Upload
azkha-avicena
View
247
Download
1
Embed Size (px)
DESCRIPTION
Biochem
Citation preview
BIOCHEMISTRY I
Midterm exam schedule (Chap 2-7)
4/24 (Fri) 8pm
No withdrawal allowed!
Key term report submission:
3/4 Chap 2. aqueous system.
3/9. Chap 3. amino acids, peptides, and proteins
3/16. Chap 4. tertiary and quaternary structure of proteins
3/23. Chap 5. protein functions
4/1. Chap 6. enzymes
4/8. Chap 7. carbohydrates and glycobiology
# Quizes
3/23 (Mon): Chapter 2 and 3
4/6 (Mon): Chapter 4 and 5
3 | Amino Acids, Peptides, Proteins
2013 W. H. Freeman and Company
CHAPTER 3 Amino Acids, Peptides,
Proteins
Structure and naming of amino acids
Structure and properties of peptides
Ionization behavior of amino acids and peptides
Methods to characterize peptides and proteins
Learning goals:
Proteins: Main Agents of Biological Function
Catalysis enolase (in the glycolytic pathway)
DNA polymerase (in DNA replication)
Transport hemoglobin (transports O2 in the blood)
lactose permease (transports lactose across the cell membrane)
Structure collagen (connective tissue)
keratin (hair, nails, feathers, horns)
Motion myosin (muscle tissue)
actin (muscle tissue, cell motility)
Proteins serve a wide range of biological functions
Amino Acids: Building Blocks of Protein
Proteins are linear heteropolymers of -amino acids
Amino acids have properties that are well-suited to carry out a variety of biological functions
Capacity to polymerize
Useful acid-base properties
Varied physical properties
Varied chemical functionality
Amino acids share many features, differing only at the R substituent
Most -amino acids are chiral
The -carbon always has four substituents and is
tetrahedral
All (except proline) have:
an acidic carboxyl group
a basic amino group
an -hydrogen connected to the -carbon
The fourth substituent (R) is unique
In glycine, the fourth substituent is also hydrogen
All amino acids are chiral (except glycine)Proteins only contain L amino acids
Absolute configuration: the configuration of four different
substituent groups around an asymmetric carbon atoms, in
relation to D-and L-glyceraldehyde
=> D, L system
Amino Acids: Atom Naming
Organic nomenclature: start from one end
Biochemical designation:
start from -carbon and go down the R-group
Amino Acids: Classification
Common amino acids can be placed in five basic groups depending on their R substituents:
Nonpolar, aliphatic (7)
Aromatic (3)
Polar, uncharged (5)
Positively charged (3)
Negatively charged (2)
These amino acid side chains absorb UV light at 270280 nm
These amino acids side chains can form hydrogen bonds.Cysteine can form disulfide bonds.
Aromatic R Groups
These amino acid side chains absorb UV light at
270-280 nm
FIGURE 3-6 Absorption of ultraviolet light by aromatic amino acids
Protein concentration measurements?
Measurement of light absorption by a spectrophotometers
used to detect and identify molecules and to measure their
concentration in solution
The fraction of the incident light absorbed by a solution
at a given wavelength is related to the thickness of the
absorbing layer (path length) and the concentration of
the absorbing species. These two relationships are combined into
the Lambert-Beer law
IoI
log = cl
Io is the intensity of the incident light
I is the intensity of the transmitted light
I/Io (the inverse of the ratio in the equation) is the transmittance
is the molar extinction coefflcient (in units of liters per mole-centimeter)
c is the concentration of the absorbing species (in moles per liter)
l is the path length of the light-absorbing sample (in centimeters)
=> absorbance, A
BOX 3-1 FIGURE 1 The principal components of a
spectrophotometer.
Absorbance A is directly proportional to the concentration
of the absorbing solute.
varies with the nature of the absorbing compound, the
solvent, and the wavelength, and also with pH if the light-
absorbing species is in equilibrium with an ionization state
that has different absorbance properties.
Cysteine can form disulfide bonds
FIGURE 3-7Reversible formation of a disulfide bond by the oxidation of two molecules of
cysteine. Disulfide bonds between Cys residues stabilize the structures of many proteins.
You should memorize
- One letter codes
- Three letter codes
- Chemical structure of 20 amino acids
Uncommon Amino Acids in Proteins
Not incorporated by ribosomes
except for Selenocysteine
Arise by post-translational modifications of proteins
Reversible modifications, especially phosphorylation, are important in regulation and signaling
Modified Amino Acids Found in Proteins
Reversible Modifications of Amino Acids
Ionization of Amino Acids
At acidic pH, the carboxyl group is protonated and the amino acid is in the cationic form.
At neutral pH, the carboxyl group is deprotonated but the amino group is protonated. The net charge is zero; such ions are called Zwitterions.
At alkaline pH, the amino group is neutral NH2 and the amino acid is in the anionic form.
Zwitterion: a dipolar ion with spatially separated positive and
negative charges.
