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Protein Primary Structure and Determination

Biochemistry lecture
by Heidi Fletcher on 1 October 2014

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Transcript of Protein Primary Structure and Determination

Protein Primary Structure and Determination
Define Protein
Protein Structure
Info
3D structure
determines
protein's function
4 levels of organization
Primary
1(knot)
AA sequence
pic
Secondary
2(knot)
Local conformation
of polypeptide
or nucleic acid w/out
regard to conformation
of its side chains
alpha-helix
Beta sheets
Tertiary
3(knot)
3D
structure
of ENTIRE
polypeptide
Quaternary
4(knot)
Spatial
arrangement
of its SUBUNITS
Primary Structure Determination
Importance for
knowing primary
structure
Frederick
Sanger
1953
1.
If more than 1
polypeptide chain
(subunit), separate
An 8 Step Strategy - chemical process
2.
Cleave Disulfide
Bridges
3.
Determine AA
composition:
the # of each AA
present
4.
Identify N- and
C- terminal resides
Subunit interactions
depend on
weak forces
use
extreme pH
8M urea
6 M guanidine-HCl
high salt conc
usually ammonium
sulfate
Performic
acid
oxidation
Sulfhydryl
reducing
agents
mercaptoethanol*
diastereomers:
dithiothreitol
Cleland's reagent
To prevent recombination
follow w/alkylating agent
like
iodoacetate
Location of bridges
determined in final step
Compelete
Hydrolysis
6 M HCl
Trp, Asn,
Gln degraded
2-4 M NaOH
Cys, Ser, Thr,
Arg degraded
Trp not harmed*
Separate fragments
by Ion exchange
chromatography
Detect by Abs or Flu
& can do quant. analysis
Example problems
N-terminal
analysis
C-terminal
analysis
Dansyl
chloride
Edman's
Degradation
PITC
Enzymatic analysis
(carboxypeptidases)
A
B
Cleaves
ANY
residue
EXCEPT
Pro
Arg
Lys
ONLY
cleaves
Arg
Lys
Speed of
enzymes
(hog pancreas)
5.
Cleave each chain
into smaller fragments
and determine sequence
of each chain
Chemical
fragmentation
Enzymatic
fragmentation
CNBr
(cyanogen bromide)
Cleaves
AFTER

Met
residues ONLY
Useful b/c Met is
a rare AA
MUST be able to
recognize product:
a homoserine lactone
Endopeptidases
(define)
Sissle bond
Pepsin
Cleaves
BEFORE AA @
Sissle bond
Rn =
Leu, Phe, Trp, Tyr
Rn-1 CANNOT = Pro
Thermolysin
Cleaves
BEFORE

AA @
Sissle bond
Rn =
Ile, Met, Phe, Trp, Tyr, Val
Rn-1 CANNOT = Pro
Elastase
Cleaves
AFTER

AA @
Sissle bond
Cleaves small neutral residues
Rn-1 =
Ala, Gly, Ser, Val
Rn CANNOT = Pro
Chymotrypsin
Cleave
AFTER

AA @
Sissle bond
Cleaves bulky hydrophobic residues
Rn-1 =
Phe, Trp, Tyr
Rn CANNOT = Pro
Trypsin
Cleaves
AFTER AA @
Sissle bond
Cleaves positively charged residues
Rn-1 =
Arg, Lys
Rn CANNOT = Pro
Clostripain
Cleaves
AFTER

AA @
Sissle
bond (Rn-1)->
Arg
Staphylococcal protease
Cleaves
AFTER

AA @
Sissle bond (Rn-1) ->
Asp, Glu
6.
Repeat step 5 using a
different cleavage procedure
to generate a different
set of fragments
7.
Reconstructing
the sequence
Use 2 or more fragments
in SEPARATE
fragmentation expts
Compare and align
OVERLAPPING
peptide seq. to get
original polypeptide chain
Mixtures from
peptide fragments
separated by HPLC
8.
Determine the positions
of disulfide bridges
Very difficult depending
on # of them
MS/MS fragmentation used
Let's Practice
Examples
One or more polypeptide chains
Monomeric protein: One polypeptide chain

Multimeric protein: More than one

Homomultimer: one kind of chain

Heteromultimer: two or more different chains
Hemoglobin, for example, is a heterotetramer
has two alpha chains and two beta chains
“Protein”
Insulin - A chain of 21 residues, B chain of 30 residues -total mol. wt. of 5,733

Glutamine synthetase - 12 subunits of 468 residues each - total mol. wt. of 600,000

Connectin proteins - alpha - MW 2.8 million!

Beta connectin - MW of 2.1 million, with a length of 1000 nm -it can stretch to 3000 nm!
Proteins - Large and Small
An incredible # of possible sequences are possible
For a protein of n residues, there are 20^n different possible sequences single polypeptide chain of 100 residues: 20^100 = 1.27 x 10^130 possible unique sequences
A quantity greater than the estimated total number of atoms (9 x 10^78) in the universe!!!
Is this an example of God’s miraculous wonders?

