BIO152 WEEK 2
Protein Structure
Protein varietee iierent
- Number an type of R groupe
- Nett charge of proteine
- Size
- Solubility
- pI
Protein eeparaton
Diierent metho e epen ing on the baeie ofo
- Charge electrophoreeie/ion exchange chromatography
- Size gel fltraton
- Solubility pI precipitaton (ppt)/ammonium eulfate ppt
- E.g. chromatography = eeparaton metho e uee to fow a mobile phaee through a
etatonary phaee
Electrophoreeie
- Separatee a mixture of proteine in a gel matrix with voltage eupply
- Catho e (+) an ano e (-) en e of gel
- Remember oppoeite chargee attract
- Loa protein mix which will eeparate baee on their charge (move to oppoeite eign of
charge at a rate proportonal to the ei e of the charge)
Ion exchange chromatography
- Matrix trape/elowe own fowing proteine of oppoeite charge
- Proteine of eame charge fow through
- Elute protein in matrix with pH change (new buier)
- Poeitvely charge ion bin e to negatvely charge hea
- Negatvely charge protein fowe through
Gel fltraton
- Mixture of large an emall proteine paeee through gel reein
- Larger proteine travel faeter (exclu e ) through reein an elute fret
- Smaller proteine are retar e (inclu e ) in reein an elute laet
- Collect fractone to purify proteine
- Reein = eepharoee bea e (eepha ex)
- Diierent pore eizee e.g. G25 1000 to 5000 Da
Ieoelectric point ppt
- Proteine are eeparate baee on iierence in eolubility in water
- Remember water ie polar (H2O)+
- The nett charge of the protein attracte water forming a hy raton ehell an
eolubilieee (aqueoue eoluton)
- If pHe = protein pI, there ie a 0 nett charge
, - Proteine leaet eoluble at pI now interact with each other rather than with water an
coaleece to precipitate (fall out of eoluton = ineoluble)
- If pH ie a juete to pI of protein, it may ppt
Ammonium eulfate ppt
- (NH4)2SO4 etripe away hy rogen ehell an ecreaeee eolubility allowing proteine to
ppt
- Diierent proteine ppt at iierent concentratone of (NH4)2SO4
Bon e
Covalent
- Dieulf e bon e
Noncovalent (weaker but iveree)
- Hy rogen bon e
- Ionic bon e
- Van er Waal interactone
- Hy rophobic interactone
Protein etructure
- Primary amino aci eequence covalent pept e bon e
- Secon ary fol ing into a helix, b eheet or ran om coil hy rogen bon e between
NH an CO groupe of pept e bon e in the backbone
- Tertary three- imeneional fol ing of a eingle polypept e chain ieulphi e bon e,
hy rogen bon e, ionic bon e, van er Waal interactone, hy rophobic interactone
- Quaternary aeeociaton of multple polypept ee to form a multmeric protein
eame ae for tertary etructure
Diierent protein etructuree
- 1°, 2°, 3°, 4°
Primary etructure
- Linear eequence of amino aci in polypept e
- 1° etructure provi ee eeeental informaton for aa content an protein pI
- Sequencing reactone uee reagente to etrip oi eingle aa at amino terminue one by
one from polypept e
- Complex proteine require eeparaton of in ivi ual polypept e chaine into emaller
fragmente (pept ee) for eequencing
- Overall 1° protein etructure ie rebuilt by overlapping pept e eequencee
Secon ary etructure
- Linear aa eequencee may juet form ran om coile, but proteine have highly organiee
etructuree
- 2° etructuree
- Alpha helix coil within a polypept e
- Beta eheet pleat between polypept ee
Alpha helix
Protein Structure
Protein varietee iierent
- Number an type of R groupe
- Nett charge of proteine
- Size
- Solubility
- pI
Protein eeparaton
Diierent metho e epen ing on the baeie ofo
- Charge electrophoreeie/ion exchange chromatography
- Size gel fltraton
- Solubility pI precipitaton (ppt)/ammonium eulfate ppt
- E.g. chromatography = eeparaton metho e uee to fow a mobile phaee through a
etatonary phaee
Electrophoreeie
- Separatee a mixture of proteine in a gel matrix with voltage eupply
- Catho e (+) an ano e (-) en e of gel
- Remember oppoeite chargee attract
- Loa protein mix which will eeparate baee on their charge (move to oppoeite eign of
charge at a rate proportonal to the ei e of the charge)
Ion exchange chromatography
- Matrix trape/elowe own fowing proteine of oppoeite charge
- Proteine of eame charge fow through
- Elute protein in matrix with pH change (new buier)
- Poeitvely charge ion bin e to negatvely charge hea
- Negatvely charge protein fowe through
Gel fltraton
- Mixture of large an emall proteine paeee through gel reein
- Larger proteine travel faeter (exclu e ) through reein an elute fret
- Smaller proteine are retar e (inclu e ) in reein an elute laet
- Collect fractone to purify proteine
- Reein = eepharoee bea e (eepha ex)
- Diierent pore eizee e.g. G25 1000 to 5000 Da
Ieoelectric point ppt
- Proteine are eeparate baee on iierence in eolubility in water
- Remember water ie polar (H2O)+
- The nett charge of the protein attracte water forming a hy raton ehell an
eolubilieee (aqueoue eoluton)
- If pHe = protein pI, there ie a 0 nett charge
, - Proteine leaet eoluble at pI now interact with each other rather than with water an
coaleece to precipitate (fall out of eoluton = ineoluble)
- If pH ie a juete to pI of protein, it may ppt
Ammonium eulfate ppt
- (NH4)2SO4 etripe away hy rogen ehell an ecreaeee eolubility allowing proteine to
ppt
- Diierent proteine ppt at iierent concentratone of (NH4)2SO4
Bon e
Covalent
- Dieulf e bon e
Noncovalent (weaker but iveree)
- Hy rogen bon e
- Ionic bon e
- Van er Waal interactone
- Hy rophobic interactone
Protein etructure
- Primary amino aci eequence covalent pept e bon e
- Secon ary fol ing into a helix, b eheet or ran om coil hy rogen bon e between
NH an CO groupe of pept e bon e in the backbone
- Tertary three- imeneional fol ing of a eingle polypept e chain ieulphi e bon e,
hy rogen bon e, ionic bon e, van er Waal interactone, hy rophobic interactone
- Quaternary aeeociaton of multple polypept ee to form a multmeric protein
eame ae for tertary etructure
Diierent protein etructuree
- 1°, 2°, 3°, 4°
Primary etructure
- Linear eequence of amino aci in polypept e
- 1° etructure provi ee eeeental informaton for aa content an protein pI
- Sequencing reactone uee reagente to etrip oi eingle aa at amino terminue one by
one from polypept e
- Complex proteine require eeparaton of in ivi ual polypept e chaine into emaller
fragmente (pept ee) for eequencing
- Overall 1° protein etructure ie rebuilt by overlapping pept e eequencee
Secon ary etructure
- Linear aa eequencee may juet form ran om coile, but proteine have highly organiee
etructuree
- 2° etructuree
- Alpha helix coil within a polypept e
- Beta eheet pleat between polypept ee
Alpha helix
