2006BiochemA chap3

Slide1 : Dr. Rita P.-Y. Chen (陳佩燁) Assistant Research Fellow Institute of Biological Chemistry Academia Sinica


Slide2 : Amino acid Chiral center Optically active: rotate plane-polarized light Amino acid in protein: L stereoisomer


Absolute configuration: D,L system : Absolute configuration: D,L system Not all L-amino acids are levorotatory (rotating polarized light to the left)


Specifying onfiguration: RS system L-amino acid has S configuration : Specifying onfiguration: RS system L-amino acid has S configuration


aliphatic amino acid structures : aliphatic amino acid structures CH CH3 CH2 CH3 Isoleucine (I) (Ile)


Side Chains with Alcohol Groups : Side Chains with Alcohol Groups Serine (Ser, S) and Threonine (Thr, T) have uncharged polar side chains Catalytic role, phosphorylation, o-linked glycosylation, hydrogen bond


Structures of aspartate, glutamate, asparagine and glutamine : Structures of aspartate, glutamate, asparagine and glutamine


Structures of histidine, lysine and arginine : Structures of histidine, lysine and arginine d-Guanido group imidazole group


Methionine and cysteine : Methionine and cysteine First a.a. Catalytic role, disulfide bond


Fig 3.4 Formation of cystine : Fig 3.4 Formation of cystine Disulfide bond


Aromatic amino acid structures : Aromatic amino acid structures phosphorylation Indole group


Proline has a nitrogen in the aliphatic ring system : Proline has a nitrogen in the aliphatic ring system Proline (Pro, P) - has a three carbon side chain bonded to the a-amino nitrogen The heterocyclic pyrrolidine ring restricts the geometry of polypeptides


Slide20 : p t


Uncommon a.a. : Uncommon a.a. Plant cell wall Collagen Collagen myosin Prothrombin Ca2+ binding protein elastin 21th a.a. Added during protein synthesis UGA codon glutathione peroxidases


Other amino acids (not constituents of proteins) : metabolite : Other amino acids (not constituents of proteins) : metabolite Key intermediate in biosynthesis of Arg and in urea cycle p843 Urea


補充教材 : 補充教材


Amino acid can act as acids and bases : Amino acid can act as acids and bases


Slide28 : 較易解離 較易解離


Slide29 : The characteristic pH at which the net electric charge is zero is called the isoelectric point or isoelectric pH, designated pI. For glycine, which has no ionizable group in its side chain, the isoelectric point is simply the arithmetic mean of the two pKa values:


Slide30 : 0 pI = (2.19+4.25)/2 = 3.22


Slide31 : 0 pI = (6+9.17)/2 = 7.59


Peptides and Proteins : Peptides and Proteins


Slide33 : SGYAL


Slide34 : +2 +1 0 -1 2.34 9.6 4.25 10.53 pI = (4.25+9.6)/2 =6.93


Slide35 : Artificial Sweetener, ex. in Diet Coke 200 times sweeter than sugar D-form a.a. substitution is bitter 苯酮尿症(Phenylketonuria) 患者不可使用, use Alatame instead


Slide36 : Fig. 3-13, p.75 催產素 血管收縮素


Glutathione : Glutathione an important water-phase antioxidant and essential cofactor for antioxidant enzymes


Protein size is varied : Protein size is varied


Polypeptides have characteristic amino acid compositions : Polypeptides have characteristic amino acid compositions


Some proteins have chemical groups other than a.a. : Some proteins have chemical groups other than a.a. Non a.a. part – prosthetic group


Protein purification : Protein purification Fractionation: protein solubility depends on temperature, pH, salt Dialysis Ultrafiltration: N2 purge, centrifugation Column chromatography Electrophoresis


Column chromatography : Column chromatography


Slide44 : Cation exchange chromatography: Protein carries postive charge (cation) Buffer pH must be lower than protein pI No sample volume limit


Slide45 : Size exclusion chromatography Big protein runs faster Sample volume is limited Column is usually long


Slide46 : Affinity chromatography Separate proteins by their binding specificities No sample volume limit


Electrophoresis: (1) SDS-PAGE : Electrophoresis: (1) SDS-PAGE Proteins migrate according to their size and shape One SDS bind for every two residues Protein is denatured, subunits will be separated cathode Anode


Slide49 : Coomassive blue staining 4 subunits


Slide51 : Electrophoresis: (2) Isoelectric focusing Determine protein pI Use ampholytes to create get with pH gradient Proteins stop migration when pH = pI


Two-dimensional electrophoresis : Two-dimensional electrophoresis


Protein sequencing : Protein sequencing Protein function depends on its sequence 20 -30 % proteins are polymorphic Most proteins contain crucial regions that are essential to their function and whose sequence is therefore conserved.


Slide57 : 1953, Frederick Sanger worked out the sequence of insulin 1958 Nobel Prize in Chemistry 1980 Nobel Prize in Chemistry: DNA sequencing b. 1918


Slide58 : Only identify the first a.a. Pehr Edman: Edman degradation


Large proteins must be sequenced in smaller segment : Breaking disulfide bond Cleaving the polypeptide chain Sequencing of peptides Ordering peptide fragments Locating disulfide bond Large proteins must be sequenced in smaller segment


Slide60 : Breaking disulfide bond


Cleaving the polypeptide chain : Cleaving the polypeptide chain


Locating disulfide bond : Locating disulfide bond Do the same thing except breaking disulfide bond See which peptide fragments are missing or which peptide fragment (longer) appears


Other methods to solve protein sequence : Other methods to solve protein sequence Translate from DNA:Genome, proteome Mass spectrometry


Slide69 : The Nobel Prize in Chemistry 1984 --for his development of methodology for chemical synthesis on a solid matrix Robert Bruce Merrifield Rockefeller University 1921-2006


Homologous proteins : Homologous proteins Paralog: homologous genes within a single species that diverged by gene duplication. Ortholog: genes in different species that derive from a common ancestor. Orthologous genes may or may not have the same function.


Molecular evolution : Molecular evolution 1960s Zuckerkandle and Pauling use nucleotide and protein sequence to explore evolution 1970s Carl Woese used ribosomal RNA sequence (archaebacteria is different from other bacteria) Not every protein is a good target (choose protein with essential function ex. cellular metabolism EF-1a) Lateral gene transfer ex. Antibiotic-resistent gene At some position, only particular amino substitutions can be tolerated Electronic search, multiple sequence alignment Gap, penalty


Slide75 : Determining how closely related the proteins are – Blosum (blocks substitution matrix) Based on short conserved blocks Unique chemical properties – higher score Each substitution has a score, based on its frequency Blosum 62 table (62% identity)


Signature sequence – useful sequence segment in taxonomy : Signature sequence – useful sequence segment in taxonomy Sequence in signature sequence might be quite distinct EF-1a/EF-Tu family


Evolutionary tree : Evolutionary tree Length of line is proportional to the number of a.a. substitution From the sum of length, we can know how close two species are From different proteins, we can obtain different evolutionary trees