Protein PDF

Protein  Contents 1. Chemical components 2. Molecular structures 3. Biological functions 4. Structure-function relationship 5. Physical and chemical properties 6. Exploration of proteins 7. Proteomics: a new frontier What are proteins? Proteins are macromolecules composed of amino ac...

Protein  Contents 1. Chemical components 2. Molecular structures 3. Biological functions 4. Structure-function relationship 5. Physical and chemical properties 6. Exploration of proteins 7. Proteomics: a new frontier What are proteins? Proteins are macromolecules composed of amino acids linked together through peptide bonds. How about proteins? the most widely distributed biomolecules the most abundant biomolecules (45% of human body) the most complex biomolecules the most diversified biological functions What do proteins do?  Section 1 Chemical Components of Proteins  Components of proteins major elements C (50~55%), H (~7%), O (19~20%), N (13~19%), S (~4%) trace elements P, Fe, Cu, Zn, I, …  The average nitrogen content in proteins is about 16%, and proteins are the major source of N in biological systems. The protein quantity can be estimated. protein in 100g sample = N per gram x 6.25 x 100  §1.1 Amino Acids The basic building blocks of proteins About 300 types of AAs in nature, but only 20 types are used for protein synthesis in biological systems. A amino group, a carboxyl group, a H atom and a R group are connected to a C atom. The C atom is an optically active center.  Amino acid + H3N - OOC C H R  Molecular weight Dalton: A unit of mass nearly equal to that of a hydrogen atom Glycine C2NO2H5 75 12×2+14+16×2+1×5=75 Alanine C3NO2H7 89 Valine C5NO2H11 117 Leucine C6NO2H13 131 isoleucine C6NO2H13 131  Amino acid + H3N - OOC C H R §1.1.a Classification The R groups, also called side chains, make each AA unique and distinctive. R groups are different in their size, charge, hydrogen bonding capability and chemical reactivity. Aas are grouped as (1) non-polar, hydrophobic; (2) polar, neutral; (3)basic; and (4) acidic Non-polar and hydrophobic AAs R groups are non-polar, hydrophobic aliphatic or aromatic groups. R groups are uncharged. AAs are insoluble in H2O.  Polar and uncharged AAs R groups are polar: -OH, -SH, and -NH2. R groups are highly reactive. AAs are soluble in H2O, that is, hydrophilic.  Basic AAs R groups have one -NH2. R groups are positively charged at neutral pH (=7.0). AAs are highly hydrophilic.  Acidic AAs R groups have –COOH. R groups are negatively charged at physiological pH (=7.4). AAs are soluble in H2O. Aspartic acid glutamic acid (Asp or D) (Glu or E)  Nomenclature Starting from the carboxyl group, and naming the rest carbon atoms sequentially in Greek letters. NH β α δ γ β α CH3 CH COO- NH2 C NH CH2 CH2 CH2 CH COO- NH3+ NH3+ α-amino-propionic acid α-amino-δ-guanidinovaleric acid (alanine) (arginine)  Special amino acids - Gly optically inactive + H3N - OOC C H H  Special amino acids - Pro Having a ring structure and imino group CH2 CHCOO- CH2 NH2+ CH2  Special amino acids - Cys active thiol groups to form disulfide bond §1.2 Peptide §1.2.a Peptide and peptide bond A peptide bond is a covalent bond formed between the carboxyl group of one AA and the amino group of its next AA with the elimination of one H2O molecule. Peptides can be extended by adding multiple AAs through multiple peptide bonds in a sequential order. dipeptide, tripeptide, oligopeptide, polypeptide AAs in peptides are called as residues. §1.2.b Biologically active peptides Glutathione (GSH) Glutamic acid cysteine glycine  H2O2 2GSH NADP+ GSH peroxidase GSH reductase 2H2O GSSG NADPH+H+ As a reductant to protect nucleic acids and proteins from toxin by discharging free radical or H2O2  Peptides Peptide hormones secreted from peptidergic neurons or – Somatostatin, Noacosapeptide, Octapeptide, – Thyrotropin-release hormone, Antidiuretic hormone Neuropeptides responsible for signal transduction – Enkephalin, Endorphin, Dynorphin, Substance P, Neuropeptide Y  thyrotropin-release hormone Pyroglutamic acid histidine prolinamide  Neuropeptide name amino acid sequence oxytocin Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2 └──S───S──┘ Vasopresin Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2 └──S────S──┘ Met-enkephalin Tyr- Gly-Gly-Phe-Met Leu-enkephalin Tyr- Gly-Gly-Phe-Leu Atrial natriuetic Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg- factor Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys- Asn-Ser-Phe-Arg-Tyr Substance P Arg-Pro-Lys-Pro-Bln-Phe-Phe-Gly-Leu-Met-NH2 Bradykinin Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg  Section 2 Molecular Structures of Proteins  Overview Proteins are composed of AAs. Distinctive properties of proteins are determined by AA compositions, AA sequences as well as the relative positions of AAs in space. Proteins need well defined structures to function properly. Their structures are organized in a hierarchy format, that is, primary, secondary, tertiary and quaternary structure. §2.1 Primary Structure The primary structure of proteins is defined as a linear connection of AAs along the protein chain. It is also called amino acid sequence. The AA sequence must be written from the N-terminus to the C-terminus. Peptide bonds are responsible for maintaining the primary structure.  Primary structure of insulin Two peptides of 21 and 30 AAs Two inter-chain -S-S- bonds One intra-chain -S-S- bond §2.2 Secondary Structure The secondary structure of a protein is defined as a local spatial structure of a certain peptide segment, that is, the relative positions of backbone atoms of this peptide segment.  Repeating units of N(-H), Cα, and C(=O) constitute the backbone. H-bonds are responsible for stabilizing the secondary structure. The side chains are not considered. α-helix β-pleated sheet β-turn (β-bend) random coil  Peptide unit Six atoms, Cα-C(=O)-N(-H)-Cα, constitute a planer peptide unit. The peptide unit is rigid due to the partial double bond property. C=O and N-H groups are in trans conformation and cannot rotate around the peptide bond.  Resonant conjugation O O－ C C N N＋ H H O H R2 0.124 1 C 0.1 Cα 0. 1 5 32 C-N: 0.149nm 46 Cα N 0.1 C＝N: 0.127nm H R1 H  Rotation of peptide unit Peptide units can rotate freely around Cα-C and Cα-N bonds to form two torsion angles ψ and ϕ.  “Beads on a string” N-terminus Cα Backbone C-terminus Peptide unit Like beads on a string, amino acids are assembled into the proteins. Linus Carl Pauling b. 1901, d. 1994 California Institute of Technology, CA The Nobel Prize in Chemistry (1954), “for his research into the nature of the chemical bond and its application to the elucidation of the structure of complex substances” The Nobel Peace Prize (1962) §2.2.a α-helix A helical conformation is right-handed. 3.6 AAs per turn and a 0.15 nm vertical distance, creating a pitch of 0.54 nm. Side chains of AA residues protrude outward from the helical backbone. The hydrogen-bonds are parallel to the helical axis. Left-hand versus right-hand The -CO group of residue (n) is H-bonded to the -NH group of residue (n+3). §2.2.b β-pleated sheet An extended zigzag conformation of protein backbones Protein backbones are arranged side- by-side through H-bonds. H-bonds are perpendicular to the backbone direction. The side chains of adjacent AAs protrude in opposite directions. The adjacent protein backbones can be either parallel or anti-parallel. §2.2.c β-turn One β-turn involves four AAs. The -CO and -NH groups of the first AA are hydrogen bonded to the -NH and -CO groups of the fourth AA, respectively. The β-turn reverses abruptly the direction of a protein backbone. H-bonds are perpendicular to the protein backbone.  Red: O Black: C Blue: N Green: R White: H The -CO and -NH groups of the first AA are hydrogen bonded to the - NH and -CO groups of the fourth AA, respectively. H-bonds are perpendicular to the protein backbone. §2.2.d Random coil There is no consistent relationship between planes. §2.2.e Motif When several local peptides of defined secondary structures are close enough in space, they are able to form a particular “super-secondary” structure. Zinc finger HLH (helix-loop-helix) HTH (helix-turn-helix) Leucine zipper §2.2.f Side chains effect Shape: Pro having a rigid ring (α– helix disrupter) Size: β-sheet needs AAs of small side chain. Leu, Ile, Trp, and Asn having bulky sides (hard to form α–helix) Charge: Too many charged AAs in a short region of one peptide is hard to form