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Determination of structure of proteins

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A comprehensive presentation on Determination of structure of proteins for MBBS, BDS, B Pharm & Biotechnology students to facilitate self- study.

Publicado en: Salud y medicina
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Determination of structure of proteins

  1. 1. Determination of structure of proteins Dr. Rohini C Sane
  2. 2. Levels of protein structure
  3. 3. Steps for determining the primary structure of protein 1. Determination of the number of polypeptide chains in a protein. 2. Determination of amino acid composition by complete hydrolysis of the polypeptide chains followed by ion –exchange chromatographic separation and identification. 3. Identification of N –terminal and C –terminal amino acids. 4. Side specific hydrolysis of polypeptide chain using specific enzymes to get a mixture of overlapping peptides. 5. Separation and purification of each of these peptides ,and then analyzing the amino acid sequence of the small peptides, and then deciphering the sequence of the whole protein. 6. Cleavage of the original polypeptide chain by a second procedure 7. Ordering peptide fragments by establishing overlaps
  4. 4. Elucidation of protein structure ❖Steps in elucidating structure of proteins : 1. Purification of proteins by various chromatography techniques : a. Partition b. Ion exchange c. Adsorption d. Gel filtration e. Affinity f. HPLC 2. Analysis of purity of purified /isolated protein by electrophoresis: i. Agar ii. PAGE iii. Iso electric focusing 3. Molecular weight determination : a) mass spectroscopy b) electrophoresis ( e.g. Agar , SDS PAGE)along with suitable Molecular weight markers
  5. 5. Determination of the number of polypeptide chains in a protein: Step 1 • Determination of number of poly peptide chains is done using Urea/ Guanidine hydrochloride which break non-covalent bonds . • Dansyl chloride binds to N-terminal amino acids of each of constituent polypeptide chain. • The tagged polypeptide chains are subjected to complete hydrolysis by boiling with 6 N HCl at 110 C for 18- 36 hours under anaerobic conditions to give mixture of amino acids. • The number and nature of the Dansyl amino acids can be determined and will indicate the number of polypeptide chains of the protein. • i.e. 2 different polypeptide chains→ 2 different Dansyl amino acids can be identified
  6. 6. Dansyl chloride in Determination of the number of polypeptide chains in a protein Dansyl chloride /Fluorescamine /Dabsyl chloride /1-fluoro 2, 4 dinitrobenzene: react with - amino group of amino acids to form colored /fluorescent derivatives . ❖Advantages of reactions : 1. Unlike ninhydrin , the intact R group of amino acids remains the part of product , so that derivatives of different amino acids can be distinguished. 2. Product of reaction are derivatives of these reagents and absorb light . Hence they facilitate the detection and quantification of amino acids Reactions of - amino group of amino acids with Product of reactions Application of reactions Fluorescamine fluorescent derivatives with intact R group of amino acids Detectionofnanogram quantitiesofaminoacids Dabsyl chloride ,Dansyl chloride, 1-fluoro 2, 4 dinitrobenzene fluorescent derivatives with intact R group of amino acids fluorescentderivativesof reactionarestableatharsh conditions.
  7. 7. H3C- N-CH3 O S O Dansyl chloride Cl Dansyl chloride binds to N-terminal amino acids (with free amino group )of each of constituent polypeptide chain.
  8. 8. Solubilizing agents for proteins
  9. 9. Determination of amino acid composition: Step 2 ❖Pure protein sample used to detect a. Quality of amino acids b. Quantity of each of amino acids Step 2 : Complete hydrolysis of the polypeptide chains ( all peptide bonds are hydrolyzed at 110 C using strong acids for 24 hr.)followed by cation-exchange chromatographic separation and identification→ determination of amino acid present and their relative numbers of each.
  10. 10. Determination of Primary structure of polypeptide
  11. 11. Acid and Alkali hydrolysis of polypeptide chains for determination of amino acid composition ❖Complete hydrolysis of proteins using acids /alkalis : Acid hydrolysis Alkali hydrolysis A Carried out using 6 N HCl & boiling at 110 ⁰ C Carried out using 4 N NaOH & boiling at 100 ⁰ C B Tryptophan destroyed Tryptophan intact (Tryptophan content can be measured ) C Asparagine gets converted Aspartate during acid hydrolysis Glutamine gets converted to Glutamate during acid hydrolysis Serine ,Threonine , Cysteine decomposed during alkali hydrolysis D Tryptophan cannot be quantitated Tryptophan can be quantitated.
