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Fundamentals of Protein Chemistry - Duke GCB

Fundamentals of Protein ChemistryAmino Acid and Peptide ChemistryTranscription and TranslationPost-Translational ModificationsClassical Analytical MethodsIn-Gel ProteolysisAmino Acids are the Basic Structural Units of ProteinsNames, Abbreviations, and Properties of The Twenty Amino AcidsNameSide Chain pKapI Twenty Amino Acid Side Chains Vary SignificantlyThe twenty naturally occurring amino acids that make up proteins can be grouped according to many criteria, including hydrophobicity, size, aromaticity, or Acids are Linked by Amide Bonds to Form Peptide ChainsAcid-Base Chemistry in Protein CharacterizationThe net charge of a peptide or Protein at any pH depends on the combined pK values for its amino acids and terminal = pK + log [A-]/[HA]pK of alpha-COOH - of alpha-NH2 - of ionizable side - isoelectric point is the pH at which there is no net is important to remember how Protein and peptide pK values affect Chemistry and separations:Chemical Modification ( Reduction / Alkylation)Proteolysis ( specificity of Glu-C)Chromatography ( Ion Exchange)2D Gel Separations (Isoelectric Focusing)Ionization for Mass Spectrometry ( MALDI-TOFMS)Peptide Chains are Linked by Disulfide Bonds Between CysteinesProtein Synthesis:Transcription & TranslationT.

TCA Precipitation: (if [protein] is >0.05 mg/mL) Chill protein in a microcentrifuge tube to 0°C . Add 1/9 volume of cold 100% w/v TCA. Vortex . Incubate at 0°C for 30-60 min. Spin down in microcentrifuge. Remove supernatant. Wash pellet 3x w/ 200 μL cold acetone (do not vortex)

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Transcription of Fundamentals of Protein Chemistry - Duke GCB

1 Fundamentals of Protein ChemistryAmino Acid and Peptide ChemistryTranscription and TranslationPost-Translational ModificationsClassical Analytical MethodsIn-Gel ProteolysisAmino Acids are the Basic Structural Units of ProteinsNames, Abbreviations, and Properties of The Twenty Amino AcidsNameSide Chain pKapI Twenty Amino Acid Side Chains Vary SignificantlyThe twenty naturally occurring amino acids that make up proteins can be grouped according to many criteria, including hydrophobicity, size, aromaticity, or Acids are Linked by Amide Bonds to Form Peptide ChainsAcid-Base Chemistry in Protein CharacterizationThe net charge of a peptide or Protein at any pH depends on the combined pK values for its amino acids and terminal = pK + log [A-]/[HA]pK of alpha-COOH - of alpha-NH2 - of ionizable side - isoelectric point is the pH at which there is no net is important to remember how Protein and peptide pK values affect Chemistry and separations:Chemical Modification ( Reduction / Alkylation)Proteolysis ( specificity of Glu-C)Chromatography ( Ion Exchange)2D Gel Separations (Isoelectric Focusing)Ionization for Mass Spectrometry ( MALDI-TOFMS)Peptide Chains are Linked by Disulfide Bonds Between CysteinesProtein Synthesis:Transcription & TranslationT.

2 Zamis, U. Wisc., Stephens RNA is synthesized in the nucleus and exits through a nuclear assemble along the mRNA and translate it into a peptide growing peptide may be directed into the lumen of the endoplasmic reticulum(ER) for : Amino Acid CodonsPhe TTTTTCLeu TTATTGCTTCTCCTACTGIle ATTAT CATAMet ATGVa lG T TGTCGTAGTGSer TCTTCCTCATCGAGTAGCProCCTCCCCCACCGThr ACTACCACAACGAla GCTGCCGCAGCGTyrTATTA CHisCATCACGln CAACAGAsn AATAACLys AAAAAGAspGATGACGluGAAGAGCys TGTTGCTrp TGGArgCGTCGCCGACGGAGAAGGGlyGGTGGCGGAGGGM olecular Biology of the Cell by Bruce Alberts, Dennis Bray, Julian Lewis, Martin Raff, Keith Roberts, and James D. Watson, Garland Publishing, NY 1994 Histology by Bergman, , Afifi and Heidger, Saunders Publishing, Philadelphia, Pa, 1995 Protein Synthesis: TranslationA Textbook of Histology by D Fawcett.