α–helix. §2.3 Tertiary Structure The tertiary structure is defined as the spatial positions of all atoms of a protein including main chain and side chain, i.e., the three-dimensional (3D) arrangement of all atoms. Four types of interactions stabilize the protein tertiary structure. hydrophobic interaction ionic interaction hydrogen bond van der Waals interaction §2.3.a Hydrophobic interaction Nonpolar molecules tend to cluster together in water, that is, aqueous environment tends to squeeze nonpolar molecules together. §2.3.b Ionic interaction A charged group is able to attract another group of opposite charges. The force is determined by Coulomb’s law. §2.3.c Hydrogen bond A hydrogen atom is shared by two other atoms. H-donor: the atom to which H atom is more tightly attached, and the other is H-acceptor. §2.3.d van der Waals force An asymmetric electronic charge around an atom causes a similar asymmetry around its neighboring atoms. The attraction between a pair of atoms increases as they come closer, until they are repelled by van der Waals contact distance. Interactions stabilizing proteins Myoglobin (Mb) Located in muscle to supply O2 1st protein in high resolution 153 AAs 75% of structure is α-helix in 8 regions. the interior almost entirely nonpolar residues Ribonuclease A pancreatic enzyme that hydrolyzes RNA 124 AAs Mainly β-sheet Highly compact and nonpolar interior 4 disulfide bonds Rhodopsin Photoreceptor protein 7 trans- membrane helices 11-cis-retinal chromophore in the pocket Residues are modified. §2.3.e Domain Large polypeptides may be organized into structurally close but functionally independent units. Fiberousis protein Methyl-accepting chemotaxin Highly conservative cytosolic domain Divergent periplasmic domain serving as a chemosensor Transducing the external signals into the cell §2.3.f Chaperon Chaperones are large, multisubunit proteins that promote protein foldings by providing a protective environment where polypeptides fold correctly into native conformations or quaternary structures.  How does chaperon work? Reversibly bind to the hydrophobic portions to advance the formation of correct peptide conformations Bind to misfolded peptides to induce them to the proper conformations Assist the formation of correct disulfide bonds §2.4 Quaternary Structure The quaternary structure is defined as the spatial arrangement of multiple subunits of a protein.  Proteins need to have two or more polypeptide chains to function properly. Each individual peptide is called subunit. These subunits are associated through H-bonds, ionic interactions, and hydrophobic interactions. Polypeptide chains can be in dimer, trimer.., as well as homo- or hetero- form.  Hemoglobin(Hb) O2 transporter in erythrocyte 2 α subunits, 141 AAs 2 β subunits, 146 AAs 4 subunits are maintained together by 8 pairs of ionic interactions. Each subunit contains one heme group. The conserved hydrophobic core stabilizes the 3D structure. Structure of hemoglobin Ionic forces among Hb subunits From primary to quaternary structure §2.5 Protein classification Constituents simple protein conjugated protein = protein + prosthetic groups Prosthetic group is non-protein part, binding to protein by covalent bond. This group can be carbohydrates, lipids, nucleic acids, phosphates, pigments, or metal ions.  Classification based on the overall shape Globular protein: long/short < 10，soluble in water; including enzymes, transportors, receptors, regulators, … Fibrous protein: highly elongated; insoluble in water; including collage, elastin, α- keratin, …  Section 3 Biological Functions of Proteins §3.1 Hemoglobin Hb can bind O2 reversibly, just like Mb. Both α and β chains are strikingly similar to that of Mb. Although only 24 of 146 AAs of their sequences are identical, 9 critical residues are conserved in sixty species. Residues on the surface are highly variable, but the nonpolar core is conserved. Structural similarity of Mb and Hb Fe-porphyrin complex Fe lies at the center of picket- fence porphyrin to form 4 coordinate bonds with 4 N atoms. Heme group The 5th coordinate of Fe is formed with histidine F8, and the 6th one is for either histidine E7 or O2. Heme group Oxygen-disassociation curve The saturation Y is defined as the fractional occupancy of all O2-binding sites. Y varies with the concentration of O2. The equilibrium constants for Hb subunits are different.  