February 3, 2014
Bovine insulin- first protein sequenced
Importance of known the sequence of amino acids in a protein:
1. Prerequisite for determining its three dimensional structure
2. Essential for understanding the proteins mechanism of action
3. Protein sequence analysis can assist in the development of diagnostic test and effective therapies as most
inherited diseases are caused by mutations in the DNA, and therefore changes in the amino acids
4. Protein sequence can be determined directly using a chemical method or indirectly by sequencing the
5. To sequence a protein directly, the protein must be broken down into fragments small enough to be
individually sequence. The sequence of the whole protein is the reconstructed from the sequence of the
overlapping fragments Identification if the N-terminal amino acids using DNFB
(Peptide hydrolysis generates individual amino acids, one being derivatized by DNFB)
In peptide hydrolysis, a peptide is treated with an acid (eg HCl) that breaks (Hydrolyze) all the amide bonds that
bind the corresponding amino acids within the peptide. This result in a mixture that contain all the amino aids
present on the peptide.
6M HCl Ala+Try+Phe+
Unknown peptide 20AA Pro+Gly+ Leu+
DNFB Pro+Gly+ Leu+
Arg+ Gln+ DNFB-LEU
Identificaction of the N-terminal amino acids using dansyl chloride (has a higher florecences)
Hydrolyzed amino acids are separated by chromatography and the corresponding dansylamino acids id detected
Disulfide bonds must be cleaved before sequencing using a reducing agent Reducing agent
Cysteine alkylation with idocacetate prevent re-formation of disulfide bond via oxidation by atmospheric O2
1. more stable product
2. prevents reformation of disulfide bond
3. keeps prptide lineal (with no loops) in the case if intermolecular discol
4. (Didn’t finish slide)
Protein cleavage (peptide fragment formation)
Chemical or enzymatic treatment to selectively cleave the protein
1. Enzymatically, using a protease
2. Chemically, using cyanogen bromide
Protesase of peptidase
1. Enzymes that cleave a peptide bond within a protein 2. They can be
a. endopeptidases- cleaved internal peptide binds
b. exopeptidases- cleave either the N- or C-terminal peptide bond
3. Peptides bond cleavage occurs by hydrolysis of the bond
4. Most proteases are specific to a particular amino acid sequence, having particular size chain
requirements (table 5.4)
February 7, 2014
February 10, 2014
Proteins: three-dimensional structure
• The first X-ray structure of a protein was that of sperm whale myoglobin 1958
• It was soon discovered that proteins are more complex than nucleic acids
• Primary, secondary, tertiary and quaternary structure
• Bacteria and viruses display a complex array of proteins on their surface (Rhinovirus)
o Avirus that effects people and animals
Primary structure: sequence of amino acids in that chain
Secondary structure: is the local spatial arrangement of a polypeptide’s backbone atoms without regard to the
conformations of its side chains.
Tertiary structure: refers to the three-dimensional structure of an entire polypeptide, including its side chains.
One complete protein.
Most proteins are one chain and do not form a quaternary structure.
Primary peptides only make a secondary structure
Peptide bonds have a rigid planar structure:
(As a consequence of resonance interaction that give that peptide bond aprox. 40%double bond character) Note cis conformation
Making a tras conformation (more
Torsional angles between peptide groups describes the polypeptide chain conformation
The peptide backbone can be drawn as a linked sequence of rigid planar peptide groups.
TorsionalAngles Only thing you needs to know are the angles
Not all possible potations are allowed
Due to steric interference of the adjacent groups.
The Ramachandran Diagram Creates populations of the more stable angles.
-Defines the alpha strand
-Defines the beta strand.
It’s a way to predict the most stable conformation of the angles.
The alpha helix (right handed)
A. 3.6 residues per turn
B. Pitch of 5.4A
C. Most a-helixes in proteins have an average of:
a. 12 residues
b. 18 Ain length
D. Amino acid side chains project outward to avoid steric interference and they are typically hydrophilic
E. Helix is stabilized by intermolecular H-bonds
Intermolecular H-bonds stabilize the helix Red: oxygen
Prolene’s break alpha helix
They are either in loops or at the top or bottom of the helix NEVER in the middle.
1. Typically a B-sheet is formed when it is interacting with another secondary structure, typically
2. Hydrogen bonding occurs between the b-sheets
3. They have a rippled or pleated appearance, for this reason they are sometimes called “pleated
4. Aset of B-sheets can be either
a. Antiparallel (has stronger bonds)
5. Typically, they are composed of hydrophobic amino acids. Pleated appearance of a b-sheet
1. Successive side chains of a polypeptide chain in a b-sheet extend to opposite sites of the sheet with a
two residue repeat distance of 7.0A
2. Within Protein up to 22 b-sheets can be present, minimum is 2.
Structure of bovine carboxypeptideaseA
-Pancreatic exopeptidase that hydrolyses peptide bonds of C-terminal residues with hydrophobic side chains.
