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1、Chapter 1Protein,Chemical componentsMolecular structuresBiological functionsStructure-function relationshipPhysical and chemical propertiesExploration of proteinsProteomics:a new frontier,Contents,Proteins are macromolecules composed of amino acids linked together through peptide bonds.,What are pro
2、teins?,What do proteins do?,Peptide Classification,Protein Shape,Fibrous proteins,Globular proteins,Membrane proteins,Protein functions,Enzymes,Regulatory Proteins,Transport Proteins,Structural Proteins,Motor protein,Signal Protein,Protein Components,Simple protein,Conjugated protein,Section 1Chemic
3、al Components of Proteins,major elementsC(5055%),H(6.97.7%),O(2124%),N(1517.6%),S(0.32.3%)trace elementsP,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
4、 in 100g sample=N per gram x 6.25 x 100,1.1 Amino Acids,The basic building blocks of proteinsAbout 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 optica
5、lly active center.,L-Amino acid,L、D-Amino acid,Molecular weight,Dalton:A unit of mass nearly equal to that of a hydrogen atom GlyC2NO2H5 75AlaC3NO2H7 89ValC5NO2H11 117LeuC6NO2H13 131IleC6NO2H13 131,Amino acid Classification,The R groups,also called side chains,make each AA unique and distinctive.R g
6、roups are different in their size,charge,hydrogen bonding capability and chemical reactivity.,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 gr
7、oups 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.,Asparti
8、c 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),Uncommon Amino Acids,Several amino acids occur only rarely in proteins,Amino Aci
9、ds Not Found in Proteins,Certain amino acids not found in proteins nonetheless are biochemically important.,1.1 The properties of the amino acid,AcidBase Chemistry of Amino AcidsAll the amino acids contain at least two dissociable hydrogens,so calledampholyte.,AAs tend to be least soluble at theiris
10、oelectric point,the pH value at which the sum of their positive and negativeelectrical charges is zero.,Isoelectric point,Ionization of Side Chains,Reactions of Amino Acids,The Ninhydrin Reaction,Carboxyl and Amino Group Reactions,Specific Reactions of Amino Acid Side Chains,Millon reaction HgNO3+Hg
11、(NO3)2+HNO3 Red Phenyl Tyr,Folin reaction Phosphomolybdate Blue Phenyl Tyr,Phosphotungstic acid Indolyl,Sakoguchi NaBrO+-Naphthol Red Guanidine Arg,Pauly Pauly agent Orange His Tyr,Section 2Chemical Structure of Proteins,Proteins are composed of AAs.Distinctive properties of proteins are determined
12、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 o
13、f 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.,Peptide and peptide bond,A peptide bond is a co
14、valent 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
15、called as residues.,Glutathione(GSH)Glutamic acidcystein glycine,Primary structure of insulin,Two peptides of 21 and 30 AAs Two inter-chain-S-S-bondsOne 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 segm
16、ent,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),Peptide unit,Six atoms,C-C(=O)
17、-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 Calif
18、ornia Institute of Technology,CAThe 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 ve
19、rtical 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,An extended zigzag conformation of protein backbonesProtein backbones are arranged side-by-si
20、de 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 hydro
21、gen 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,2.3 Tertiary Structure,The tertiary structure is defined as the spatial positions of all atoms of a protein,i.
22、e.,the three-dimensional(3D)arrangement of all atoms.,Four types of interactions stabilize the protein tertiary structure.hydrophobic interaction ionic interaction hydrogen bondvan der Waals interaction,2.3.a Hydrophobic interaction,Nonpolar molecules tend to cluster together in water,that is,aqueou
23、s environment tends to 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
24、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,Inter
25、actions stabilizing proteins,Myoglobin(Mb),Located in muscle to supply O2153 AAs 75%of structure is-helix in 8 regions.the interior almost entirely nonpolar residues,Ribonuclease,A enzyme that hydrolyzes RNA124 AAs Mainly-sheetHighly compact and nonpolar interior4 disulfide bonds,2.4 Quaternary Stru
26、cture,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
27、 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 AAs4 subunits are maintained together by 8 pairs of ionic interactions.Each subunit contains one heme group.The conserved hydrophobic core stabiliz
28、es the 3D structure.,Hemoglobin(Hb),Structure of hemoglobin,Section 3Structure-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
29、one has the biological functions.,3.1 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
30、 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.,Homologous proteins,tuna-heart photosynthetic denitrifying mitochondria bacterium bacterium,Structures of Cytochrome C,Proteins having s
31、imilar 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 nucleas
32、e,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
33、.,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.,
34、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 cellsclinical test:The shape of the red cells was very irregular,large number of thin,elongated,sickle-shaped
35、 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
36、 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 st
37、ructure 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 functio
38、ns.,3.2 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 ch
39、anges of prion protein,PrPc PrPsc,Section 4Physical and ChemicalProperties 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 b
40、y pK,the ionization constant of the ionizable groups.,Isoelectric point,4.1 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 prot
41、ein 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,histonepI 7.4:acidic proteins:pepsin,4.2 Colloid property,Diameter:1100nm,in the range of colloid;Hydrophilic groups on the surface fo
42、rm a hydration shell;Hydration shell and electric repulsion make proteins stable in solution.,positively charged(hydrophobic),Instable protein(deposition),dehydration,dehydration,dehydration,Precipitation of protein colloid,negatively charged(hydrophobic),positively charged(hydrophilic),negatively c
43、harged(hydrophilic),The denatured proteins tend to-decrease in solubility;-increase the viscosity;-lose the biological activity;-lose crystalizability;-be susceptible to enzymatic digestion.,4.3 Protein denaturation,The process in which a protein loses its native conformation under the treatment of
44、denaturants is referred to as protein denaturation.,Cause of denaturation the disruption of hydration shell and electric repulsionDenaturants physical:heat,ultraviolet light,violent shaking,chemical:strong acids,bases,organic solvents,detergents,Applicationssterilization,lyophilization,Renaturation,
45、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,The denatured proteins expose their side chains or the inner part to the aqueous environment,which ca
46、uses the proteins aggregated and separated out from the aqueous solution.,Protein precipitation,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 ir
47、reversible process.,Protein coagulation,4.4 UV absorption,Trp,Tyr,and Phe have aromatic groups of resonance double bonds.AAs have a strong absorption at 280nm.Both free and incorporated AAs show this absorption.,Section 5Exploration of Protein,5.1 Isolation and purification,Homogenization and centri
48、fugation Dialysis Precipitation Chromatography Electrophoresis,Rupture the plasma membrane to release the intracellular components into the buffered solutionSonication,French pressure,mechanical grinding,Chemical reagents,lysozymes,5.1.a Homogenization,Centrifugation,Because of the differences in si
49、ze 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,5.1.b Dialysis
50、,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.Dialysis can be used for protein purification,desalting,and condensation.,5.1.c Precipitation,Adding a large quantity