Binding behavior of Hb Hb has a lower affinity for O2 than Mb (lower P50). The O2–binding to the 1st subunit enhances the O2–binding to the 2nd and 3rd subunits. Such process further enhances the O2–binding to the 4th subunit significantly. Hb binds O2 in a positive cooperative manner, which enhances the O2 transport.  Local structural change Upon oxygenation, the Fe atom is moved into the porphyrin plane, leading to the formation of a strong bond with O2.  CO and O2 binding Hb forces CO to bind at an angle due to steric hindrance of His E7, which weakens the binding of CO with the heme.  Conformational changes The quaternary structure of Hb changes markedly upon oxygenation (αβ subunit shifts by 0.6nm and rotates by 15°). Global structural change The quaternary structure of Hb changes markedly for the tense (T) form to the relaxed (R) form upon oxygenation.  Allosteric effect The behavior that the lignad-binding to one subunit causes structural changes and stimulate the further binding to other subunits is termed as allosteric effect. The protein is allosteric protein, and the substrate is allosteric effector. Allosteric effect can be influenced by activators as well as inhibitors.  Concerted versus sequential Ο2 Ο2 α1 α2 Ο2 Ο2 β1 β2 Ο2 non-oxygenized Hb Ο2 Ο2 （T conformation） oxygenized Hb （R conformation） Ο2 Ο2 Ο2 Ο2 Ο2 Ο2 Ο2 Ο2 Ο2 Ο2 Ο2 §3.2 Collagen insoluble fibers that have high tensile strength 25% of total protein weight of human body consisting of three chains of same size (285kd) Collagen in different organisms Tissue Content Bone 88.0 Calcaneal tendon 86.0 Skin 71.9 Cornea 68.1 Cartilage 46-63 Ligament 17.0 Aorta 12-24 Liver 3.9  Unusual components AA components Gly (1/3), proline (1/4), 4-hydroxyproline (1/10), 5-hydroxylysine (1%) AA sequences (Gly-Pro-Y)n or (Gly-X-Hyp)n X and Y can be any AAs. n can be as high as a few hundreds.  Unusual triplex Each helix is L- handed and 3 AAs per turn. Three helixes wind together through H- bonds in the right- handed form. Unusual helical conformation (0.312 nm versus 0.15nm)  Intermolecular cross-link Lys at N- and C-termini and Hly in helical regions are responsible for the cross-link. The linkage varies with the physiological function and the tissue age. 30 genes encode for collagens, and 8 post- translational modifications are needed collagen maturation. Type of collagens Diseases and collagen Structure of hemoglobin From primary to quaternary structure  Section 4 Structure-Function Relationship of Proteins §4.1 Primary Structure and Function Primary structure is the fundamental to the spatial structures and biological functions of proteins. For a protein of particular sequence, many conformers are possible, but only the correct one has the biological functions. 1. Proteins having similar amino acid sequences demonstrate the functional similarity. 2. Proteins of incorrect structures have no proper biological functions, even their amino sequences are remained in a right order. 3. The alternation of key AAs in a protein will cause the lose of its biological functions.  Sequences of Cytochrome C ________10 ________20 ________30 ________40 ________50 ________60 ________70 ________80 ________90 _______100 ____ Human GDVEKGKKIF IMKCSQCHTV EKGGKHKTGP NLHGLFGRKT GQAPGYSYTA ANKNKGIIWG EDTLMEYLEN PKKYIPGTKM IFVGIKKKEE RADLIAYLKK ATNE Chimpanzee GDVEKGKKIF IMKCSQCHTV EKGGKHKTGP NLHGLFGRKT GQAPGYSYTA ANKNKGIIWG EDTLMEYLEN PKKYIPGTKM IFVGIKKKEE RADLIAYLKK ATNE Monkey GDVEKGKRIF IMKCSQCHTV EKGGKHKTGP NLHGLFGRKT GQASGFTYTE ANKNKGIIWG EDTLMEYLEN PKKYIPGTKM IFVGIKKKEE RADLIAYLKK ATNE Macaque GDVEKGKKIF IMKCSQCHTV EKGGKHKTGP NLHGLFGRKT GQAPGYSYTA ANKNKGITWG EDTLMEYLEN PKKYIPGTKM IFVGIKKKEE RADLIAYLKK ATNE Cow GDVEKGKKIF VQKCAQCHTV EKGGKHKTGP NLHGLFGRKT GQAPGFSYTD ANKNKGITWG EETLMEYLEN PKKYIPGTKM IFAGIKKKGE RADLIAYLKK ATNE Dog GDVEKGKKIF VQKCAQCHTV EKGGKHKTGP NLHGLFGRKT GQAPGFSYTD ANKNKGITWG EETLMEYLEN PKKYIPGTKM IFAGIKKKGE RADLIAYLKK ATKE Grey whale GDVEKGKKIF VQKCAQCHTV EKGGKHKTGP NLHGLFGRKT GQAVGFSYTD ANKNKGITWG EETLMEYLEN PKKYIPGTKM IFAGIKKKGE RADLIAYLKK ATNE Horse GDVEKGKKIF VQKCAQCHTV EKGGKHKTGP NLHGLFGRKT GQAPGFSYTD ANKNKGITWK EETLMEYLEN PKKYIPGTKM IFAGIKKKTE RADLIAYLKK ATNE Cytochrome C is a protein which can be found in all aerobic organisms.  