Baixe Aula estrutura de proteinas e outras Notas de aula em PDF para Bioquímica, somente na Docsity! Primary structure Tertiary Quaternary structure structure Amino acid residues Polypeptide chain Assembled subunits 8 Conformation 2,000 x 5 À c aHelix 900 x 11 À Native globular form 130 x 30 À DONT x — X NH HN X =| x DA x X (a) (d) o o Nº Ca / N CH CH, a c C PNL NAO CH-c7 ç ç Joca; — N+ — ENE NG cH “Feó cH N Ea / CN *N=C end d d c cH, £Z Ns / As CH; A “en” CH; CH “cm, (b) Edge view cH Nona | FA pa c £ScH CH, Histidine Plane of residue porphyrin ring system 1.0 0.5 pO9 (kPa) (b) 10 Myoglobin B subunit of hemoglobin 1.0 0.8 0.6 0.4 0.2 pO, in pÔ, in tissues lungs High-affinity state Transition from low- to high- affinity state Low-affinity state 4 8 12 16 0 l l l 1 0 2 4 6 8 10 pOa (kPa) an EO A Cro IT Õ R Õ Õ amino denninal Carbamino-terminal residue residue Reservoir Solution (mobile phase) Solid porous matrix (stationary phase) Porous support ô 6% — Effluent (a) Key: O Large net positive charge O Net positive charge O Net negative charge O Large net negative charge Polymer beads with negatively charged functional groups Protein mixture is added to column containing cation exchangers. Proteins move through the ô column at rates determined by their net charge at the [2] e! [6 pH being used. With cation exchangers, proteins with amore negative netcharge 1 234 5 6 move faster and elute earlier. (a) Protein mixture is added to column containing cross-linked polymer. Protein molecules separate by size; larger molecules 3 ja alo! pass more freely, appearing im the earlier fractions. É 123456 (b) “9 | Mixture of * )| macromolecules Electrophoresis ———————>» Direction of electrophoresis | Porous gel l Na O—S—O— (CH), CHs | O Sodium dodecyl sulfate (SDS) l | EM j 060 14041 MN IA AA E O + E] mm o Ud log M, Unknown protein | | | | | Relative migration (b) A Purification Table for a Hypothetical Enzyme* Fraction Total Procedure volume protein Activity Specific activity or step (ml) (mg) (units) (units/mg) . Crude cellular extract 1,400 10,000 100,000 10 . Precipitation with ammonium sulfate 280 3,000 96,000 32 . lon-exchange chromatography 90 400 80,000 . Size-exclusion chromatography 80 100 60,000 . Affinity chromatog- raphy 6 3 45,000 *AII data represent the status of the sample after the designated procedure has been carried out. Activity and specific activity are defined on page 137. Homogenate Salt lon exchange Molecular Affinity fractionation chromatography exclusion | chromatography chromatography mm mm É mm (A) Low pH esto º a “e High pH (=) + +———— +—— (+) (B) Low pH High pH () (1) Isoelectric focusing gel is placed on SDS polyacrylamide gel. Second dimension SDS polyacrylamide gel electrophoresis First dimension Decrecaing P) Isoelectric focusing 0) DD) , O = po Decreasing is . ça ns — Es a E RR Le Es — | E mm o e a e [=== E A = L E ge|s apruejApeAjod-sas 5 tu (A) CH, CH; Sa 7 N CH: CO N PN N=N PN SO,CI CH; — — SOsCl Dansyl chloride Dabsyl chloride Disulfide bond V (cystine) + Fo CHysH CHOH doa CH9SH Dithiothreitol (DTT) A NH o O=C | I I | Etta Op SH— OH HG CHa SE HS CH OH C=0 O HN C=0 HN. s Cysteic acid + É acetylation residues by e | iodoacetate + E = HO—CH;—S—CH>—COO” “000-—CHa—S—CHs—CH C—0 HN Acetylated Y cysteine residues The Specificity of Some Common Methods for Fragmenting Polypeptide Chains Treatment* Cleavage pointst Trypsin Lys, Arg (C) Submaxillarus protease Arg (C) Chymotrypsin Phe, Trp, Tyr (C) Staphylococcus aureus V8 protease Asp, Glu (C) Asp-N-protease Asp, Glu (N) Pepsin Phe, Trp, Tyr (N) Endoproteinase Lys C Lys (C) Cyanogen bromide Met (C) *AIl except cyanogen bromide are proteases. All are available from commercial sources. tResidues furnishing the primary recognition point for the protease or reagent; peptide bond cleavage occurs on either the carbonyl (C) or the amino (N) side of the indicated amino acid residues. Glass | Sample Mass capillary solution spectrometer High | voltage Vacuum interface EE (a) Relative intensity (%) 47,342 100 - 50+ 50 - 100 + 0E===É = 5 L 40+ 47,000 48,000 y a 50 - 30+ 25 - | 0 M : y u Ú da nm a 800 1,000 1,200 1,400 1,600 mz (b) Collision MS-1 cell MS-2 Detector FE NO NO kk SE nn Electrospray Separation Breakage ionization | b Ri Rê O Rº Dol H H | H H lo 40 HN—C—C—N—C—C—-N-C—CIN—-C—-C—No-C—COl H ll H H | H o” R2 O R$ O y Ri Rê O Ró LI H HI H H | 40 HN—C—C—N—C—C—N—C— Ce eN—C—C—-N—-C— CL H lo H H | H o”