Molecular Biology & Biochemistry 694:407& 115:511Methods of Protein AnalysisSept. 16th, 2005, LectureSpecial thanks for many slides in this lectureagain goes to Dr. Gabriel Fenteany, Dept. ofChemistry, University of Illinois at Chicago(www.chem.uic.edu/fenteany/teaching/452).So…..Let us say you have an impuresolution containing a proteinof interest.Q: How do you (a) analyzewhat you have and (b) purifywhat you want?Polyacrylamide Gel Electrophoresis (PAGE)Note: proteins are usually mixed with adetergent, sodium dodecylsulfate (SDS),and a tracking dye to make the sample.The SDS binds to the protein and gives itsize-dependent negative charge andconsistent hydrodynamic properties.Polyampholyte Character of aTetrapeptide and Isoelectric PointsIsoelectric Point (pI), pH atwhich molecule has net zerocharge, determined usingcomputer program for knownsequence or empirically (byisoelectric focusing)Group pKa-NH3+ 9.7Glu -COOH 4.2Lys -NH3+ 10.0-COOH 2.2Isoelectric FocusingElectrophoresis through polyacrylamide gel inwhich there is a pH gradient.Two-Dimensional Gel Electrophoresis• Separate proteins based on pI in 1st dimension• Separate proteins based on molecular weight in2nd dimensionFigure 5.11The solubility ofmost globularproteins ismarkedlyinfluenced by pHand ionic strength.This figure showsthe solubility of atypical protein asa function of pHand various saltconcentrations.“Salting Out”: Ammonium SulfatePrecipitation in Protein FractionationCentrifugationCentrifugation Methods•Differential (Pelleting) – simplemethod for pelleting large particlesusing fixed-angle rotor (pellet atbottom of tube vs. supernatantsolution above)•Zonal ultracentrifugation (e.g.sucrose-gradient) – swinging-bucketrotor•Equilibrium-density gradientultracentrifugation (e.g. CsCl) –swinging-bucket or fixed-angle rotorLow-speed, high-speed, orultracentrifugation: differentspin speeds and g forcesZonal Centrifugation: Sucrose-Gradient Preparative CentrifugationSeparates by sedimentation coefficient(determined by size and shape of solutes)Column ChromatographyFlow-throughEluate15ChromatographyLiquid flowLiquid flow4:37990909Time 1 2 3 4 5Separation according to: -molecular weight/ size-charge-hydrophobicity-affinitySample containing proteins or peptidesPositively-charged basic residues (K, R, & H)Negatively-charged acidic residues (E & D)Hydrophobic “patch”Ligand binding pocket(active site)ca. 40 ÅMacromoleculardimensions:Proteins are Amphiphilic Macro-Ions>>> The charged groups, hydrophobic regions, size, and solvation affect the biophysical properties of the protein and largely determine its purification behavior.Different Types of Chromatography•Gel filtration/size exclusion - separates by size (molecularweight) of proteins• Ion exchange (cation exchange and anion exchange) -separates by surface charge on proteins– Cation exchange: separates based on positive charges ofsolutes/proteins, matrix is negatively charged– Anion exchange: separates based on negative charges ofsolutes/proteins, matrix is positively charged• Hydrophobic interaction - separates by hydrophobicity ofproteins•Affinity - separates by some unique binding characteristicof protein of interest for affinity matrix in columnIon-Exchange ChromatographyIon Exchange (IEX) ChromatographyGel Filtration ChromatographyGel Filtration (GF) ChromatographyThe principle of gel filtration -- excluded volume[Note: gel filtration chromatography is also sometimescalled “size exclusion chromatography”]Vo = “void volume”Vt = “bed volume”Ve = “elution volume”Vi = Vt - VoAffinity Chromatography• Sanger's results established that all of the molecules of agiven protein have the same, unique sequence and thatthe polypeptide chain is unbranched (apart from disulfidecrosslinks at some cysteines).• Proteins can be sequenced in two ways:- real amino acid sequencing (the “classic” approach)- sequencing the corresponding DNA of the gene (orcDNA copy of the mRNA), then inferring theprotein sequence using the genetic code.Frederick Sanger was the first.In 1953, he sequenced the two chains of insulin.Protein SequencingPrimary Structure of Bovine InsulinFirst protein to be fully sequenced;Fred Sanger, 1953. For this, he won hisfirst Nobel Prize (his second was for theSanger dideoxy method of DNAsequencing).• 1. If more than one polypeptide chain, separate.• 2. Cleave (reduce) disulfide bridges• 3. Determine composition of each chain• 4. Determine N- and C-terminal residues• 5. Cleave each chain into smaller fragments anddetermine the sequence of each chain• 6. Repeat step 5, using a different cleavage procedureto generate a different set of fragments• 7. Reconstruct the sequence of the protein from thesequences of overlapping fragments• 8. Determine the positions of the disulfide crosslinksDirect Determination of a Protein Sequence An Eight Step StrategyComment re previous slide:Obviously, it is easier to just sequence the gene for aprotein rather than laboriously sequence the protein itselfby direct methods. This is why most (>99%) of proteinsequences generated today are from indirect, inferentialnucleic acid sequencing data. However, for special cases,e.g., characterizing a new protein isolated from a naturalsource, direct sequencing is still required. Even in thiscase, though, often only partial sequencing is required --enough to generate a unique peptide “fingerprint” -- thenthe rest of the sequence can be obtained by looking it upin a database of protein sequences generated from DNAsequencing data from the organism from which theprotein was isolated.Figure 5.32 Proteins of the human genome grouped according to their molecular function. Thenumbers and percentages within each functional category are enclosed in parentheses. Notethat the function of more than 40% of the proteins encoded by the human genome remainsunknown. Considering those of known function, enzymes (including kinases and nucleic acidenzymes) account for about 20% of the total number of proteins; nucleic acid-binding proteins ofvarious kinds, about 14%, among which almost half are gene-regulatory proteins (transcriptionfactors). Transport proteins collectively constitute about 5% of the total; and structural proteins,another 5%. (Adapted from figure 15 in Venter, J.C., et al., 2001. The sequence of the human genome.Science 291:1304-1351.)Example ofinferring proteinsequence
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