Recommended problems from the end of chapter 4: 3,4,6,7,8,12,13,15,19.
For problem 13, use [protein]=50 µM, not mM, and the extinction coefficient below. Answer is 6 tyrosine/protein
Also work on this: A. A protein contains a single tryptophan and no tyrosine residues. The extinction coefficient of trp at 280 nm is 5690 M-1 cm-1, and the absorbance is 1.2 in a 1 cm cuvette, what is the concentration (in µM) of a homogeneous protein solution?
B. A different protein contains 9 tyrosine and no tryptophan residues. The absorbance of a homogeneous solution at 280 nm is 0.6 in a 1 cm cuvette, and the extinction coefficient of tyrosine at 280 nm is 1280 M-1cm-1. What is the concentration of the protein?
Amino Acids
Amino acids are the building blocks of proteins,
OR,
proteins are polymers of amino acids.
1 General properties of amino acids
pKR
pK2 pK1
•There are 20 R groups that occur in the vast majority of proteins •Amino acids have characteristic pK1, pK2, and pKR. •pK 1 are around 2.2, so at physiological pH the carboxyl groups are deprotonated. •pK 2 are around 9.4, so at physiological pH the amino groups are protonated. •Therefore, an amino acid can act as an acid or base, depending on the pH of the solution - substances with this property are called AMPHOLYTES. •Amino acids also are ZWITTERION or DIPOLAR as they carry ions of opposite charges at physiological pH. They have characteristics of ionic compounds - high melting temperatures, they readily dissolve in water and other polar solvents, but not in organic solvents.
Peptide bond formation - a condensation reaction catalyzed by the ribosome
R1 R2
Water is eliminated
R1 R2
2 20 amino acids can generate a large variety of proteins
In principle, the 20 amino acids can generate 202 = 400 dipeptides 203 = 8000 tripeptides 20100 = 1.27 x 10130 different proteins of 100 amino acids
Most combinations are not made (the number of atoms in the universe is estimated to be on the order of 1079).
Some amino acids and amino acid derivatives do not occur in proteins but are biologically important, e.g., epinephrine, serotonin, histamine.
see amino acids worksheet
3 Amino acids are weak electrolytes
All amino acids have at least 2 dissociable protons, and they behave as weak acids in aqueous solutions.
pKR
pK2 pK1
Amino acids are weak electrolytes
Example: titration of glutamic acid with NaOH
4 Amino acids are weak electrolytes
Example: titration of lysine with NaOH
The isoelectric point of amino acids
•The pH at which a molecule carries no net charge is called the pI. This also is called the isoelectric point. •This property is relevant for predicting the charge of an amino acid at a particular pH. •For amino acids it generally holds that: pK + pK pI = i j 2
where pKi and pKj are pK1 and pK2 for amino acids that do not have an ionizable side chain. € For amino acids that have an ionizable side chain, one of these values can be pKR, depending on the amino acid. To determine which pKa values to use, identify the ionizable groups on the amino acid, and then the overall protonated states that give an overall charge of 0 (see previous slides). Then, the mean of the two flanking pKa values is the pI. For example, for Lysine the mean of pK2 and pKR gives the pI.
5 Spectrophotometry of amino acids All amino acids absorb infrared light. The aromatic amino acids absorb ultraviolet light: phenylalanine, tyrosine, and tryptophan. Beer’s law can be used to quantify the concentrations of protein solutions.
A = ε × c × l
where A is the absorbance, ε is the extinction coefficient (M-1cm-1), € c is the concentration (M), and l is the light path length (cm).
Usually, the absorbance at 280 nm is taken, and the contribution of tryptophans and tyrosines is calculated. You must know how many trp and tyr residues are in the protein to calculate the protein concentration.
Fluorescence spectroscopy of amino acids
Tryptophan fluoresces, absorbing at 280 nm and emitting fluorescent light at ~340 nm. This can be very useful for studying protein interactions with other biomolecules.
Tryptophan fluorescence may be quenched by an interacting molecule, e.g., a nucleic acid or another protein. Monitoring the quenching of steady-state fluorescence can yield the equilibrium constants for the formation/dissociation of the complex. Time-resolve data can yield the on-and-off-rates of a protein-nucleic acid complex formation.
6 Stereochemistry of amino acids
C is the chiral center The two non-imposable molecules are enantiomers.
These molecules can rotate about the plane of polarization of plane- polarized light. Unless probed by asymmetric molecules or plane- polarized light, these molecules are chemically and physically indistinguishable.
In the fisher projections, horizontal bonds point above the page, and vertical bonds point below the page. Note that the central C is sometimes omitted in the fisher projection.
Stereochemistry of amino acids
The α carbon is the chiral center of every amino acid except glycine.
7 Stereochemistry of amino acids
If optically-active molecules rotate clockwise about the plane of polarized light, they receive the prefix (+) If optically-active molecules rotate counter-clockwise about the plane of polarized light, they receive the prefix (-). The specific rotation of a sample is related to the concentration and given by
rotation (o) [α]25 = D optical path length (dm)×concentration (g•cm−3) where the superscript 25 refers to the temperature in °C and the subscript D refers to the monochromatic light that is usually employed in polarimetry. € 25 If [α]D > 0, the molecule rotates in the D (dextrorotatory) direction and is designated the prefix (+)
€ 25 If [α]D < 0, the molecule rotates in the L (levorotatory) direction and is designated the prefix (-) €
Stereochemistry of amino acids
The fisher convention for glyceraldehydes (the D and L system) can be related to protein- derived amino acids. It can be shown that all protein-derived amino acids are of the L configuration.
8 Stereochemistry of amino acids •The stereochemistry of amino acids is not terribly useful because the value of α cannot be predicted on the basis of structural information, especially for molecules containing multiple chiral centers (for example, threonine) •Another nomenclature system you may encounter is the S and R (by Cahn, Ingold, and Prelog). The 4 groups surrounding the chiral center are ranked according to the arbitrary priority system: atoms of higher atomic number are ranked above those of lower atomic number. •If any of the first atoms are of the same element, the priority of their groups is established by the second, third, etc. atom out from the chiral center. For example, an OH group is ranked higher than an CH4 group because the oxygen has a higher atomic number. A CH2OH group is ranked higher than a CH3 group because the second atom on the CH2OH group is oxygen, which is ranked higher than hydrogen. •The order for some common functional groups is
SH > OH > NH2 > COOH > CH2OH >CHO > C6H5 > CH3 > H. •The prioritized groups are assigned the letters W > X > Y > Z. •The configuration of the chiral center is established by viewing from the asymmetric center towards the Z group (the lowest priority). If the order of the groups WXY is clockwise, the configuration of the center is R. If the order of the groups WXY is counterclockwise, the configuration of the center is S.
The point of all this is that all L-amino acids are (S)-amino acids, except L-cysteine is (R)-cysteine.
Stereochemistry of amino acids
Other terms you may encounter: Meso-compounds do not have a optical activity because they have an internal mirror symmetry. Racemic mixtures (or solutions) of molecules do not exhibit optical activity because there are equal numbers of each enantiomeric pair.
In biological systems, there are distinct preferences for particular stereoisomers, e.g., the L-amino acids and the D-aldoses. The findings of non-racemic mixtures of organic compounds, therefore, may be indicative of a biosynthetic pathway. Do optically active mixtures in extraterrestrial samples (meteorites, for example) indicate that their origin was biological?
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