中国医科大学蛋白质生物化学英文ppt课件

Chapter 1 Protein,Chemical components Molecular structures Biological functions Structure-function relationship Physical and chemical properties Exploration of proteins Proteomics: a new frontier,Contents,Proteins are macromolecules composed of amino acids linked together through peptide bonds.,What are proteins?,the most widely distributed biomolecules the most abundant biomolecules (45% of human body) the most complex biomolecules the most diversified biological functions,How are about proteins?,What do proteins do?,Section 1 Chemical Components of Proteins,major elements C (5055%), H (7%), O (1920%), N (1319%), S (4%) trace elements P, Fe, Cu, Zn, I, ,Components of proteins,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.,L-Amino acid,Molecular weight,Dalton: A unit of mass nearly equal to that of a hydrogen atom Gly C2NO2H5 75 Ala C3NO2H7 89 Val C5NO2H11 117 Leu C6NO2H13 131 Ile C6NO2H13 131,1.1.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.,R groups are non-polar, hydrophobic aliphatic or aromatic groups. R groups are uncharged. AAs are insoluble in H2O.,Non-polar and hydrophobic AAs,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.,R groups have one -NH2. R groups are positively charged at neutral pH (=7.0). AAs are highly hydrophilic.,Basic AAs,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.,-amino-propionic acid -amino-guanidinovaleric acid (alanine) (arginine),Special amino acids - Gly,optically inactive,Special amino acids - Pro,Having a ring structure and imino group,Special amino acids - Cys,active thiol groups to form disulfide bond,1.2 Peptide,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.,1.2.a Peptide and peptide bond,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 cystein glycine,GSH peroxidase,GSH reductase,NADPH+H+,NADP+,As a reductant to protect nucleic acids and proteins from toxin by discharging free radical or H2O2,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,Peptides,thyrotropin-release hormone,Pyroglutamic acid histidine prolinamide,Neuropeptide,Section 2 Molecular Structures of Proteins,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.,Overview,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), C, and C(=O) constitute the backbone. H-bonds are responsible for stabilizing the secondary structure. The side chains are not considered. -helix -pleated sheet -turn (-bend) random coil,Peptide unit,Six atoms, C-C(=O)-N(-H)-C, 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,Rotation of peptide unit,“Beads on a string”,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),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.,2.2.a -helix,Left-hand versus right-hand,The -CO group of residue n is H-bonded to the -NH group of residue (n+4).,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.b -pleated sheet,One -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 -turn reverses abruptly the direction of a protein backbone. H-bonds are perpendicular to the protein backbone.,2.2.c -turn,2.2.d Random coil,There is no consistent relationship between planes.,2.2.e Motif,Zinc finger HLH (helix-loop-helix) HTH (helix-turn-helix) Leucine zipper,When several local peptides of defined secondary structures are close enough in space, they are able to form a particular “super-secondary” structure.,2.2.f Side chains effect,Shape: Pro having a rigid ring (helix disrupter) Size: -sheet needs AAs of small side chain. Leu, Ile, Trp, and Asn having bulky sides (hard to form helix) Charge: Too many charged AAs in a short region of one peptide is hard to form helix.,2.3 Tertiary Structure,The tertiary structure is defined as the spatial positions of all atoms of a protein, 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.,A charged group is able to attract another group of opposite charges. The force is determined by Coulombs law.,2.3.b Ionic interaction,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.,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.,2.3.d van der Waals force,Interactions stabilizing proteins,Myoglobin (Mb),Located in muscle to supply O2 1st protein in high resolution 153 AAs 75% of structure is -helix in 8 regions. the interior almost entirely nonpolar residues,Ribonuclease,A pancreatic enzyme that hydrolyzes RNA 124 AAs Mainly -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 singles 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.,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,How does chaperon work?,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.,O2 transporter in erythrocyte 2 subunits, 141 AAs 2 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.,Hemoglobin(Hb),Structure of hemoglobin,Ionic forces among Hb subunits,From primary to quaternary structure,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.,2.5 Protein classification,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, -keratin, ,Section 3 Biological Functions of Proteins,Hb can bind O2 reversibly, just like Mb. Both and 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.,3.1 Hemoglobin,Structural similarity of Mb and Hb,Fe lies at the center of picket-fence porphyrin to form 4 coordinate bonds with 4 N atoms.,Fe-porphyrin complex,The 5th coordinate of Fe is formed with histidine F8, and the 6th one is for either histidine E7 or O2.,Heme group,Heme group,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.,Oxygen-disassociation curve,Hb has a lower affinity for O2 than Mb (lower P50). The O2binding to the 1st subunit enhances the O2binding to the 2nd and 3rd subunits. Such process further enhances the O2binding to the 4th subunit significantly. Hb binds O2 in a positive cooperative manner, which enhances the O2 transport.,Binding behavior of Hb,Upon oxygenation, the Fe atom is moved into the porphyrin plane, leading to the formation of a strong bond with O2.,Local structural change,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 ( subunit shifts by 0.6nm and rotates by 15).,The quaternary structure of Hb changes markedly for the tense (T) form to the relaxed (R) form upon oxygenation.,Global structural change,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.,Allosteric effect,Concerted versus sequential,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,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,pK curve,Structure of hemoglobin,Concerted versus sequential,Model comparison,Section 4 Structure-Function Relationship of Proteins,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.,4.1 Primary Structure and Function,Proteins having similar amino acid sequences demonstrate the functional similarity. Proteins of incorrect structures have no proper biological functions, even their amino sequences are remained in a right order. The alternation of key AAs in a protein will cause the lose of its biological functions.,Sequences of Cytochrome C,Cytochrome C is a protein which can be found in all aerobic organisms.,tuna-heart photosynthetic denitrifying mitochondria bacterium bacterium,Structures of Cytochrome C,Proteins having similar amino acid sequences demonstrate the functional similarity. Proteins of incorrect structures have no proper biological functions, even their amino acid sequences are remained in a right order. 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),The denatured protein remains its primary structure, but no biological function. Only the correct form has the enzymatic activity.,The renatured protein will restore its functions partially or fully depending upon the correctness of the refolded structure.,Proteins having similar amino acid sequences demonstrate the functional similarity. Proteins of incorrect structures have no proper biological functions, even their amino sequences are remained in a right order. The alternation of key AAs in a protein will cause the lose of its biological functions.,Sickle-cell of anemia,Patients 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.,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).,Identifying the cause,Identifying the cause,Sequence analysis showed the difference in AA sequence. Hb A : Val-His-Leu-Thr-Pro-Glu-Glu-Lys- Hb S : Val-His-Leu-Thr-Pro-Val -Glu-Lys- This is the first case of molecular disease identified in history. Further studies showed that the AA variation is due to the gene mutation.,Difference in primary structure of Hb,Proteins will experience multiple processed 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.,4.2 Spatial Structure and Function,A transmissible, inheritable neural disease, destroying brain tissues by converting them to a spongy appearance the conformational changes of prion protein (PrP) PrPc: -helix, water soluble PrPsc: -sheet, water insoluble,Mad cow disease and prion proteins,Structural changes of prion protein,PrPc PrPsc,Section 5 Physical and Chemical Properties of Proteins,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.,Isoelectric point,5.1 Amphoteric,pH=pI,pHpI,pHpI,amphoteric,cation,anion,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).,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: 1100nm, in the range of colloid; Hydrophilic groups on the surface form a hydration shell; Hydration shell and electric repulsion make proteins stable in solution.,positively charged (hydrophobic),Instable pr