Protein Geometry
Chapter 5
Covalent structures of proteins
Proteins function as:
1. Enzymes:biological catalysts
2. Regulators of catalysishormones
3. Transport and store i.e. O2, metal ions sugars, lipids, etc. 4. Contractile assemblies
Muscle fibers
Separation of chromosomes etc.
5. Sensory
6. Cellular defense immuoglobulins Antibodies
Killer T cell Receptors
7. Structural Collagen Silk, etc.
There are four levels of protein structure
1.
Primary structure
1
= Amino acid sequence, the linear order of
AA’s.
Remember from the Nterminus to the Cterminus
There are four levels of protein structure
2.
Secondary structure
2
= Local spatial alignment of amino acids
without regard to side chains.
Usually repeated structures
3.
Tertiary Structure
3
= the 3 dimensional structure of an entire
peptide.
4.
Quaternary Structure
4
two or more peptide chains associated
with a protein.
Spatial arrangements of subunits.
Chapter 5.3 is how to determine a protein’s primary
structure.
Insulin was the first protein to be sequenced
F. Sanger won the Nobel prize for protein
sequencing.
It took 10 years, many people,
and it took 100 g of protein!
Today it takes one person several days to
sequence the same insulin.
Steps towards protein sequencing
Above all else, purify it first!! Chapter 5.3 then 5.1 and 5.2
1. Prepare protein for sequencing
a. Determine number of chemically different polypeptides.
b. Cleave the protein’s disulfide bonds.
c. Separate and purify each subunit.
2. Sequencing the peptide chains:
a. Fragment subunits into smaller peptides
50
AA’s in length.
b. Separate and purify the fragments
c. Determine the sequence of each fragment.
3. Organize the completed structure.
a. Span cleavage points between sets of peptides
determined by each peptide sequence.
b. Elucidate disulfide bonds and modified amino
acids.
At best, the automated instruments can sequence about 50
amino acids in one run!
How many peptides in protein?
Bovine insulin should give 2 Nterminii and 2 C
terminii
Nterminus
1Dimethylamino naphthalene5sulfonyl chloride
Dansyl chloride
Disadvantage with the Dansyl-chloride method is that you must use 6M HCl to cleave off the derivatized amino acid, this also cleaves all other amide bonds (residues) as well.
Edman degradation has been automated as a
method to sequence proteins. The PTH-amino acid
is soluble in solvents that the protein is not. This fact
is used to separate the tagged amino acid from the
remaining protein, allowing the cycle of labeling,
degradation, and separation to continue.
Even with the best chemistry, the reaction is about
98% efficient. After sufficient cycles more than one
amino acid is identified, making the sequence
Demonstration of Edman
degradation
Use your CD disk- install it and run
CH
NH C NH CH NH CH O
O Rn-2 Rn-1 Rn
C O
C O
CH
NH C NH CH O
Rn-2 Rn-1 C O
H3N CH O Rn
C O O
H2O Carboxypeptidase
Carboxypeptidase cleavage at the C-terminus
Carboxypeptidase A Rn R, K, P
Rn-1 P
Cleavage of disulfide bonds
Permits separation of polypeptide chains Prevents refolding back to native structure Performic acid oxidation
Changes cystine or cysteine to Cystic acid Methionine to Methionine sulfone
2-Mercaptoethanol, dithiothreitol, or dithioerythritol Keeps the equilibrium towards the reduced form
Amino acid composition
The amino acid composition of a peptide chain is determined by its complete hydrolysis followed by the quantitative analysis of the liberated amino acids.
Acid hydrolysis (6 N HCl) at 120 oC for 10 to 100 h
destroys Trp and partially destroys Ser, Thr, and Tyr. Also
Gln and Asn yield Glu and Asp
Base hydrolysis 2 to 4 N
NaOH at 100 oC for 4 - 8 h.
Amino acid analyzer
In order to quantitate the amino acid residues after hydrolysis,
each must be derivatized at about 100% efficiency to a compound that is colored. Pre or post column derivatization can be done.
CH
CH
O O
+ HS CH
2 CH2 OH + H3N CH O
R C O
N CH O
R C O
CH2 CH2 OH
S
o-Phthalaldehyde (OPA)
2-mercaptoethanol Amino acid
These can be separated using HPLC in an
Amino acid compositions are indicative
of protein structures
Leu, Ala,Gly, Ser, Val, Glu, and Ile are the most
common amino acids
His, Met, Cys, and Trp are the least common.
