoproteins found in the cell membrane of nerve tissue • Normal conformation PrP (Prion Protein) - lots of a helix · Disease-causing form PrPsc enriched in 3 sheets aggregates into multimeric complexes, which are resistant to degradation Seen as insoluble plaques in brain tissue • Spontaneous refolding of PrP into PrPsc is extremely rare · Ingested Prpsc travel around the body until they come into contact with nerve tissue · Presence of Prpsc acts as a template for misfolding of native prion proteins Mı (a) (b) Normal PrP Abnormal PrPsc Figure: Biochemistry-Campbell and Farrell

Denaturation: Disrupting Protein Structure

Denaturation: Disrupting Protein Structure Examples of physical conditions or chemical agent(s) that can disrupt protein structure: · HEAT increase in vibrations in a protein, disrupting tertiary structure • EXTREMES OF pH electrostatic interactions interrupted · DETERGENTS, UREA, GUANIDINE HYDROCHLORIDE disrupt hydrophobic interactions · THIOL AGENTS, REDUCING AGENTS reduce and thereby disrupt disulphide bonds (a) Albumen Water-soluble insoluble (b) Protein Thermal Irreversible Denaturation Native albumen · : - SH Denaturation Crosslinking - : S-S http://spie.org/x86653.xml