  12. 12. Determination of amino acid composition • Complete hydrolysis of proteins using acids /alkalis is followed by gel filtration of for separation of amino acids from acid/ alkali hydrolysate of protein. • Identification of amino acids is done using chromatography /Gel filtration. • UV spectrophotometry /colorimetric analysis is used for quantitation of amino acids. • Enzyme hydrolysis : Pepsin ,Chymotrypsin ,Trypsin are used for smaller fragments (peptides) • Chemical cleavage can be carried out Cyanogen bromide . • Pronase : is a mixture of non-specific enzymes that cause complete hydrolysis of proteins.
  13. 13. Gel filtration for amino acid determination Identification of amino acids is done using chromatography /Gel filtration. UV spectrophotometry /colorimetric analysis is used for quantitation of amino acids.
  14. 14. Chromatography for amino acid determination : comparison of RF value with standard amino acids Rf value of each amino acids is its characteristic property. Thus ,the amino acids in the unknow mixture can be identified fairly well by comparing their Rf values with those of pure amino acids.
  15. 15. Identification of amino acids is done using chromatography Rf value of each amino acids is its characteristic property. Thus ,the amino acids in the unknow mixture can be identified fairly well by comparing their Rf values with those of pure amino acids.
  16. 16. Identification of N -terminal and C-terminal amino acids of a polypeptide chain: Step3 • Primary sequence of protein means its complete amino acid sequence . • To find out complete sequence one starts with finding out N-terminal and C- terminal amino acid of given peptide .
  17. 17. Identification /detection of C-terminal amino acid a polypeptide chain using carboxy peptidase: End group analysis of protein ❖Detection of C-terminal amino acid using carboxy peptidase : 1. The polypeptide is incubated with Enzyme carboxy peptidase. 2. Enzyme carboxy peptidase hydrolyzes the first peptide bond from C- terminal liberating C-terminal amino acid residue in free form and leaving a peptide with n-1 amino acids. 3. The liberated C-terminal amino acid in free form can be identified/detected by chromatography using standard /pure amino acids (comparing Rf value) .
  18. 18. Schematic representation for identification /Detection of C-terminal amino acid of a polypeptide using carboxy peptidase 1 2 3 4 1 2 3 4 I . C –terminal end amino acid residue reacts with enzyme Carboxy peptidase  II. HydrolysiswithacidtoreleaseC –terminalendaminoacidresidue  TheliberatedC-terminalamino acidinfreeformcanbe identified/detectedbyion- exchange chromatographyusing standardaminoacids(comparing Rfvalue). I II Carboxy peptidase A or B ❖ C –terminal end amino acid residue can be identified by Carboxy peptidase A and B. Carboxy peptidase A : will not act if C –terminal end amino acid residue is Arginine , Proline or Lysine . Carboxy peptidase B : will act only if the penultimate amino acid residue is Proline. C-terminalendaminoacidresidue
  19. 19. Detection of N-terminal amino acid :End group analysis of protein ❖Detection of N-terminal amino acid: 1. Sanger’s method for determination of N-terminal amino acid : using 2,4 di fluoro 2,4 dinitrobenzene(FDNB)which binds to N- terminal end amino acid followed by acid hydrolysis & chromatography for separation of N-terminal end amino acid DNP derivative(yellow colored 2, 4dinitrophenyl derivative) . 2. Dansyl chloride for determination of N-terminal amino acid : Dansyl chloride binds to N-terminal amino acids of each of constituent polypeptide chain and N-terminal amino acids can be identified using chromatography .
  20. 20. DetectionofN-terminalaminoacidofapolypeptideusingSanger’sreagent 1. Peptide (e.g. N=10 amino acids) is reacted with Sanger’s reagent( 1-fluoro- 2,4 dinitro benzene FDNB) (N-terminal amino acid =aa1 , C -terminal amino acid = aa 10 ) FDNB + H2N –aa1—CO –NH -----–NH –aa 10– COOH DNP- HN –aa1—CO –NH –NH –aa 10– COOH ( Dinitrophenylderivativeofpeptide) 2. DNP derivative of peptide is treated in acid. This results in the breakdown of every peptide bond and forms DNP- HN –aa1– COOH + aa2 + aa3 + aa4 + aa5+aa6+aa7 +aa8+aa9+aa10   DNP derivative of N-terminal amino acid free amino acids 3. Detection of DNP derivative of N-terminal amino acid by chromatographic comparison with authentic DNP derivative of different amino acids .
