Download Steps in Protein Sequencing Separate Fragments and Sequence

Lecture 12—BCH 4053—Summer 2000
Slide 1
Review: Steps in Protein
Sequencing
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Cleave disulfide bonds, if any
Separate and purify peptides
Determine amino acid composition of
each peptide
Determine N-terminal and C-terminal
residues
Cleave each polypeptide into smaller
fragments, enzymatically or chemically.
Slide 2
Review, con’t.: Steps in Protein
Sequencing
6. Determine amino acid composition and
sequence of each fragment.
7. Repeat step 5 and 6with a different
cleavage procedure.
8. Reconstruct overall sequence by overlap
of fragments from 6 and 7.
9. Determine positions of disulfide bridges
Slide 3
Separate Fragments and
Sequence
• Automated Edman degradation generally used to
sequence the individual fragments.
– (Sometimes it may not be necessary to separate a few
peptides before carrying out the automated sequencing.
See your extra credit problem.)
• Fragmentation by Mass Spectrometry
– Several mass spectrometric methods have been
developed as very sensitive ways to carry out
sequencing, particularly of peptide mixtures. (See
Figures 5.23 and 5.24)
Lecture 12, Page 1
Slide 4
Reconstruct the Sequence from
Overlaps
GWASGNA must be from the
carboxyl-terminal peptide,
because trypsin doesn’t cleave at
alanine.
• Trypsin fragments:
– AEGPK + GWASGNA + LCFATR
• Chymotrypsin fragments
– ASGNA +
AEGPKLCF + ATRGW
• Overlap
– AEGPK LCFATR GWASGNA
– AEGPKLCF ATRGW ASGNA
• See Figure 5.25 for more extensive example.
Slide 5
Determining Disulfide Linkages
• Fragment the polypeptide with trypsin before
cleaving the disulfide bonds.
• Separate fragments by paper electrophoresis in
one dimension
• Treat paper with performic acid
• Separate in the second dimension by the same
electrophoresis
• Isolate “off-diagonal” peptides and sequence
– (See Figure 5.26)
Slide 6
Sequence Databases
• Several electronic databases contain
sequence information for proteins. Most
sequences have come from DNA gene
sequencing.
These are just a few of the
databases on the web. Find
more by searching on “protein
sequences” with your web
browser.
– GenBank
– Protein Information Resource
– Swiss Protein Database
Lecture 12, Page 2
Slide 7
Protein Homology
• Similar proteins from different species have
similar sequences.
• Sequence similarity gives clues to evolution
– A phylogenetic tree has been developed just from
comparing sequences of cytochrome c from many
organisms. (See Figure 5.29)
• Myoglobin and hemoglobin subunits have high
degree of homology, and are evolutionarily
related. (See Figure 5.30)
Slide 8
Protein Homology, con’t.
• Sequence similarity is sometimes found
between proteins that are no longer
functionally similar.
– Egg white lysozyme and human lactalbumin
• See Figure 5.32
Slide 9
Peptide Synthesis
• Chemical synthesis requires first blocking
reactive functional groups, then reagents to
form the peptide bond.
• The process has been automated for
machines.
• For your own interest, read this section of
the chapter, but we will not cover it.
Lecture 12, Page 3
Slide
10
CHAPTER 6
Proteins: Secondary, Tertiary, and
Quaternary Structure
Slide
11
Levels of Protein Structure
• Primary (sequence)
• Secondary (ordered structure along peptide
bond)
• Tertiary (3-dimensional overall)
• Quaternary (subunit relationships)
Slide
12
Forces Contributing to Overall
Structure
• Strong (peptide bond, disulfide bond)
• Weak
– Hydrophobic (40 kJ/mol)
– Ionic bonds (~20 kJ/mol)
• Figure 6.1
– Hydrogen bonds (~12-30 kJ/mol)
– Dispersion (van der Waals) (0.4-4 kJ/mol)
Lecture 12, Page 4
Slide
13
Effect of Sequence on Structure
• Sufficient information for folding into
correct 3-dimensional structure is in the
sequence (primary structure) of the protein
– Experiments of Anfinsen and White on
Ribonuclease
• However—the “folding problem” is one of
the major unsolved problems of
biochemistry and structural biology
Slide
14
Secondary Structure
• Folding probably begins with nucleation sites
along the peptide chain assuming certain stable
secondary structures.
• Planarity of the peptide bond restricts the number
of conformations of the peptide chain. Rotation is
only possible about the
– C(alpha)-N bond (the Φ (phi) angle)
– C(alpha)-C bond (the Ψ (psi) angle)
• See Figure 6.2
Slide
15
Steric Constraints on Φ and
ΨAngles
• Examine the effects of rotation about the Φ
and Ψ angles using Kinemage
– Download Kinemage
– Download Peptide file
• Note that some angles are precluded by
orbital overlap:
– Figure 6.3
Lecture 12, Page 5
Slide
16
Ramachandran Map
• Plot of Φ versus Ψ angle for a peptide bond
is called a Ramachandran Map
• Ordered secondary structures have repeats
of the Φ and Ψ angles along the chain.
– See Figure 6.4
Slide
17
Some Common Secondary
Structures
• Alpha Helix (Figure 6.6)
– Residues per turn: 3.6
• 13 atoms in a turn (3.613 helix)
– Rise per residue: 1.5 Angstroms
– Rise per turn (pitch): 3.6 x 1.5 A = 5.4 A
– Φ = -60 degrees; Ψ = -45 degrees
• Discuss polyglutamate and polylysine
• Two proteins with substantial alpha helix structure
(Figure 6.7)
• Other helix structures (310 and 4.4 16 helices)
Slide
18
Common Secondary Structures,
con’t.
• Beta Sheet (or “pleated sheet”)
– See Figure 6.10
• Can be Parallel or Antiparallel
– See Figure 6.11
• Parallel sheets usually large structures
– Hydrophobic side chains on both sides
• Antiparallel sheets often smaller
– Hydrophobic side chains on one side
Lecture 12, Page 6
Slide
19
Common Secondary Structures,
con’t.
• Beta-Turn
– See Figure 6.12
• Beta-Bulge
– See Figure 6.13
Lecture 12, Page 7