Natural polypeptides are limited in size and composition and most contain 100-1000 residues
Protein Structure
Many proteins are composed of more than one identical or different polypeptide chain
Each chain is called a subunit

Proteins are essential for life and involved in molecular transformations, cell structure, cellular transport , cellular signaling, DNA regulation, etc.
These functions can best be understood in terms of the 3D structure of proteins

The 3D structure (shape) determines the protein’s functions (the AA sequence determines the 3D structure)

AA SEQ --> 3D STRUCTURE/SHAPE --> PROTEIN FUNCTION
Protein Structure
4 levels of protein organization:

1. Primary structure (1)
(focus of the rest of this lecture)
2. Secondary structure (2)

3. Tertiary structure (3)

4. Quaternary structure (4)
Protein Structure
1. Primary structure (1): The AA sequence for a protein and the base sequence for a nucleic acid

2. Secondary structure (2): The local conformation of polypeptide or nucleic acid’s backbone atoms w/out regard to the conformation of its side chains
Usually refers to -helices, -sheets, and turns

3. Tertiary structure (3): 3D structure of the entire polypeptide or polynucleotide chain

4.Quaternary structure (4): Most proteins are composed of 2 or more polypeptide chains known as subunits
4^knot structure refers to the spatial arrangement of its subunits
1, 2, 3 and 4 structures
Primary (1), Secondary (2), Tertiary (3) and Quaternary (4)
Structural hierarchy of proteins
How do we elucidate the AA sequence?
What information does it tell us?
Protein function (3D structure, but AA sequence determines 3D structure)
Encoded by the nucleotide sequence of DNA
A form of genetic information
Inherited diseases occur as a result caused by mutations from a change in an AA in the protein’s sequence
Treatment
Makes protein unique for identification
Importance for knowing Primary structure
In 1953, Frederick Sanger elucidated for the 1st time the complete AA sequence of a protein
He sequenced the 2 chains of insulin (A = 21 residues and B = 30 residues)
Sanger's results established that all of the molecules of a given protein have the same sequence







1 structure of bovine insulin
Proteins can be sequenced in two ways:
- real amino acid sequencing
- sequencing the corresponding DNA in the gene
Primary structure determination
How is 1 structure determined?
1. Determine which amino acids are present
(amino acid analysis)

2. Determine the N- and C- termini of the
sequence (a.a sequencing)

3. Determine the sequence of smaller peptide
fragments (most proteins > 100 a.a)

4. Some type of cleavage into smaller units
necessary
Primary Structure Determination
Primary Structure Determination
Iodoacetic acid: prevents reformations of disulfide bonds through oxidation by O2
2-Mercaptoethanol: most often used
Performic acid Oxidation
Cleavage of disulfide bonds
Determine Amino Acid Composition:
the # of each AA residue present
1. Complete Hydrolysis
6 M HCl for 10-100h @ 100-120 C
Trp, Asn and Gln degraded
2 - 4 M NaOH for 4-8h @ 100 C
Cys, Ser, Thr, Arg degraded
primarily for Trp determination
2. Separation by Ion Exchange Chromatography
3. Detection by Absorbance or Fluorescence
used for quantitative analysis
Step 3:
Asp-Trp-Val-Arg-Asn-Ser-Phe-Cys-Gln-Gly-Pro-Tyr-Met
Asp-Trp-Val-Arg-Asn-Ser-Phe-Cys-Gln-Gly-Pro-Tyr-Met
Asp-Trp-Val-Arg-Asn-Ser-Phe-Cys-Gln-Gly-Pro-Tyr-Met

What AA’s would be liberated on its complete acid hydrolysis?





What AA’s would be liberated on its complete alkaline hydrolysis?
Consider the following peptide:
o-Phthalaldehyde (OPA) reacts with amino acids to yield highly fluorescent adducts that can be separated on reverse-phase HPLC. Automation permits analysis in ca. 1 h at a sensitivity of 1 pmol of each amino acid.
Amino Acid Analysis
N-terminal analysis:
Dansyl chloride: reacts w/ all 1 amines, followed by acid hydrolysis

Edman's degradation: reagent is Phenylisothiocyanate (PITC) and reacts w/ N-terminal amino groups and is released from the rest of polypeptide

AA sequence can be determined for an entire polypeptide chain from the N-terminus inward by subjecting the polypeptide chain to repeated cycles of the Edman degradation and, after every cycle, identifying the newly liberated PTH-amino acid

Technique is automated
Step 4: Identify N- and C-terminal residues
The Edman Degradation Reaction
C-Terminus Identification
Trypsin- Cleaves @ C-terminal of (+) charged side chains
Chymotrypsin- Cleaves @ C-terminal of aromatics
You need to KNOW these!
Specificity of Endopeptidases
Peptide Digestion
Multiple cleavage techniques (chemical or enzymatic) can establish sequence order in a single chain
Overlap Peptides Define Sequence Order
Met residue
Cleaves @ C-terminal of INTERNAL methionines
Cleavage by CnBr
After cleavage, mixture of peptide fragments produced.
• Can be separated by HPLC or other chromatographic techniques
• Use different cleavage reagents to help in 1˚ determination
Determining Protein Sequence
Sequence analysis of catrocollastatin-C, a 23.6 kD protein from the venom of Crotalus atrox
State the cleavage pattern of the following
polypeptides by the indicated agents:

A. Ser-Ala-Phe-Lys-Pro by chymotrypsin
B. Thr-Cys-Gly-Met-Asn by CNBr
C. Leu-Arg-Gly-Asp by carboxypeptidase A
D. Val-Trp-Lys-Pro-Arg-Glu by trypsin
Let’s Practice
Problem
Garrett and Grisham
Chapter 5: 2-9
Practice Problems
Gives the % or ratio of the AA present in protein
Review
How proteins are made

*Alanine occurs most frequently in proteins overall.
Practice Problems
Garrett and Grisham, 4th ed
2-9

I.
i.
ii.
a.
a.
b.
i.
ii.
iii.
iv.
1.
a.
CAUTION MUST BE TAKEN!
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