  21. 21. Reaction of an amino acid with Sanger’s reagent(1-fluoro-2,4 dinitrobenzene=FDNB) H R R F NO2 + H- N+ -C- COO- COO--C- N NO2 H H HF H H  Sanger’s reagent (1-fluoro-2,4dinitrobenzene) =FDNB NO2 NO2 1 4 2 3 6 5 -Amino acid   2,4 dinitrophenylaminoacid
  22. 22. Sanger’s method for determination of amino acid sequence
  23. 23. Side specific hydrolysis of polypeptide chain using specific enzymes to get a mixture of peptides : step 4 • With the amino –terminal and carboxy terminal residues identified, another sample of polypeptide chain is fragmented into smaller pieces, short peptides having on the average 10-15 amino acid residues each. • Digestive enzyme Trypsin is commonly employed for partial hydrolysis as it catalyzes hydrolysis regardless of length or amino acid sequence of polypeptide chain. • The number of fragments produced by trypsin cleavage can be predicted from total number of lysine or arginine residues in original polypeptide. • These fragments are separated and amino acid sequence of each fragment is elucidated .
  24. 24. Specificity of enzymes used for partial hydrolysis of polypeptide into smaller fragments Enzymes Hydrolysis of a peptide bond contributed by alpha carboxyl group of Trypsin Lysine or Arginine Chymotrypsin Phenylalanine , Tyrosine , Tryptophan, or Leucine Pepsin Phenylalanine , Tyrosine , Tryptophan ,several others Cyanogen bromide ( CNBr) hydrolyses of a peptide bond contributed by alpha carboxyl group of Methionine .
  25. 25. Enzymatic hydrolysis of peptide bond in primary structure determination
  26. 26. Degradationofpolypeptideintosmallerfragmentsbypartialhydrolysisusingenzyme:step4
  27. 27. Determinationofprimarystructureofeachfragment/peptide involvesfivesteps 1. Very long chain proteins , the chain is fragmented into small peptides with overlapping sequences. 2. This is done by subjecting polypeptide chain to hydrolysis with two or more different site specific enzymes . 3. Each of the fragments /small peptides produced by trypsin are separated from each other (purified )by ion-exchange chromatography on a column. Paper electrophoresis and chromatography are carried out in two dimensions to yield peptide map. 4. Sequence of each of peptide is analyzed by Edman’s degradation and the whole sequence of polypeptide deciphered as if fitting in the parts of a jigsaw puzzle. 5. The position of disulphide bonds can be determined by cleaving the cleaving the native protein sample to get fragments with intact S-S bonds. These fragments are then identified .
  28. 28. A map of the peptides obtained after cleavage of a protein with Trypsin Schematic diagram Origin paper electrophoresis in this direction Paper chromatography in this direction
  29. 29. Limitation of Enzymatic cleavage of peptide bonds using Trypsin and Chymotrypsin
  30. 30. Identifying the sequences of the peptide fragments : step 5 • Separation and purification of each of these peptides ,and then analyzing the amino acid sequence of the small peptides, and then deciphering the sequence of the whole protein. • The Edman’s degradation method labels and removes only the amino acid residue ,while leaving all other peptide bonds intact.
  31. 31. Edman’s degradation method for determination of amino acid sequence of peptide fragment ❖The purified polypeptide / small fragments( 10- 20 residues) obtained after partial enzyme hydrolysis of polypeptide is subjected to Edman’s degradation technique for determination of amino acid sequence. ❖Edman’s degradation method (using phenyl isothiocyanate –PTH which binds to N-terminal end amino acid followed by hydrolysis & chromatography for separation of N-terminal end amino acid phenyl carbamoyl derivative (PTC) + remaining fragment of peptide (n-1 amino acids ) →”subtractive method ” • Cycle is repeated several times for complete sequence determination. ➢Using Edman’s degradation technique , amino acid sequencing can be completed within a few hours by automatic machines .