3 Chapman and Hall, 1994 Histology by Bergman, , Afifi and Heidger, Saunders Publishing, Philadelphia, Pa, 1995 Protein Synthesis: TranslationAs a peptide grows into the ER, it is folded and ER has multiple compartments in which specific binding proteins and enzymes act on the new Protein Textbook of Histology by D Fawcett. Chapman and Hall, 1994 Molecular Biology of the Cell by Bruce Alberts, Dennis Bray, Julian Lewis, Martin Raff, Keith Roberts, and James D. Watson, Garland Publishing, NY 1994 Protein Folding, Modification, and TransportA newly synthesized Protein can be transported from the ER to the Golgi Apparatus, another complex series of compartments in which modifications are made. The Golgi is important for determining the disposition of Post-Translational ModificationProtein modifications performed by extra-translational be definitively predicted from DNA sequenceCan involve very complex systems of enzymesIn some cases, consensus sites of modification can be identified Ubiquitous in eukaryotes Frequently critical for:initiation or modulation of biological activitytransport, secretionProtein Post-Translational ModificationProteolytic Cleavage N-term Met of all proteins removed by aminopeptidasesN-terminal Acylationformyl, acetyl, myristyl, etc.

4 By acyltransferasesGlycosylationAsn, Ser, and ThrSulfationTyrPhosphorylationSer, Thr, and TyrCarboxyl Terminal AmidationHydroxylationPro, Lys, AspN-MethylationLys, Arg, His, GlnCarboxylationGlu, AspModifications introduced by usMet [O], Cys- list maintained by Ken MitchelhillProtein MutationsSingle nucleotide changes can result in amino acid substitutionsSingle amino acid substitutions can result in alterations in: Protein 3D structureMolecular WeightIsoelectric PointProteolysis by specific enzymesMolecular weights of proteolytic fragmentsProtein Prospector, UCSF MS of Mass Shifts due to Single AA Analytical Methods for Protein CharacterizationAmino Acid AnalysisAcid Hydrolysis followed by derivatization and HPLCD etermines the precise molar ratios of amino acids presentCan also be used to accurately determine concentrationAsp/Asn and Glu/Gln are not distinguishedCysteine and Tryptophan are problematic in some methodsAmino-Terminal Sequencing by Edman DegradationVery sensitiveStandard method- still best approach to NH2-terminusVery steep learning curve to do it wellBlocked proteins cannot be analyzedMixtures are challengingPeptides with long repeats are problematicPTMs are often missed but can be dealt withOften not competitive with MS for internal sequenceRobust Analytical Methods for Protein CharacterizationPolyacrylamide Gel ElectrophoresisA crude measure of molecular weight and purityAnalytical or

5 Preparative separationsCoupled with Blotting- sensitive & selective detectionIsoelectric FocusingAnalytical or preparative separationsUsed for mapping disease markers ( CGDs)Variety of pH gradientsAutomated, high throughput instrumentsTwo Dimensional IEF PAGEO rthogonal separations- large separation spaceDetection of small changes in complex samplesSeparation of post-translationally modified proteinsDynamic Range problems due to sample loading capacityIsoelectric FocusingImmobilized pH gradient gel stripsMany can be run in parallel for greater reproducibilityIn a pH gradient, under an electric field, a Protein will move to the position in the gradient where its net charge is immobilized pH gradient is created in a polyacrylamide gel strip by incorporating a gradient of acidic and basic buffering groups when the gel is are denatured, reduced, and alkylated, and loaded in a visible sample is soaked into the gel along its entire length before the field is is determined by the slope of the pH gradient and the field Gel ElectrophoresisPolyacrylamide gels may be have many different constant or gradient may be run directly, or are denatured, reduced, and , with a visible dye added, are loaded in wells cut into the top of the capacity depends on gel size and 2D IEF/PAGE, the gel strip from IEF is loaded into a single large weight standards are often run to calibrate the separation, the gel is removed from the rig and stained, or bands are blotted onto a Methods for Protein CharacterizationDenaturation-dissociates and unfolds proteinsChaotropes: Urea, GuanidineDisulfide Bond CleavageReducing Agents.

6 Dithiothreitol, beta-mercaptoethanolCysteine Alkylation-prevents reoxidation to form disulfidesAlkylating Agents: Iodoacetamide, VinylpyridineChemical Methods for Protein Characterization: ProteolysisChemical Methods:Acid Hydrolysis, various [H+], time, tempCyanogen Bromide cleavage, C-term to Methionine gives a homoserine lactone at MetEnzymes:TrypsinC-term to Lys, ArgpH to Y, F, W, H, LpH aureus V8 proteaseC-term to GlupH 8S. aureus V8 protease C-term to Glu, AsppH 5 Achromobacter protease (Lys-C)C-term to LyspH 8 Arg-CC-term to ArgpH 8 Asp-NN-term to AsppH 8 ThermolysinN-term to L, I, M, F, WpH of Proteolysis is a very powerful weaponSpecificity of these methods is variable: some excellent, some almost noneLadder Sequencing, sometimes done using MALDI-TOF/MSManual Edman Degradation, Carboxypeptidase Y digestion, Acid HydrolysisChemical Methods for Protein Characterization: A Basic Protocol for Denaturation & Proteolysis1.

