Ionic properties of amino acids impart ionic properties to proteins

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Ionic properties of amino acids impart ionic
properties to proteins

• in general these are SURFACE properties (i.e.
  charged sidechains are on solvent-exposed outside
  of folded structure)
• affect protein-ligand binding (e.g. DNA-binding
  proteins) or catalysis
• average charge on protein is an important
  consideration in the design of a purification
  process

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pKa3
pKa2
pKa1
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pI isoelectric point--the pH at which the net
charge is 0.
For amino acids with no ionizable functional
group, pI (pK1 pK2)/2
For amino acids with an ionizable functional
group, the average of the 2 pKs surrounding the
isoelectric form of the amino acid determines
the pI.
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pKa3
The isoelectric form of asp occurs between pK1
and pK2.
pKa2
pI 2.0 3.9 2 2.95
pKa1
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Other Properties of Amino Acids

• Stereochemistry (all biosynthetic proteins made
  up of L-isomer)
• Hydropathy (partitioning between polar and
  nonpolar solvents as indicator of polarity) (see
  Table 6-2 in VVP p 150 Take Note p58)
• these two properties are major determinants of
  peptide conformation

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See VVP Fig 4-3
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VVP Fig 6-3 p 126
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Example of a protein sequence
N-terminus

• MANSKINKQL DKLPENLRLN GRTPSGKLRS FVCEVCTRAF
  ARQEHLKRHY
• RSHTNEKPYP CGLCNRCFTR RDLLIRHAQK IDSGNLGETI
  SHTKKVSRTI
• TKARKNSASS VKFQTPTYGT PDNGGSGGTV LSEGEWQLVL
  HVWAKVEADV
• AGHGQDILIR LFKSHPETLE KFDRFKHLKT EAEMKASEDL
  KKHGVTVLTA
• LGAILKKKGH HEAELKPLAQ SHATKHKIPI KYLEFISEAI
  IHVLHSRHPG
• DFGADAQGAM NKALELFRKD IAAKYKELGY G

C-terminus
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VVP page 150
nonpolar
polar
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VVP Fig 6-1 p 125
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VVP Fig 5-1 p 94
C-termini
N-termini
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(Rasmol)
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VVP Fig. 5-1 Bovine Insulin
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VVP Fig. 5-3 ELISA
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VVP Fig. 5-4 Salting Out
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VVP Fig. 5-5 Ion Exchange Chromatography
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VVP Fig. 5-6 Gel Filtration (SEC)
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VVP Fig. 5-7 Affinity Chromatography
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VVP Fug, 5-8 Purification of Stapylococcal
Nuclease
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Stryer Fig. 4.7 PAGE
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Animation http//www.whfreeman.com/lodish/con_ind
ex.htm?03 choose animations and then SDS gel
electrophoresis
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Stryer Fig. 4.9 Coomassie blue stained SDS gel.
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VVP Fig. 5-9 PAGE
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Stryer Fig. 4.8
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VVP Fig. 5-10 SDS-PAGE of Salmonella proteins
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VVP Fig. 5-10 MW vs. Mobility in SDS-PAGE
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Stryer Fig. 4.36 Western blot.
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VVP Fig. 5-11 Density Gradient Ultracentrifugation
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Stryer Fig. 4.14
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Stryer Fig. 4.6 MALDI-TOF
Matrix-assisted laser desroption-ionization- Time
of Flight
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Stryer Fig. 4.17
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Determining Primary Structure
1. Determine aa composition
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Stryer Fig. 4.18 Ion-exchange chromatography
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Stryer p. 92 Agents for N-terminal analysis
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Stryer Fig. 4.20
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Stryer Fig. 4.21
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Animation Edman Degradation http//www.wiley.com
/college/fob/quiz/quiz05/f5-15.html
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Stryer Fig. 4.22 Separation of PTH-aa by HPLC.
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2. Partially digest intact protein with
specific agents.
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Stryer Fig. 4.23
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Stryer Fig. 4.24
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3. Sequence the fragments and
align overlapping sequences.
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Stryer Fig. 4.25
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or ASKFGKYN ?
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or ASKFGKYN ?
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or ASKFGKYN ?
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VVP Fig. 5-13 Dansyl Chloride Rxn
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VVP Fig. 5-14 Amino Acid Analysis
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Edman Sequencing VVP Fig 5-15 p 113
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VVP Fig. 5-16 Overlapping fragments
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VVP Fig. 5-17 Determine positions of S-S
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VVP Fig. 5-18 Phylogenetic tree of Cyt C
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(Figs 19 and 20)
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