Structures of Cytochrome C tuna-heart photosynthetic denitrifying mitochondria bacterium bacterium 1. Proteins having similar amino acid sequences demonstrate the functional similarity. 2. Proteins of incorrect structures have no proper biological functions, even their amino acid sequences are remained in a right order. 3. The alternation of key AAs in a protein will cause the lose of its biological functions. Bovine nuclease 124 AAs, 4 disulfide bonds (105 possibilities)  Only the correct form has the enzymatic activity. The denatured protein remains its primary structure, but no biological function. The renatured protein will restore its functions partially or fully depending upon the correctness of the refolded structure. 1. Proteins having similar amino acid sequences demonstrate the functional similarity. 2. Proteins of incorrect structures have no proper biological functions, even their amino sequences are remained in a right order. 3. The alternation of key AAs in a protein will cause the lose of its biological functions.  Sickle-cell of anemia Patient’s symptoms: Cough, fever and headache, a tinge of yellow in whites of eyes, visible pale mucous membrane, enlarged heart, well developed physically, anemic, much less RD cells. clinical test: The shape of the red cells was very irregular, large number of thin, elongated, sickle-shaped and crescent-shaped forms.  Identifying the cause pI of sickle-cell Hb was higher than normal one by 0.23, which is equivalent to 2 to 4 net positive charges per Hb molecule. (1949, Pauling) 2-D electrophoresis showed only one peptide of 28 digested Hb peptides is different (1954, Ingram). Difference in primary structure of Hb Sequence analysis showed the difference in AA sequence. This is the first case of molecular disease identified in history. Further studies showed that the AA variation is due to the gene mutation. §4.2 Spatial Structure and Function Proteins will experience multiple processes to become correctly folded, that is, having a correct structure. The incorrect protein structure may lead to function alternation or diseases. A particular spatial structure of a protein is strongly correlated with its specific biological functions. Protein conformational diseases Some proteins aggregate with each other after misfolding, and often form amyloid fiber precipitates resistant to proteolytic enzymes, which are toxic and pathogenic, manifesting as pathological changes of protein amyloid fiber precipitates. Mad cow disease  Mad cow disease and prion proteins A transmissible, inheritable neural disease, destroying brain tissues by converting them to a spongy appearance. the conformational prion changes of prion protein (PrP) – PrPc: α-helix, water soluble Brain – PrPsc: β-sheet, tissue water insoluble Structural changes of prion protein normal abnormal It turns normal protein into infectious protein, thus leading to the occurrence of disease. PrPc PrPsc  Section 5 Physical and Chemical Properties of Proteins §5.1 Amphoteric Isoelectric point AAs in solution at certain pH are predominantly in dipolar form, fully ionized but without net charge due to -COO- and - NH3+ groups. This characteristic pH is called isoelectric point, designated as pI. pI is determined by pK, the ionization constant of the ionizable groups.  Different AAs have different pI Amino acid pI M.W. Amino acid pI M.W. Glycine 5.97 75 cystein 5.07 121 Alanine 6.00 89 methionine 5.74 149 Valine 5.96 117 asparagine 5.41 132 Leucine 5.98 131 glutamine 5.65 146 cystein 5.60 119 Isoleucine 6.02 131 aspartic 2.97 133 Phenylalan 5.48 165 acid ine glutamic 3.22 147 Proline 6.30 115 acid tryptophan 5.89 204 Lysine 9.74 146 serine 5.68 105 Arginine 10.76 174 tyrosine 5.66 181 Histidine 7.59 155  Side-chains of a protein have many ionizable groups, making the protein either positively or negatively charged in response to the pH of the solution. The pH at which the protein has zero net- charge is referred to as isoelectric point (pI). At the isoelectric point, the protein has the lowest solubility and is most likely to form precipitates.  