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Transcript of Proteins
Amino Acids, Peptides, and Proteins
Outline
Amino Acids
Chemical structure
Acid-base properties
Stereochemistry
Non-standard amino acids
Formation of Peptide Bonds
The building blocks of proteins Also used as single molecules in biochemical
pathways 20 standard amino acids (-amino acids) Two functional groups:
carboxylic acid group amino group on the alpha () carbon
Have different side groups (R) Properties dictate behavior of AAs
Amino Acids
R side chain
| H2N— C —COOH
|H
Both the –NH2 and the –COOH groups in an amino acid
undergo ionization in water.
At physiological pH (7.4), a zwitterion forms Both + and – charges
Overall neutral
Amphoteric Amino group is protonated
Carboxyl group is deprotonated
Soluble in polar solvents due to ionic character
Structure of R also influence solubility
Zwitterions
Classification of Amino Acids
Classify by structure of R Nonpolar
Polar
Aromatic
Acidic
Basic
Nonpolar Amino Acids
Hydrophobic, neutral, aliphatic
Nonpolar Amino Acids
Hydrophobic, neutral, aliphatic
Polar Amino Acids
Hydrophilic, neutral, typically H-bond
Polar Amino Acids
Hydrophilic, neutral, typically H-bond
Disulfide Bonds
Formed from oxidation of cysteine residues
Aromatic Amino Acids
Bulky, neutral, polarity depend on R
Aromatic Amino Acids
Bulky, neutral, polarity depend on R
Acidic and Basic Amino Acids
Acidic R group = carboxylic
acid Donates H+ Negatively charged
Basic R group = amine Accepts H+
Positively charged His ionizes at pH 6.0
Acidic and Basic Amino Acids
Acidic R group = carboxylic
acid Donates H+ Negatively charged
Basic R group = amine Accepts H+
Positively charged His ionizes at pH 6.0
Remember H3PO4 (multiple pKa’s)
AAs also have multiple pKa’s due to multiple ionizable
groups
Acid-base Properties
pK1 ~ 2.2(protonated below 2.2)
pK2 ~ 9.4(NH3
+ below 9.4)
pKR
(when applicable)
Table 3-1
Note 3-letter and 1-letter
abbreviations
pH and Ionization
Consider glycine:
Note that the uncharged species never forms
H3N CH C
H
OH
O
H3N CH C
H
O
O
H2N CH C
H
O
O
OH-
H3O+
OH-
H3O+
Glycine ion at acidic pH
(charge = 1+)
Zwitterion of glycine (charge = 0)
Glycine ion at basic pH
(charge = 1-)
Titration of Glycine
pK1
[cation] = [zwitterion]
pK2
[zwitterion] = [anion]
First equivalence point Zwitterion Molecule has no net charge pH = pI (Isoelectric point)
pI = average of pKa’s = ½ (pK1 + pK2)
pIglycine = ½ (2.34 + 9.60) = 5.97
Animation
pI of Lysine
For AAs with 3 pKa’s, pI = average of two relevant pKa values
Consider lysine (pK1 = 2.18, pK2 = 8.95, pKR = 10.53):
Which species is the isoelectric form?
So, pI = ½ (pK2 + pKR)
= ½ (8.95 + 10.53) = 9.74
Note: pKR is not always higher than pK2 (see Table 3-1 and Fig. 3-12)
H3N CH C
CH2CH2CH2CH2NH3+
OH
O
H3N CH C
CH2CH2CH2CH2NH3+
O
O
H2N CH C
CH2CH2CH2CH2NH3+
O
O
H2N CH C
CH2CH2CH2CH2NH2
O
O
pK1 pK2 pKR
Learning Check
Would the following ions of serine exist at a pH above, below, or at pI?
H3N CH C
CH2
O
O
OH
H3N CH C
CH2
OH
O
OH
H2N CH C
CH2
O
O
OH
Stereochemistry of AAs
All amino acids (except glycine) are optically active
Fischer projections:
D and L Configurations
d = dextrorotatory l = levorotatory D, L = relative to glyceraldehyde
Importance of Stereochemistry
All AA’s found in proteins are L geometry
S enantiomer for all except cysteine
D-AA’s are found in bacteria
Geometry of proteins affects reactivity (e.g
binding of substrates in enzymes)
Thalidomide
Non-standard Amino Acids
AA derivatives Modification of AA after
protein synthesized
Terminal residues or R
groups
Addition of small alkyl
group, hydroxyl, etc.