  32. 32. Detection of N-terminal amino acid of a peptide using Edman’s reagent ❖Another reagent for Detection of N-terminal amino acid of a peptide is Edman’s reagent (phenyl isothiocyanate –PTC) . (N-terminal amino acid =aa1 , C -terminal amino acid = aa 10 ) • Steps in this detection are : Peptide ( e. g .N=10 amino acids) is reacted with Phenyl isothiocyanate(PTC) Phenyl isothiocyanate + H2N –aa1—CO –NH -----–NH –aa 10– COOH  PTC - HN –aa1—CO –NH -----–NH –aa 10– COOH  Phenyl isothiocyanate (PTC ) peptide→ treated with acid  Break down of only the first peptide bond and forms PTH - HN –aa1—CO + H2N –aa2—CO –NH -----–NH –aa 10– COOH  Phenylthiohydrantoin derivative of N-terminal amino acid intact peptide with remaining 9 amino acids 
  33. 33. Schematic representation of Edman’s degradation process for determination of primary structure of peptide /protein fragment 1 2 3 4 1 2 3 4 2 3 4 2 3 4 A . N-terminalendaminoacidresidueforms covalentbondwithEdman’sreagent  B. Hydrolysis with acid to release N –terminal end amino acid residue  C. Label next N –terminal end amino acid residue reacts with Edman’s reagent (phenyl isothiocyanate –PTC)  D. Hydrolysis with acid to release next N –terminal end amino acid residue  Edman’sdegradationprocessrepeatedfor determinationofprimarystructureof protein(10-30AA) A B C D (Phenyl isothiocyanate –PTC)=Edman's reagent N-terminal endamino acidresidue
  34. 34. Edman’s degradation method for determination of amino acid sequence
  35. 35. Comparison of Sanger’s reagent ( 1-fluoro-2,4 dinitro benzene FDNB) and Edman’s reagent (Phenyl isothiocyanate –PTC) in the determination of amino acid sequence of a protein Sanger’s reagent Edman’s reagent -N=C=S Protein labelling followed by hydrolysis Polypeptide (N- terminal amino acid) Phenylthiohydrantoin (PTH) derivative of N-terminal amino acid Identified by chromatography O R1 II I C CH -N NH C II S Protein labelling followed by hydrolysis Free amino acids Dinitro phenyl derivative of N-terminal amino acid Identified by chromatography -FO2N- NO2 O2N- NO2 R -N-CH-COO H
  36. 36. Comparison of Sanger’s method and Edman’s degradation method for determination of amino acid sequence
  37. 37. Advantages of Edman's reagent over Sanger’s reagent • Edman's reagent (phenyl iso thio cyanate –PTC ) is advantageous over Sanger’s reagent( 1-fluoro-2,4 dinitro benzene FDNB) in that only the N- terminal amino acid is cleaved leaving rest of the peptide intact so that the resulting peptide can again react with reagent to form PTH derivative of the new N-terminal amino acid. • Thus , Edman's reagent is helpful in automatic sequential identification of several amino acids from N-terminal. • In the automatic chromatographic analysis of amino acids on cation – exchange resin ,the elution is carried out with different buffers of successively higher pH . • The effluent is caught in small volumes and the amino acid content is automatically analyzed. • The area under each peak of curve is proportional to the amount of each amino acid in the mixture .
  38. 38. Cleavage of the original polypeptide chain by a second procedure : step 6 • In order to establish the order of the peptide fragments formed by trypsin, another sample of intact polypeptide is cleaved into small fragments by using chemical cyanogen bromide. • Specificity of Cleavage by cyanogen bromide : a peptide bonds in which the carbonyl group contributed by Methionine residue(8Met→9peptidefragments) • The peptidefragmentsarenowseparatedfromeachotherbyelectrophoresisor chromatography. • EachfragmentissubjectedtoEdman’sdegradationasinstep5toidentifyitsaminoacid sequence. • Twosetsofpeptidefragments(actionoftrypsinandcyanogen bromide) are analyzed to detect overlaps in order to established amino acid sequence.