COOH COO- COO- + OH- + OH- P P P + H+ + + H+ NH3+ NH3 NH2 cation amphoteric anion pH < pI pH = pI pH > pI pI of most protein is ~ 5.0, and negatively charges in body fluid (pH7.4) pI > 7.4: basic proteins: protamine, histone pI < 7.4: acidic proteins: pepsin §5.2 Colloid property Diameter: 1~100nm, in the range of colloid; - - Hydrophilic groups - + + + - - - on the surface form a - + - + - - hydration shell; - - + - +- -+ Hydration shell and + -- - - - + - + electric repulsion - + - make proteins stable - in solution.  Precipitation of protein colloid + + － － + acid base － － + + base acid － － + + －－ positively charged isoelectric point negatively charged (hydrophilic) (hydrophilic) (hydrophilic) dehydration dehydration dehydration + + + － － base acid － + + － － + －－ － + + positively charged Instable protein negatively charged (hydrophobic) (deposition) (hydrophobic) §5.3 Protein denaturation The process in which a protein loses its native conformation under the treatment of denaturants is referred to as protein denaturation. The denatured proteins tend to - decrease in solubility; - increase the viscosity; - lose the biological activity; - lose crystalizability; - be susceptible to enzymatic digestion.  Cause of denaturation the disruption of hydration shell and electric repulsion Denaturants physical: heat, ultraviolet light, violent shaking, … chemical: strong acids, bases, organic solvents, detergents, … Applications sterilization, lyophilization Protein denaturation ruptures H-bonds and disulfide bonds, and creates a “random coil” conformation.  Renaturation Once the denaturants are removed, the denatured proteins tend to fold back to their native conformations partially or fully. The renatured proteins can restore their biological functions.  Renaturation When the conditions of denaturation are intense and persistent, the denaturation of proteins is usually irreversible.  Protein precipitation The denatured proteins expose their side chains or the inner part to the aqueous environment, which causes the proteins aggregated and separated out from the aqueous solution.  Protein coagulation When the denatured proteins become insoluble fluffy materials, heating denatured proteins will turn them into a hard solid which are not soluble even strong acids and bases are applied. Coagulation is an irreversible process. §5.4 UV absorption Trp, Tyr, and Phe have aromatic groups of resonance double bonds. Proteins have a strong absorption at 280nm. Both free and incorporated AAs show this absorption.  This feature can be used to strong absorption at 280nm distinguish between proteins and nucleic acids.  §5.5 Coloring reactions (1). Biuret reaction: peptide bonds and Cu2+ under the heating condition to form red or purple chelates. Used for determine the hydrolysis of proteins, since free amino acids do not react. (2) Ninhydrin reaction: Amino acids can react with ninhydrin to form a chemicals having maximal adsorption at 570 nm. Used for quantifying the free amino acids.  Summary The Physical and Chemical Properties of Proteins Amphoteric Colloid property Protein denaturation UV absorption Coloring reactions  Section 6 Exploration of Protein §6.1 Isolation and purification Homogenization and centrifugation Dialysis Precipitation Chromatography Electrophoresis §6.1.a Homogenization Rupture the plasma membrane to release the intracellular components into the buffered solution – Sonication, French pressure, mechanical grinding – Chemical reagents, lysozymes  Centrifugation Because of the differences in size and shape, proteins will sediment gradually under the centrifugal force until the sedimentation force and buoyant force reach the balance. The sedimentation behavior is described in sedimentation coefficient (S) which is proportional to the molecular weight. Differential centrifugation Differential centrifugation homogenate 600 g，3 min Pellet supernatant (nuclei) 6,000 g，8 min Pellet supernatant (mitochondria, chloroplasts, lysosomes, peroxisomes) 40,000 g，30 min Pellet supernatant (plasma membrane, fragments of Golgi and ER) 100,000 g，90 min Pellet supernatant (ribosomal subunits) (cytosol) Rate-zone centrifugation  §6.1.b Dialysis Proteins, as macromolecules, cannot