FIGURE 3-9 Nonionic and zwitterionic forms of amino acids.
Cation Zwitterion Anion
FIGURE 3-10 Titration of an amino acid.
Chemical Environment Affects pKa Values
-carboxy group is much more acidic than in carboxylic acids-amino group is slightly less basic than in amines
Amino acids can act as buffers
Amino acids with uncharged side chains, such as glycine, have two pKa values:
The pKa of the -carboxyl group is 2.34
The pKa of the -amino group is 9.6
It can act as a buffer in two pH regimes.
Buffer Regions
Amino acids carry a net charge of zero at a specific pH (the pI)
Zwitterions predominate at pH values between the pKa values of the amino and carboxyl groups
For amino acids without ionizable side chains, the Isoelectric Point(equivalence point, pI) is
At this point, the net charge is zero
AA does not migrate in electric field
2
21 pKpKpI
Ionizable side chains can show up in titration curves
Ionizable side chains can be also titrated
Titration curves are now more complex
pKa values are discernable if two pKa values are more than two pH units apart
How to Calculate the pI When the Side Chain is Ionizable
Identify species that carries a net zero charge
Identify pKa value that defines the acid strength of this
zwitterion: (pK2)
Identify pKa value that defines the base strength of this
zwitterion: (pK1)
Take the average of these two pKa values
What is the pI of histidine?
Formation of Peptides
Peptides are small condensation products of amino acids
They are small compared to proteins (Mw < 10 kDa)
Peptide ends are not the same
Numbering (and naming) starts from the amino terminus
AA1 AA2 AA3 AA4 AA5
Naming peptides: start at the N-terminus
Using full amino acid names
Serylglycyltyrosylalanylleucine
Using the three-letter code abbreviation
Ser-Gly-Tyr-Ala-Leu
For longer peptides (like proteins) the one-letter code can be used
SGYAL
Sample problem
Draw the chemical structure of the following peptide at pH 7.
CHEMIST
11. Net Electric Charge of Peptides A peptide has the sequence
GluHisTrpSerGlyLeuArgProGly
(a)What is the net charge of the molecule at pH 3, 8, and 11? (Use
pKa values for side chains and terminal amino and carboxyl groups as
given in Table 31.)
(b) Estimate the pI for this peptide.
p.111
Peptides: A Variety of Functions
Hormones and pheromones insulin (think sugar)
oxytocin (think childbirth)
sex-peptide (think fruit fly mating)
Neuropeptides substance P (pain mediator)
Antibiotics polymyxin B (for Gram bacteria)
bacitracin (for Gram + bacteria)
Protection, e.g., toxins amanitin (mushrooms)
conotoxin (cone snails)
chlorotoxin (scorpions)
Proteins are:
Polypeptides (covalently linked -amino acids) + possibly:
cofactors functional non-amino acid component metal ions or organic molecules
coenzymes organic cofactors NAD+ in lactate dehydrogenase
prosthetic groupscovalently attached cofactors heme in myoglobin
other modifications
Polypeptide size and number varies greatly in proteins
Classes of Conjugated Proteins
What to Study about Peptides and Proteins
What is its sequence and composition?
What is its three-dimensional structure?
How does it find its native fold?
How does it achieve its biochemical role?
How is its function regulated?
How does it interacts with other macromolecules?
How is it related to other proteins?
Where is it localized within the cell?
What are its physico-chemical properties?
A mixture of proteins can be separated
Separation relies on differences in physical and chemical properties
Charge
Size
Affinity for a ligand
Solubility
Hydrophobicity
Thermal stability
Chromatography is commonly used for preparative separation
Protein separation
Crude extract
released proteins into a solution after breaking open the cells
Separate proteins into different fractions
(old fashion) Protein solubility: pH, temperature, salt concentration
(now) column chromatography
Column Chromatography
Separation by Charge
5. Separation of Amino Acids by Ion-Exchange Chromatography Mixtures of
amino acids can be analyzed by first separating the mixture into its components
through ion-exchange chromatography. Amino acids placed on a cation-exchange
resin (see Fig. 317a) containing sulfonate (-SO3- ) groups flow down the column
at different rates because of two factors that influence their movement: (1) ionic
attraction between the sulfonate residues on the column and positively charged
functional groups on the amino acids, and (2) hydrophobic interactions between
amino acid side chains and the strongly hydrophobic backbone of the polystyrene
resin. For each pair of amino acids listed, determine which will be eluted first from
an ion-exchange column by a pH 7.0 buffer.