  39. 39. Orderingpeptidefragmentsbyestablishingoverlaps:step7 • In determination of primary structure of protein , several methods (enzymatic or chemical)are simultaneously employed .This results in the formation of Overlapping peptides .This is due to the specific action of different sites in the polypeptide. • The amino acid sequences of each fragment obtained from the original polypeptides by two cleavage procedure is examined in order to find out peptides from the second fragmentation whose sequences establish continuity or overlaps between fragments obtained from the first fragmentation. • Overlapping peptides obtained by the second fragmentation yield the correct order of peptide fragments produced in first cleavage . This is very useful in determining accurate amino acid sequence without any possible errors . • Sometimes third /fourth cleavage using chymotrypsin/pepsin are needed in case of failure of second procedure to establish overlaps .
  40. 40. Placing peptide fragments in their proper order with overlaps
  41. 41. AutomationofEdman’sdegradationmethodfordeterminationofaminoacidsequence
  42. 42. Role of retention time in identification of amino acids after their separation
  43. 43. Anionic site schematic diagram SO3 - Na + SO3 - Na + + N + H 3 - CHR-COOH  Amino acid SO3 -N + H 3 -CHR-COOH SO3 -N + H 3 -CHR-COOH + 2 Na + Resin particle cation exchange Resin particle Mechanismofcationexchangeinionexchangechromatographyindeterminationofamino acidsequence
  44. 44. Mechanism of cation exchange in ion exchange chromatography in an amino acid analyzer • The negatively charged sulfonate (SO3 -) groups attract and bind cations such as H+ , Na + or cationic forms of amino acids . • At pH 3.0 most amino acids are cations but differ in net strength of their positive charges and thus in degree to which they can displace Na + from the fixed anionic groups . • Lysine would be bound most tightly because of its two N + H3 groups whereas Glutamic acid and Aspartic acid would be bound least tightly since they have least amount of positive charge at pH 3.0. • The binding of amino acids to ion exchange resins is also affected by their degree of adsorption or their solubility in the resin particles .
  45. 45. Sequenator ❖Sequenator : is an automatic machines to determine the amino acid sequence in polypeptide ( with 100 residues) ❖It is based on the principle of Edman’s degradation . Amino acids are determined sequentially from N –terminal end . ❖The PTH –amino acid liberated is identified by high performance liquid chromatography( HPLC). ❖ Sequenator : takes about two hours to determine each amino acid.
  46. 46. Chromatographic analysis of amino acids on cation exchange resin Amino acids eluted at 150 cm column pH 3.25 and 0.2 N Sodium citrate Amino acids eluted at 150 cm column pH 4.25 and 0.2 N Sodium citrate Amino acids eluted at 15 cm column pH 5.28 and 0.35 N Sodium citrate 1.Aspartic acid 6.Glycine 15.Lysine 2.Threonine 7.Alanine 16.Histidine 3.Serine 8.Cysteine 17.NH3 4.Glutamic acid 9.Valine 18.Arginine 5.Proline 10.Methionine 11.Isoleucine 12.Leucine 13.Tyrosine 14.Phenylalanine
  47. 47. Elution profile of standard amino acids of PTH ( 20 pmoles) Using Edman’s degradation technique , amino acid sequencing can be completed within a few hours by automatic machines .