This is the tertiary structure of bovine
*Protein at the core is a metal
Reverse turns, or beta-turns (bends) are required to connect units with secondary structures
February 14, 2014
Two antiparallel strands had only 4 amino acids in the loop
Beta-turns (bends) in polypeptides
Hydrogen pointing outword and hydrogen bond pointing inward There is a 180 degree flip of the peptide bond linking residues 2 and 3
Residue 2 is almost always a Pro in both type I and II turns
Residue 3 is almost always a Gly in type II turns.
-because of steric hindrance with bigger proteins
*Important to know where these bonds are
Protein Families or types (depending on their morphology)
a. Often have protective, connective or supporting roles in living organism
b. Have a structural purpose
c. Most common ones are
a. Have enzymatic functions
Alpha-keratin is a coiled coil (power point online)
-not an enzyme
-we don’t call it an alpha-helix because it’s more compressed
-two polypeptides mixed together create the a-keratin helix
interactions of two a-keratin helic subunits Right-handed helix Right-handed helix
Left handed coil
Need to be hydrophobic in order for a and d to interact
Higher order structure of a alpha-keratin Macrofibrils
*Changes in amino acid composition (amino acid sequence) and the number of fibers and how they are
packed determines weather or not its going to be hair, horns or feathers.
-horn has more Cystine
Higher order structure of an alpha keratin
1. a-keratins are rick in Cys residues, which form disulfide bonds that cross-link adjacent
2. the number of disulfide bonds (high or low), determine if the a-keratin is hard or soft
3. these disulfide bonds can be reductively cleaved using mercaptants
4. to curl or relax hair, one must first apply a reducing agent to break the disulfide bonds, then
curl or relax the hair while applying an oxidizing agent to re-introduce the disulfide bonds at
5. For example:Ammonium Thioglycolate – reducing agents
hydrogen peroxide- oxidizing agent.
Collagen is a tripled helix
1. Most abundant protein in vertebrates
2. consist of strong insoluble fibers which are present in bone, teeth, cartilages, skin and blod vessels
3. collagen is mostly composed of Gly (30-40%), Pro (30-15%)
4. Collagen also contain the following modified amino acid residues to lower amounts:
5. 4hydroxyprolyl , 3hydroxyprolyl, 5hydroxyprolyl 6. NOT an a-helix.. it’s a triple helix
7. left handed helix
8. right handed coil
Know these structures and the numbering
*Know the table of enzymes
Collagen is a left handed coil
1. collagen peptide sequence consists of repeating triplets of Gly-X-Y over 100, residues where X is often
a proline and Y is 4-hydroxyproline
2. the presence of Pro prevents the formation of a real a-helix, instead an extended left-handed helix (3
residues per turn) is form
4. On every 3 residue the helix passes through the center of the triple helix assembly (triple coil), which is
also crowded that only Gly fits.
Both collagens and keratins are cross-linked
1. That is one of the reason why they have different hardness
2. Keratin contains Cys
3. Collagen does not contain Cys
4. Collagen does not contain Cys groups, but contains other amino acids such a Lys that can be chemically
cross-linked by lysyl oxidase
*collagen does not have disulfide bonds.
*Know that is the case of the collagen that cross link that are that of lysine and histadines.
*Look at figure legends
1. Contains a repetitive amino acid sequence
2. Mostly one type of secondary structure
3. Mostly absent of coils
4. Involved in protective connective and supportive roles with no catalytic activity
1. Contains several types of regular secondary structures
2. Mostly involve in catalysis (enzymes)
3. The secondary structures are connected by loops or coils
4. Loops or coils are undefined structures with stable conformation.
February 15, 2014 Table 6-1 Propensities of amino acids residues for a helical and b sheets conformations
**(know which amino acids have a helix or b sheets)
-p value is the probability of that conformation.
-If a sequence has a lot of alanine then its probably going to form an a helix.
Protein Tertiary Structure
1. x-ray crystallography is mainly used to determine the position of atoms (electron density
within a protein
2. the technique relies on the diffraction of electron when they interact with proteins in a crystal,
3. x-rays interact with the electrons in the protein creating an electron density map
4. in order to obtain good diffraction and therefore be able to more accurately determine the
structure of the protein , good crystal must be obtained.
5. (azurin, flavodoxin, rubredoxin) three different proteins
-in order to have a good reaction you need to have good crystals.
-crystals deffract the electrons
X-ray diffraction pattern of sperm whale myoglobin
-the a-helix was determined with this x-ray crystallography technique.