pass through the semipermeable membrane containing pores of smaller than protein dimension, thus large proteins and small molecules can be separated. Application Dialysis can be used for protein purification, desalting, and condensation. §6.1.c Precipitation Adding a large quantity of salts, such as Ammonia sulfate, into the protein solution will neutralize the surface charges and the destruct the hydration shell of proteins, causing them to precipitate. Acetone has the similar function. Application An efficient way to concentrate proteins §6.1.d Chromatography When a protein solution (called as mobile phase) passes through a stationary phase, proteins can interact with the stationary phase due to the differences in size, charge, and affinity, making the different proteins flow through the stationary phase at different speeds.  Elution buffer Protein mixture Solid phase capable of reacting with proteins to be separated Protein 1 Protein 2 OD280nm Elution volume  Type of chromatography Ion exchange: based on the ionic interactions Affinity: based on the binding strengths Filtration: based on the protein sizes Hydrophobicity: based on the hydrophobic forces Ion-exchange chromatography More negatively charged proteins bind to the solid phase tightly, and stronger elution buffer is needed to elute them out the column ＝ － ＝ － － ＋ ＋ ＝ ＋ ＋ ＝ ＋ ＋ ＝ － ＝ ＋ ＋ ＝ ＋ ＋ ＝ － ＋ ＋ ＋ ＋ ＝ ＝ ＋ ＋ ＝ － ＝ － － － － Ionic exchange column with Less negatively charged positive charge proteins bind to the solid phase loosely, and weak elution buffer can be used to elute them out the column Affinity chromatography Exchange column with the ligands for binding special proteins Proteins having Proteins having weak binding strong binding affinity with the affinity with the ligads ligads  Gel filtration Proteins are separated based on their sizes and shapes. The stationary phase is of semi-uniform pores. When the protein solution flows through porous beads, smaller proteins can enter the pores and stay there for a longer period, but larger proteins flow directly through the column, resulting in the separation of proteins. It is also called molecular sieve or size exclusion.  Gel filtration Small proteins can enter the porous beads, and have a longer stationary time Porous beads Large proteins that allow the small are unable to enter proteins enter the porous beads will pass by and flow out directly §6.2 Electrophoresis Analysis Application Used mainly for determination of proteins SDS-PAGE = Sodium dodecyl sulfate polyacrylamide gel electrophoresis IFE = isoelectric focusing electrophoresis 2D = two dimensional electrophoresis  §6.2.a SDS-PAGE Sodium dodecyl sulfate is a kind of detergent to denature the proteins Anionic SDS bind to protein in the ratio of 1 SDS per 2 AAs. large number of negative charges completely mask the original charge of the protein The protein-SDS complex is roughly charged proportional to the mass. The smaller the protein, the faster the moving speed in the electric field.  The gel polymer material for protein discrimination is composed of methylenebisacrylamide and acrylamide. The pore size can be controlled by changing the concentration of cross-linking reagent. The gel with different concentration of cross-linking reagent can be used for different size proteins. § 6.2.b Isoelectric focusing Depend upon the electric properties of proteins The charged proteins, either positively or negatively, will migrate in the electric field. The proteins having net zero charges stop moving in the electric field. Principle of IFE §6.2.c 2D electrophoresis 1st dimension: isoelectric focusing electrophoresis （pl） 2nd dimension: SDS-PAGE (MW) Application A high throughput approach to identify proteins Protein extraction IEF SDS-PAGE Each spot corresponds to a protein, and thousands of proteins can be separated. §6.3 Composition and Sequence Composition Determination of terminal residues Edman degradation Sequencing strategy Mass spectroscopy Deduction form DNA sequences §6.3.a Composition analysis Hydrolyzing the purified protein samples in an evacuated and sealed tube by heating it in 6 M HCl at 100 °C. Analyzing the AA components using chromatography (Alaa, Argb, Asnc, Aspd, … Valz) Chromatography of AA §6.3.b Terminal residues The amino-terminal residue reacts with fluorodinitrobenzene or dabsyl chloride to form a stable product which can be analyzed using chromatography. The carboxyl-terminal residue can react with fluorodinitrobenzene or dabsyl chloride to form a stable product. N-terminal reaction The N-terminus is the starting point of a protein and is crucial for its structure and function. The separated N- terminal amino acids are usually sent to high- performance liquid chromatography (HPLC) for analysis.  §6.3.c Edman degradation 1. Labeling the N-terminal residue with a fluorophore. 2. Cleaving the labeled residue without breaking the peptide bonds of the rest part of the peptide. 3. Determining the N-terminal residue with chromatography. 4. Repeating the same procedure to the rest peptide until the whole sequence is determined. First cycle of Edman degradation H O H O N C S H2N C C N C C R1 H R2 phenyl isothiocyanate labeling H S H H O H O N C N C C N C C R1 H R2 releasing S H O H O N C H2N C C N C C R2 H R3 C N H O C H R1 Edman degradation §6.3.d Overlapping approach 1. Cleaving a protein into small peptides by chemical or enzymatic methods, and purify these peptides. 2. Sequencing each peptide using Edman degradation approach. 3. Overlapping peptide fragments to arrange them in a right order, and accomplishing the AA sequencing.  Cleavage of peptides Cleavage reagent Cleavage site Cyanogen bromide Met (C) O-Iodosobenzoate Try (C) Hydroxylamine Asp-Gly Trypsin Lys and Arg (C) Clostripain Arg (C) Staphylococcal protease Asp and Glu (C) Thrombin Arg (C)  Overlapping approach 1. Tryptic cleavage generates two peptides Gly-Phe-Val-Glu-Arg, Val-Phe-Asp-Lys 2. Chymotryptic cleavage generates three peptides Val-Phe, Val-Glu-Arg, Asp-Lys-Gly-Phe 3. Overlapping the sequenced peptides Tryptic peptide Tryptic peptide Val - Phe - Asp - Lys - Gly - Phe - Val - Glu - Arg Chymotryptic peptide  §6.3.e Mass spectroscopy Newly developed approach applied to biology and medicine areas Revolutionized bioanalytical technique Offering a fast, high accuracy, and high throughput determination for analyzing peptides and proteins. Matrix-aided ionization 1. Deposit samples on a plate. 2. Introduce a beam of laser. 3. Ionize samples. 4. Analyze ionized molecules. 5. Determine the AA sequence.  Fragmentation of peptide xn+3 yn+3 xn+2 yn+2 xn+1 yn+1 O O O O O —N—C—C —N—C—C—N—C—C —N—C—C —N—C—C H Ri H Ri+1 H Ri+2 H Ri+3 H Ri+4 ai+1 bi+1 ai+2 bi+2 ai+3 bi+3  From MS to AA sequence Separation of ions with different mass to charge ratios (m/z) under the action of electric and magnetic fields. Based on the information of molecular and fragment ions in the mass spectrum, the molecular weight and structural composition of the analyte can be analyzed. §6.3.f Deduction from DNA sequence Isolate the genes encoding the protein DNA sequencing mRNA sequencing Determine the AA sequence according to the 3-letter genetic codons §6.4 Structure Determination Circular dichroism spectroscopy X-ray crystallography Nuclear magnetic resonance spectroscopy Prediction based on the protein sequence homology Computer simulation  Section 7 Proteomics: A New Frontier Proteomics  Proteomics 1994, Australian scientist Marc Wilkins proposed the concept of proteomics, which characterizes all the proteins that the genome can express. In 1997, the concept of proteomics emerged, which mainly studies all proteins within cells, tissues, or organs. In 2001, the International Human Proteome Organization (HUPO) was officially established, promoting the development of proteomics research.  Proteomics a comprehensive knowledge about all the proteins of a cell at a specific given time.  Objectives Biological process – The overall process toward which this protein contributes Molecular function – The biological activity the protein accomplishes Cellular component – The location of protein activity  Proteomics approaches Separation Sequence determination 3D-structure Functionality Expression regulation Post-translation modification Proteomics analysis reveals differential protein expression in the bone marrow of acute myeloid leukaemia （AML ） patients. Changed proteins in CSF of patients in Japanese encephalitis （JE） subgroups