  48. 48. Comparison of Sanger’s reagent ( 1-fluoro-2,4 dinitro benzene FDNB) and Edman’s reagent (Phenyl isothiocyanate –PTC) in the determination of amino acid sequence of a protein Sanger’s reagent Edman’s reagent -N=C=S Protein labelling followed by hydrolysis Polypeptide (N- terminal amino acid) Phenylthiohydrantoin (PTH) derivative of N-terminal amino acid Identified by chromatography O R1 II I C CH -N NH C II S Protein labelling followed by hydrolysis Free amino acids Dinitro phenyl derivative of N-terminal amino acid Identified by chromatography -FO2N- NO2 O2N- NO2 R -N-CH-COO H
  49. 49. Finger printing method (Ingham's Technique) ❖Finger printing method ( Ingham's Technique): • Help to easily identify any qualitative abnormalities in protein structure • Protein is digested with into small peptides by Trypsin. • The mixture of peptides are separated by chromatography.( Peptide mapping ) • The position of the peptide containing the altered amino acid is found different when compared to peptide map of the normal polypeptide . • e.g. Beta chain of hemoglobin HbA and HbS (sickle cell anemia)
  50. 50. Use of a peptide map in determining a mutation in hemoglobin ( e.g sickle cell hemoglobin with altered amino acid sequence):1 1. Human hemoglobin cleaved with trypsin to yield a mixture of peptides 2. A mixture of peptides spotted on Whatman filter paper No1 3. Electrophoresis of a mixture of peptides is carried out in one direction of paper square 4. Drying of the Whatman filter paper after electrophoretic separation of peptides 5. Chromatography separation of the peptides in the another direction 6. The sequential combination of two processes/techniques effectively separate complex mixture of peptides 7. Analysis for identification
  51. 51. Use of a peptide map in determining a mutation in hemoglobin ( e.g sickle cell hemoglobin with altered amino acid sequence):2 Human hemoglobin cleaved with trypsin to yield a mixture of peptides A mixture of peptides spotted on Whatman filter paper No1 Electrophoresisofamixtureofpeptidesiscarriedoutinonedirectionofpapersquare DryingoftheWhatmanfilterpaper afterelectrophoreticseparationofpeptides Chromatography separation of the peptides in the another direction Thesequentialcombinationoftwoprocesses/techniques effectivelyseparate acomplex mixtureofpeptides→analysisforidentification
  52. 52. A map of the peptides obtained after cleavage of a protein with Trypsin Schematic diagram Origin paper electrophoresis in this direction Paper chromatography in this direction
  53. 53. Peptide map for Beta chain of hemoglobin HbA and HbS (sickle cell anemia) sixth amino acid in beta() chain of HbA1 (Glutamic acid )replaced by Valine in HbS
  54. 54. Recent trends in study of higher level of protein structure ❖Techniques to study higher level of protein structure : 1. X-ray diffraction 2. Ultra violet light spectroscopy 3. Optical rotary dispersion 4. Circular dichroism 5. Nuclear magnetic resonance(NMR) 6. DNA sequencing
  55. 55. PrincipleofX-raydiffractioninstudyofhigherlevelofproteinstructure ❖X-ray diffraction study is possible only on crystallized pure proteins. ❖Principle : A beam of X-ray is diffracted by the electrons around each atom and the intensity of diffracted beam is detected by photographic plate or collected by an electronic device.
  56. 56. Nuclear magnetic resonance(NMR) in study of higher level of protein structure • Nuclear magnetic resonance(NMR) spectroscopy: measures the absorbance of radiofrequency of atomic nuclei . By measuring the radiofrequency at which a particular of atomic nucleus absorbs energy, the functional group present in the molecule can de predicated . • Two –dimensional Nuclear magnetic resonance(NMR) spectroscopy permits a study of three-dimensional conformation of the protein in solution. • It also permits to study the alteration in conformation of the protein in solution during binding with another ligand.
  57. 57. Steps of DNA sequencing in study of higher level of protein structure RoughsequencingofaproteinusingEdman’sdegradationmethod(primarystructureofaprotein) Synthesisofsmalllengtholigonucleotideprimersbasedontheaminoacidsequenceofaprotein Amplification of appropriate gene by polymerase chain reaction (PCR) Correct DNA clone is obtained DNA sequencing of DNA clone Identification of encoded protein using knowledge of genetic code ( Thus ,DNA sequencing help to study of primary structure of protein)
  58. 58. Reverse sequencing technique ❖DNA ( the genetic material) determines the sequence of amino acids in polypeptide chain. • By analyzing the nucleotide sequence of DNA that codes for proteins, it is possible to translate the nucleotide sequence into amino acid sequence. ❖Limitation of Reverse sequencing technique : fails to identify disulfide bonds and post-translational modification ( the amino acids after the protein is synthesized)
  59. 59. Factors affecting Gene expression and Proteomics ❖Proteomics:thestudyoftheentiregalaxyofproteinsproducedbyacellunderdifferent conditions.Itaimsatstudyingtheproteinstructure.Severalfactorsregulate‘on’and‘off’ mechanismofgeneduringgrowthanddevelopmentofcell/tissue. A single gene expression Cell type Organ type Time Enviro nment Stimuli Phase ofcell cycle Post translational modification Functional native protein Proteomics attempt to study this multifaceted picture in toto.

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