CHMI-2227EL Study Guide - Final Guide: Mushroom Poisoning, Hemoglobin C, Xeroderma Pigmentosum
27 Jun 2018
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CHMI-2227 Exam Review
Determining the Amino Acid Sequence of a protein
Step 1: Separation of chains (intramolecular forces)
-Separation here is achieved with
-Heat
-Extreme pH: using 8 M urea or 6 M guanindine HCl
-High salt concentration (usually ammonium sulfate)
Step 2: Cleavage of Disulphide bridges between/within polypeptide chains
-First: Use Performic acid oxidation, which converts all cysteine residues to cystic acid
residues
-Second: Use Sulfhydryl reducing agents, which is done to prevent the cysteine
residues from recombining (mercaptoe-ethanol)
-Third: Use an alkylating agent, like iodoacetate, to form a stable S-carboxymethyl
derivative
Step 3: Identify the N- and C- terminal residues
-N- terminal analysis (2 methods are widely used):
-The general 3 steps followed are:
-1. Label all amino groups
-2. Hydrolyze the peptides to amino acids
-3. Determine the labeled amino acid
-1. Edman’s reagent phenylisothiocyanate (PITC)
-PITC reacts with the N-terminal amino group of a peptide under alkaline
conditions (pH =9), and then is treated with trifluoroacetic acid, to release the N-
terminal amino acid residue from the chain, as a thiozolinone derivative
-Then the derivative is treated with weak acid to give a stable product called
phylthiohydantoin (PTC-derivative), which is then identified by chromatography
-Here the remainder of the polypeptide is still be intact and can be treated again
with PITC to react with the new N-terminal, so using this method it is possible to
obtain the entire sequence of peptides
-Disadvantages of the Edman degradation is that peptides longer then around 50
residues can not be sequenced completely
-2. Dabsyl chloride
-When we have a chain longer than 50 residues we can use Dabsyl chloride
-This reacts with N-terminal to produce dabsyl polypeptide, which is then
hydrolized (so made acidic) to separate the N-terminal, and we then use
chromatography to identify it
-C-terminal analysis
-No reliable method that exists for sequencing the C-terminal amino acid of peptides
-Carboxypeptidase A will cleave all amino acid residues except Pro, Arg, and Lys
(AA with NH2+ on the R chain)(when they are at the C terminal)
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-Carboxypeptidase B only works on C-terminal Arg and Lys residues
Step 5: Fragmentation of the Chains
-1) Enzymatic fragmentation
-5 Enzymes can be used for this, and they will all cleave on the C-side of specific
amino acids
-1) Trypsin: cleaves Lys and Arg (positive charged R groups AA)
-2) Chymotrypsin: cleaves Phe, Tyr, Trp (aromatic AA)
-3) Clostripain: cleaves only at Arg
-4) Endoproteinase: Cleaves only Lys
-5) Staphylococcal protease: Cleaves Glu, and Asp (acidic AA)
-2) Chemical fragmentation
-Cyanogen bromine (CNBr): highly selective cutting agent, cleaves the the C-side of
every Met reside in a protein, which is useful because proteins usually have only a
few methionine residues
-Peptides resulting from cleavage of intact proteins are usually separated by
chromatography
Step 6: Reconstructing the Sequence
-1) Sequence all the peptides produced by Edman degradation
-2) Compare and align overlapping peptide sequences to learn the sequence of the
original polypeptide chain
-3) Compare cleavage by trypsin and staphylococcal protease on a typical peptide
Protein Sequence
-3-D Structural properties of proteins are uniquely dependant on the order of amino
acids in the polypeptide chain
-Many genetically heritable diseases are caused by a change of 1 or more amino
acids in the sequence of one protein
Secondary Structure
-The amino acid chains with proteins can fold and coil in repeating patterns
-The most common secondary structures are the α-helix and β-pleated sheet
-These patterns are maintained by H-bonds
-The peptide bond C-N cannot rotate as it is shorter then a normal single C-N bond,
and exhibits partial double bond character in a resonance structure
-The N-Cα bond can rotate with bond angle (phi) Φ
-The Cα-C bond can rotate with bond angle (psi) Ψ
The Alpha Helix
-The α-helix is the dominant structure in fibrous proteins (like α-keratins in hair and
skin), and the average content of α-helix in globular proteins is about 26%
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-The α-helix observed in proteins is almost aways right handed, this means
that the backbone turns clockwise along the axis from the N-terminal
-α-helix is most stable when the structure contains amino acid with
uncharged side chains
-Several factors can disrupt an α-helix
-Proline (has a +1 charge) creates a bend because of the restricted
rotation due to its cyclic structure, and its α-amino group has no N
available for h-bonding
-Strong repulsion caused by bulky side chains
-Glycine’s R-group (H) is so small, so the polyptide chain may be too
flexible
-In α-helix there are:
-3.6 amino acid/turn, and each residue advance the helix by 1.5 A along the axis
-The distance between corresponding points per turn (rise per turn or pitch) is 3.6 x
1.5 A = 5.4 A
-Each peptide bond is trans and planar
-C=O (carbonyl bond) of each peptide bond is h-bonded to N-H
(amio group) of 4 amino acids away (like in picture) and are
parallel to the axis
-phi = -60 degrees, psi = -45 degrees
-The R groups of amino acids extend outwards perpendicular to
the axis of the helix
The β-pleated sheet
-These structures are pleated because C-C bonds are tetrahedral and
cannot exist in straight lines
-2 types of β-pleated sheets
-Parallel: where chains run in the same direction from the N-terminal
to the C-terminal of the molecule
-Antiparallel: where chains run in the opposite direction so N->C, C-
>N
-Here polypeptide chains lie adjacent to one another
-R groups alternate first, above then below the plane of the beta sheet
-Each peptide bond is trans and planar
-C=O and N-H groups of each peptide bonds are perpendicular to the axis
-The H bonding between peptide chains give rise to repeated zig-zag
-β-pleated sheets are found in both fibrous and globular proteins
-β-turns/bends (or hair pin turns), are the part of a polypeptide chain where the chain abruptly
reverse its direction
-This involves 4 amino acids and are often stabilized by H-bonds between the 1st and 4th
amino acids
-Glycine and proline frequently occurs in β-turn sequences, because glycine with no side
chains are flexible, and cyclic proline forms a natural turn
Protein Folding in Tertiary Structure
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Document Summary
Determining the amino acid sequence of a protein. Extreme ph: using 8 m urea or 6 m guanindine hcl. Step 2: cleavage of disulphide bridges between/within polypeptide chains. First: use performic acid oxidation, which converts all cysteine residues to cystic acid residues. Second: use sulfhydryl reducing agents, which is done to prevent the cysteine residues from recombining (mercaptoe-ethanol) Third: use an alkylating agent, like iodoacetate, to form a stable s-carboxymethyl derivative. Step 3: identify the n- and c- terminal residues. N- terminal analysis (2 methods are widely used): Pitc reacts with the n-terminal amino group of a peptide under alkaline conditions (ph =9), and then is treated with tri uoroacetic acid, to release the n- terminal amino acid residue from the chain, as a thiozolinone derivative. Then the derivative is treated with weak acid to give a stable product called phylthiohydantoin (ptc-derivative), which is then identi ed by chromatography.