(a) Asp and Lys
(b) Arg and Met
(c) Glu and Val
(d) Gly and Leu
(e) Ser and Ala
p.111
Separation by Size
Separation by Affinity
Modern chromatographic methods employing HPLC
High pressure pump, higher quality chromatographic materials
=> Reducing the transit time on the column, limit diffusional spreading
Electrophoresis for Protein Analysis
Separation in analytical scale is commonly done by electrophoresis
Electric field pulls proteins according to their charge
Gel matrix hinders mobility of proteins according to their size and shape
SDS PAGE: Molecular Weight
SDS sodium dodecyl sulfate a detergent
SDS micelles bind to and unfold all the proteins
SDS gives all proteins an uniformly negative charge
The native shape of proteins does not matter
Rate of movement will only depend on size: small proteins will move faster
SDS-PAGE can be used to calculate the molecular weight of a protein
10. Subunit Composition of a Protein A protein has a molecular
mass of 400 kDa when measured by gel filtration. When subjected
to gel electrophoresis in the presence of sodium dodecyl sulfate
(SDS), the protein gives three bands with molecular masses of 180,
160, and 60 kDa. When electrophoresis is carried out in the
presence of SDS and dithiothreitol, three bands are again formed,
this time with molecular masses of 160, 90, and 60 kDa. Determine
the subunit composition of the protein.
p. 111
Answer The protein has four subunits, with molecular masses of
160, 90, 90, and 60 kDa. The
two 90 kDa subunits (possibly identical) are linked by one or
more disulfide bonds.
Isoelectric focusing can be used to determine the pI of a protein
Isoelectric focusing and SDS-PAGE are combined in 2D electrophoresis
Quantification of unseparated proteins
Activity: total units of enzymes in a solution
Specific activity: the number of enzyme units per milligram of
total protein
Specific activity (activity/total protein) can be used to assess protein purity
Levels of structure in proteins
-Primary structure
: a description of all covalent bonding (peptide bonding, -S-S-)
: sequence of amino acid residues
-Secondary structure
: particular structural arrangements of amino acids giving rise to
recurring structural patterns
-Tertiary structure
: 3D folding of a polypeptide chain
- Quaternary structure
: arrangement in space of 2 or more polypeptide subunits
FIGURE 3-23 Levels of structure in proteins.
The function of a protein depends on its amino acid sequence
Each type of protein has a unique amino acid sequence.
Amino acid sequence determines 3D structure and ultimately
its function.
If primary structure alters, the function may also be
changed.
Functionally similar proteins from different species often
have similar amino acid sequence.
But, 20-30% of the proteins in humans are polymorphic.
The amino acid sequences of millions of proteins have been determined
Determination of amino acid sequence from polypeptide
chain is difficult
Amino acid sequence in protein is related to nucleotide
sequence
DNA sequence => decoding genetic code => protein amino
acid sequence
Protein Sequencing
It is essential to further biochemical analysis that we know the sequence of the protein we are studying
Actual sequence generally determined from DNA sequence
Edman Degradation (Classical method)
Successive rounds of N-terminal modification, cleavage, and identification
Can be used to identify protein with known sequence
Mass Spectrometry (Modern method)
MALDI MS and ESI MS can precisely identify the mass of a peptide, and thus the amino acid sequence
Can be used to determine post-translational modifications
Edmans Degradation
FIGURE 3-27 Cleaving proteins and sequencing and ordering the
peptide fragments.
Amino acids sequences can also be deduced by other methods
Mass spectrometry
Analytes
Ionization in vacuum: generating charged analytes
apply them into an electric/magnetic field
paths through the field are a function of mass-to-charge ratio (m/z)
Matrix-assisted laser desorption/ionization mass spectrometry
(MALDI MS)
Light absorbing matrix: laser light
protein ionization and desorption into vacuum
Electrospray ionization mass spectrometry
(ESI MS)
Analytes pass through a charged needle kept at high electric potential
dispersing the solution into a find mist of charged microdroplets.
Electrospray mass spectrometry of a protein.
A protein solution is dispersed into highly charged droplets by passage
through a needle under the influence of a high-voltage electric field. The
droplets evaporate, and the ions (with added protons in this case) enter
the mass spectrometer for m/z measurement.
Electrospray mass spectrometry of a protein
The spectrum generated (b) is a family of peaks, with each successive
peak (from right to left) corresponding to a charged species increased by
1 in both mass and charge. Inset: a computer-generated transformation of
this spectrum.
Tandem MS (MS/MS)
To sequence short polypeptide
peptide separation by first MS
fragmentation by high-energy impact with collision gas
b-type ion: charge retained on N-terminal side
y-type ion: charge retained on C-terminal side
measure m/z ratio by second MS
BOX 3-2 FIGURE 2a Obtaining protein sequence information
with tandem MS.
Obtaining protein sequence information with tandem MS.
Protein Sequences as Clues to Evolutionary Relationships
Sequences of homologous proteins from a wide range of species can be aligned and analyzed for differences
Differences indicate evolutionary divergences
Analysis of multiple protein families can indicate evolutionary relationships between organisms, ultimately the history of life on Earth
Chapter 3: Summary
In this chapter, we learned about:
The many biological functions of peptides and proteins
The structures and names of amino acids found in proteins
The ionization properties of amino acids and peptides
The methods for separation and analysis of proteins
Quiz: 3/23 (Mon)
Chap 2 and 3