Ratios of polar to non-polar amino acids are
indicative of globular or membrane proteins.
Long peptides have to be broken to shorter
ones to be sequenced
Endopeptidases cleave proteins at specific sites within the chain.
NH CH C
O Rn-1
NH CH C
O Rn
Scissile Bond
Trypsin Rn-1 = positively charged residues R, K; Rn P
Chymotrypsin Rn-1 = bulky hydrophobic residues F, W, T; Rn P
Thermolysin Rn = I, M, F, W, T, V; Rn-1 P
Specific chemical cleavage reagents
Cyanogen Bromide Rn-1 = M
Cleave the large protein using i.e trypsin, separate fragments and sequence all of them. (We do not know the order of the
fragments!!)
How to assemble a protein sequence
1. Write a blank line for each amino acid in the
sequence starting with the N-terminus.
2. Follow logically each clue and fill in the blanks.
3. Identify overlapping fragments and place in
sequence blanks accordingly.
4. Make sure logically all your amino acids fit into
the logical design of the experiment.
1 2 3 4 5 6 7 8 9 10 11 12 13 14
H
3N
_
_
_
_
_
_
_
_
_
_
_
_
_
_
COO
There are a variety of ways to purify peptides
All are based on the physical or chemical properties
of the protein.
Size
Charge
Solubility
Chemical specificity
Hydrophobicity/ Hydrophylicity
Peptide mapping: digest protein with an appropriate
agent, then separate using two dimensional paper
chromatography
Digested Peptide from normal (HbA) and Sickle cell anemia (Hbs) hemoglobins
HbA V H L T P E E K
HbS V H L T P V E K
1 2 3 4 5 6 7 8
Red blood cells :
(a) normal
(b) sickle cell
Deoxyhemoglobin aggregates and deforms cell. Primary
structure changes dictate quaternary structure.
Why did the problem not die out?
Homozygotic Heterzyatic Homozygotic
normal
sickle cell trait sickle cell
gets malaria resistant gets sickle cell
to malaria
Species variation in homologous proteins
The primary structures of a given protein from
related species closely resemble one another. If one
assumes, according to evolutionary theory, that
related species have evolved from a common
ancestor, it follows that each of their proteins must
have likewise evolved from the corresponding
ancestor
.A protein that is well adapted to its function, that is,
one that is not subject to significant physiological
improvement, nevertheless continues to evolve
.Homologous proteins
(evolutionarily related proteins)
Compare protein sequences:
Conserved residues, i.e invariant residues reflect
chemical necessities.
Conserved substitutions, substitutions with similar
chemical properties Asp for Glu, Lys for Arg, Ile for Val
Phylogenetic tree
Indicates the ancestral relationships among the
organisms that produced the protein.
Each branch point indicates a common ancestor.
Relative evolutionary distances between neighboring
branch points are expressed as the number of amino
acid differences per 100 residues of the protein.
PAM units
or
PAM values differ for different
proteins.
Although DNA mutates at an
assumed constant rate. Some proteins cannot accept
Mutation rates appear constant in time
Although insects have
shorter generation times
than mammals and
many more rounds of
replication, the number
of mutations appear to
be independent of the
number of generations
but dependent upon time
Cytochrome c amino acid differences between
Evolution through gene duplication
Many proteins within an organism have sequence similarities with other proteins.
•These are called gene or protein families.
•The relatedness among members of a family can vary greatly. •These families arise by gene duplication.
•Once duplicated, individual genes can mutate into separate genes. •Duplicated genes may vary in their chemical properties due to
mutations.
Hemoglobin:
• is an oxygen transport protein
•it must bind and release oxygen as the cells require
oxygen
Myoglobin:
• is an oxygen storage protein
The globin family history
1. Primordial globin gene acted as an Oxygen-storage protein.
2. Duplication occurred 1.1 billion years ago.
lower oxygen-binding affinity, monomeric protein.
3. Developed a tetrameric structure two and two
chains increased oxygen transport capabilities.
4. Mammals have fetal hemoglobin with a variant
chain i.e. 22).
5. Human embryos contain another hemoglobin 22.
6. Primates also have a chain with no known unique
Chimpanzee
human are about 99% the same amino
acid sequences in proteins!
However:
•Rapid divergence with few mutational changes suggest
altered control of gene expression.
•Controlling the amount, where, and when a protein is
made.