7 TCA precipitation :(if [ Protein ] is > mg/mL) Chill Protein in a microcentrifuge tube to 0 C Add 1/9 volume of cold 100% w/v TCA. Vortex Incubate at 0 C for 30-60 minSpin down in microcentrifuge. Remove supernatantWash pellet 3x w/ 200 L cold acetone (do not vortex) Air dry pellet >30 min2. Redissolving the sample in a chaotrope:Redissolve Protein in 50 L of fresh 8 M M Amm. efficient digestion, [ Protein ] of > g/mL is required. (Final volume = 4x the volume of urea added)-adjust as necessary3. Reduction and alkylation of cysteines:Add 5 L (or 1/10 volume) of 45 mM DTTI ncubate at 50 C for 15 min. Cool to room temperatureAdd 5 L of 100 mM iodoacetamideIncubate in the dark at room temperature for 15 minBarbara Merrill, Bayer Methods for Protein Characterization: A Basic Protocol for Denaturation & Proteolysis4. Trypsin digestion:Add 140 L of ddH2O, vortex.

8 Check that pH is between and added should be a 1:25 weight:weight ratio of protease to sample Concentration of trypsin should be such that 1 to 5 L is added to sample Incubate at 37 C for 24 h. Stop digest by freezingNotes:Trypsin used should be treated with TLCK to inhibit residual is made up at 1 or 5 mg/mL in 1 mM HCl. Aliquots can be stored frozen for up to 3 mos (use once and discard).This protocol can be used for chymotrypsin or Achromobacterprotease (Lys-C) Final [urea] of 1 M is suitable for Staph. AureusV-8 protease (Glu-C) or Asp-N protease In the case of Asp-N a 1:50 to 1:100 w:w ratio should be used; digest for 16 hBarbara Merrill, Bayer Methods for Protein Characterization: In-Gel ProteolysisWhy In-Gel Digestion gel piece behaves like a sponge. It shrinks and swells in response to addition of aqueous or organic gel piece shrunk with organic solvent will suck in an aqueous buffer containing reagents, thus bringing them inside the matrix to access the intact Protein is trapped in the gel, so many chemical steps can be performed without significant of the peptides resulting from proteolysis within the gel freely diffuse out of the Methods for Protein Characterization: In-Gel Proteolysis1.

9 Destaining the Gel Band:Mince gel spot or band with razor blade (cut into 1 mm cubes).Wash gel pieces with 50% acetonitrile in 50 mM Ammonium bicarbonate pH ml depending on the volume of gel pieces; wash 20 min. on rotator. If volume of pieces is large (greater than 500 ul) do 2 washes at 20 min pieces with neat in speed B. Moyer, GlaxoSmithKline, after Femmtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry , M. Wilm, A. Shevchenko, T. Houthaeve, S. Breit, L. Schweigerer, T. Fotsis, M. Mann; Nature, 1996, Reduction and alkylation:Buffer:6 M guanidine M ammo. Bicarb. PH mM EDTATo 200 ul of above buffer, add 2 ul of 2 M DTT & ul 2- or 4-vinyl pyridineAdd just enough to cover the re-swollen gel piecesIncubate 15 min at 37C or 30 min RTRemove excess reduction and alkylation mix; wash gel pieces 2 times with 1 ml aliquots of 50 mM ammonium bicarbonate pH with neat acetonitrile.

10 Swell in 50 mM AmBic pH with neat acetonitrile. Swell in 50 mM AmBic pH with neat acetonitrile. Dry in Methods for Protein Characterization: In-Gel ProteolysisMary B. Moyer, GlaxoSmithKline, after Femmtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry , M. Wilm, A. Shevchenko, T. Houthaeve, S. Breit, L. Schweigerer, T. Fotsis, M. Mann; Nature, 1996, Proteolysis:Swell in 50 mM AmBic pH containing ng/ul enough trypsin solution to cover fully swollen gel piecesDigest for 16 18 Analysis:For MALDI-TOF/MS: an aliquot may be taken from the supernatant around the gel piecesFor LC/MS: all the volume plus an additional extraction of the pieces in 50 mM AmBic pH may be combinedChemical Methods for Protein Characterization: In-Gel ProteolysisMary B. Moyer, GlaxoSmithKline, after Femmtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry , M.


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