D-AAs Bacteria
Organic Chem Review
Carboxylic acid + amine = ?
Structure of amino acid
R C OH
O
+ H2N R R C NH
O
+ H2Oheat
R
H2N C CO2H
H
R
The Peptide Bond
Chain of amino acids = peptide or protein Amino acid residues connected by peptide bonds Residue = AA – H2O
The Peptide Bond
Non-basic and non-acidic in pH 2-12 range due to delocalization of N lone pair
Amide linkage is planar, NH and CO are anti
C
O
N
H
O
N
HRigid
restricted rotation
Polypeptides
Linear polymers (no branches) AA monomers linked head to tail Terminal residues:
Free amino group (N-terminus) Draw on left
Free carboxylate group (C-terminus) Draw on right
pKa values of AAs in polypeptides differ slightly from pKa values of free AAs
Naming Peptides
Name from the free amine (NH3+)
Use -yl endings for the names of the amino acids The last amino acid with the free carboxyl group (COO-)
uses its amino acid name
(GA)
Amino Acid Ambiguity
Glutamate (Glu/E) vs. Glutamine (Gln/Q) Aspartate (Asp/D) vs. Asparagine (Asn/N) Converted via hydrolysis Use generic abbreviations for either
Glx/Z Asx/B
X = undetermined or nonstandard AA
Write the name of the following tetrapeptide using amino acid names and three-letter abbreviations.
Learning Check
CH CH3
CH3
H3N CH C
O
N
H
CH C
O
N
H
CH C
O
N
H
CH C O-
OCH CH2
CH2
S
CH3
CH2
SH
CH3
Learning Check
Draw the structural formula of each of the following peptides.A. Methionylaspartic acid
B. Alanyltryptophan
C.Methionylglutaminyllysine
D.Histidylglycylglutamylalanine
Outline (part II)
Sections 3.3 and 3.4 Separation and purification Protein sequencing
Analysis of primary structure
Protein size
In general, proteins contain > 40 residues Minimum needed to fold into tertiary structure
Usually 100-1000 residues Percent of each AA varies Proteins separated based on differences in
size and composition Proteins must be pure to analyze, determine
structure/function
Factors to control
pH Keep pH stable to avoid denaturation or chemical degradation
Presence of enzymes May affect structure (e.g. proteases/peptidase)
Temperature Control denaturation (0-4°C) Control activity of enzymes
Thiol groups Reactive Add protecting group to prevent formation of new disulfide bonds
Exposure to air, water Denature or oxidize Store under N2 or Ar Keep concentration high
General Separation Procedure
Detect/quantitate protein (assay) Determine a source (tissue) Extract protein
Suspend cell source in buffer Homogenize
Break into fine pieces Cells disrupted Soluble contents mix with buffer Centrifuge to separate soluble and insoluble
Separate protein of interest Based on solubility, size, charge, or binding ability
Solubility
Selectively precipitate protein Manipulate
Concentration of salts Solvent pH Temperature
Concentration of salts
Adding small amount of salt increases [Protein]
Salt shields proteins from each other, less
precipitation from aggregation Salting-in
Salting out Continue to increase [salt] decreases [protein]
Different proteins salt out at different [salt]
Other Solubility Methods
Solvent Similar theory to salting-out Add organic solvent (acetone, ethanol) to interact with
water Decrease solvating power
pH Proteins are least soluble at pI Isoelectric precipitation
Temperature Solubility is temperature dependent
Chromatography
Mobile phase Mixture dissolved in liquid or
solid
Stationary phase Porous solid matrix
Components of mixture
pass through the column
at different rates based on
properties
Types of Chromatography
Paper Stationary phase = filter paper
Same theory as thin layer chromatography (TLC)
Components separate based on polarity
High-performance liquid (HPLC) Stationary phase = small uniform particles, large surface area
Adapt to separate based on polarity, size, etc.
Hydrophobic Interaction Hydrophobic groups on matrix
Attract hydrophobic portions of protein
Types of Chromatography
Ion-exchange Stationary phase =
chemically modified to
include charged groups
Separate based on net
charge of proteins
Anion exchangers Cation groups (protonated
amines) bind anions
Cation exchangers Anion groups (carboxylates)
bind cations
Types of Chromatography
Gel-filtration Size/molecular exclusion
chromatography Stationary phase = gels
with pores of particular size
Molecules separate based on size
Small molecules caught in pores
Large molecules pass through
Types of Chromatography
Affinity Matrix chemically
altered to include a molecule designed to bind a particular protein
Other proteins pass through
UV-Vis Spectroscopy
Absorbance used to
monitor protein
concentrations of each
fraction
= 280 nm Absorbance of aromatic
side groups
Electrophoresis
Migration of ions in an electric field
Electrophoretic mobility (rate of movement) function of
charge, size, voltage, pH
The positively charged proteins move towards the negative
electrode (cathode)
The negatively charged proteins move toward the positive
electrode (anode)
A protein at its pI (neutral) will not migrate in either direction
Variety of supports (gel, paper, starch, solutions)
Protein Sequencing
Determination of primary structure Need to know to determine 3D structure Gives insight into protein function Approach:
Denature protein Break protein into small segments Determine sequences of segments
End group analysis
Identify number of terminal AAs Number of chains/subunits
Identify specific AA
Dansyl chloride/dabsyl chloride Sanger method (FDNB) Edman degradation (PITC)
Bovine insulin
Dansyl chloride
Reacts with primary amines N-terminus
Yields dansylated polypeptides Dansylated polypeptides
hydrolyzed to liberate the modified dansyl AA
Dansyl AA can be identified by chromatography or spectroscopy (yellow fluorescence)
Useful method when protein fragmented into shorter polypeptides
N
SO2
Cl
+
HN CH
R
C
O
N
SO2
H2N CH
R
C
O
HCl +H3O+
HN CH
R
C
O
OH
N
SO2
+ other free AAs
Dabsyl chloride and FDNB
Same result as dansyl chloride
Dabsyl chloride
1-Fluoro-2,4-dinitrobenzene (FDNB) Sanger method
SN
NN O
O
Cl
Edman degradation
Phenylisothiocyanate (PITC) Reacts with N-terminal AA to produce a phenylthiocarbamyl (PTC) Treat with TFAA (solvent/catalyst) to cleave N-terminal residue Does not hydrolyze other AAs Treatment with dilute acid makes more stable organic compound
Identify using NMR, HPLC, etc. Sequenator (entire process for proteins < 100 residues)
Fragmenting Proteins
Formation of smaller segments to assist with
sequencing
Process: Cleave protein into specific fragments
Chemically or enzymatically
Break disulfide bonds
Purify fragments
Sequence fragments
Determine order of fragments and disulfide bonds
Cleaving Disulfide Bonds
Oxidize with performic acid
Cys residues form cysteic acid
Acid can oxidize other
residues, so not ideal
H C
O
O OH
Cleaving Disulfide Bonds
Reduce by mercaptans (-SH) 2-Mercaptoethanol
HSCH2CH2OH
Dithiothreitol (DTT)
HSCH2CH(OH)CH(OH)CH2SH
Reform cysteine residues
Oxidize thiol groups with
iodoacetete (ICH2CO2-) to
prevent reformation of disulfide
bonds
Hydrolysis
Cleaves all peptide bonds Achieved by
Enzyme Acid Base
After cleavage: Identify using chromatography Quantify using absorbance or fluorescence
Disadvantages Doesn’t give exact sequence, only AAs present Acid and base can degrade/modify other residues Enzymes (which are proteins) can also cleave and affect results
Enzymatic and Chemical Cleavage
Enzymatic Enzymes used to break
protein into smaller peptides
Endopeptidases Catalyze hydrolysis of
internal peptide bonds
Chemical Chemical reagents used to
break up polypeptides Cyanogen bromide (BrCN)
An example
PRIMARY STRUCTURE
The sequence of amino acids
MIL1 sequence:>gi|7662506|ref|NP_056182.1| MIL1 protein [Homo sapiens]MEDCLAHLGEKVSQELKEPLHKALQMLLSQPVTYQAFRECTLETTVHASGWNKILVPLVLLRQMLLELTRLGQEPLSALLQFGVTYLEDYSAEYIIQQGGWGTVFSLESEEEEYPGITAEDSNDIYILPSDNSGQVSPPESPTVTTSWQSESLPVSLSASQSWHTESLPVSLGPESWQQIAMDPEEVKSLDSNGAGEKSENNSSNSDIVHVEKEEVPEGMEEAAVASVVLPARELQEALPEAPAPLLPHITATSLLGTREPDTEVITVEKSSPATSLFVELDEEEVKAATTEPTEVEEVVPALEPTETLLSEKEINAREESLVEELSPASEKKPVPPSEGKSRLSPAGEMKPMPLSEGKSILLFGGAAAVAILAVAIGVALALRKK
length: 386amino acids© Anne-Marie Ternes
PRIMARY STRUCTURE The numbers of amino acids vary
(e.g. insulin 51, lysozyme 129, hemoglobin 574, gamma globulin 1250)
The primary structure determines the folding of the polypeptide to give a functional protein
Polar amino acids (acidic, basic and neutral) are hydrophilic and tend to be placed on the outside of the protein.
Non-polar (hydrophobic) amino acids tend to be placed on the inside of the protein
© 2007 Paul Billiet ODWS
SECONDARY STRUCTURE
The folding of the N-C-C backbone of the polypeptide chain using weak hydrogen bonds
© Science Student
© Text 2007 Paul Billiet ODWS
SECONDARY STRUCTURE
This produces the alpha helix and beta pleating The length of the helix or pleat is determined by certain amino acids
that will not participate in these structures (e.g. proline)
© Dr Gary Kaiser © Text2007 Paul Billiet ODWS
TERTIARY STRUCTURE
The folding of the polypeptide into domains whose chemical properties are determined by the amino acids in the chain
MIL1 protein
© Anne-Marie Ternes © 2007 Paul Billiet ODWS
TERTIARY STRUCTURE
This folding is sometimes held together by strong covalent bonds (e.g. cysteine-cysteine disulphide bridge)
Bending of the chain takes place at certain amino acids (e.g. proline)
Hydrophobic amino acids tend to arrange themselves inside the molecule
Hydrophilic amino acids arrange themselves on the outside
© 2007 Paul Billiet ODWS
© Max Planck Institute for Molecular GeneticsChain B of Protein Kinase C
When the polypeptide folds into a three-
dimensional shape, it is called a protein
The three-dimensional shape of a protein is called
its tertiary structure
Myoglobin Binds oxygen
Found in the muscles Acts as a storage site
for oxygen Makes up the dark meat in
chicken
QUATERNARY STRUCTURE
Some proteins are made of several polypeptide subunits (e.g. hemoglobin has four)
Protein Kinase C
© Max Planck Institute for Molecular Genetics
© Text 2007 Paul Billiet ODWS
QUATERNARY STRUCTURE
These subunits fit together to form the functional protein
Therefore, the sequence of the amino acids in the primary structure will influence the protein's structure at two, three or more levels
© 2007 Paul Billiet ODWS
Shape of the protein is important for its function
Ex. Insulin = 51 amino acids
Shape of the protein is important for its function
Ex. Insulin = 51 amino acids
Result
Protein structure depends upon the amino acid sequence
This, in turn, depends upon the sequence of bases in the gene
© 2007 Paul Billiet ODWS
PROTEIN FUNCTIONS
Protein structure determines protein function Denaturation or inhibition which may change
protein structure will change its function Coenzymes and cofactors in general may
enhance the protein's structure
© 2007 Paul Billiet ODWS
Types of ProteinsTypeType FunctionFunctionCommunication Cell signaling
Ex. Hormones in the bloodstream
Defense Protection from infectionEx. Antibodies in the bloodstream
Structure Mechanical supportEx. Collagen in skin & keratin in hair/nails
Storage Stores nutrientsEx. Albumin in egg whites
Contractile MovementEx. Actin and myosin in muscles
Transport Carries other moleculesEx. Hemoglobin
Hormones Chemical messengersEx. Growth hormone stimulates bone growth
Enzymes Speed up chemical reactionsEx. Catalase
Fibrous proteins
Involved in structure: tendons ligaments blood clots(e.g. collagen and keratin)
Contractile proteins in movement: muscle, microtubules (cytoskelton, mitotic spindle, cilia, flagella)
© 2007 Paul Billiet ODWS
Just for fun facts:
Your hair is composed of all -helix
Spider webs are all -pleated sheets
Globular proteins
most proteins which move around (e.g. albumin, casein in milk)
Proteins with binding sites: enzymes, hemoglobin, immunoglobulins, membrane receptor sites
© 2007 Paul Billiet ODWS
Antibodies are Produced by B Lymphocytes
Antibodies are Proteins that Recognize Specific Antigens
Epitopes: Antigen Regions that Interact with Antibodies