Key Concepts Properties of the 20 amino acids that occur in peptides and proteins

are crucial to the structure and function of proteins.o stereochemistryo relative hydrophobicity or polarityo hydrogen bonding propertieso ionization propertieso other chemical properties

Condensation of 2 amino acids forms the peptide bond, the amide linkage holding amino acid residues in peptide and protein polymers.

Properties of the peptide bond have major consequences in terms of the 3-dimensional structures of proteins

There's an excellent website on amino acids being developed here in the Department of Biochemistry and Molecular Biophysics; parts of it are still under construction, but there are links to various very useful parts of it here in these notes, and indeed parts of it may be used in class.

BASICS

Proteins are polymers of -amino acids:  There are 20 different amino acids found in proteins and they differ by

the nature of the R group. 

Both the -amino group (amino group substituent on the C) and the -carboxyl group (carboxyl substituent on the C) are ionizable.

o -COOH group:  a weak acid, can DONATE its proton, with a pKa of about 2-3. What's the conjugate base form of the carboxyl group? Which form is charged, and is it a positive or a negative charge?

o -NH2 group:  a weak base (there's an unshared pair of electrons on the N; the neutral amino group can ACCEPT a proton). What's the conjugate acid form of the amino group? Which form is charged, and is it a positive or a negative charge?

o pKas of -amino and -carboxyl groups are different for different amino acids, and also are altered if they're the terminal groups on a chain of amino acids, i.e., a peptide or protein.

The nonionic form shown above does NOT occur in water. WHY NOT?

Predominant form in H2O is the zwitterion:   .  

Stereochemistry of the amino acids -carbon is asymmetric (has four different substituents) except for one

amino acid, for which the R group is a hydrogen atom. amino acids occur as enantiomers (nonsuperimposable complete

mirror images) L-amino acids are the naturally occurring enantiomers found in all

proteins There are naturally occurring D-amino acids, but not in proteins (found

in some bacterial cell wall peptide structures, in some peptide antibiotics, etc.) (D_L)

Perspective formulas show stereochemistry; projection formulas CAN be written "correctly", with convention that horizontal bonds project out of paper and vertical bonds behind plane of paper, but often biochemists use projection formulas casually (inaccurately), knowing that if it's in a protein, it's always an L-amino acid. (See also Fig. 5-3 in Lehninger Principles.)

Fig. 5-4 (Nelson & Cox: Lehninger Principles of Biochemistry): Absolute configurations of D-glyceraldehyde as the reference compound for -amino acids.  D- and L- apply only to the absolute configuration around the chiral carbon; 2 of the 20 amino acids (threonine and isoleucine) have a second chiral center, requiring the RS system to describe their structures accurately, but we aren't going to worry about using the RS system here.

 

Which of the amino acids does NOT have a chiral center, so has no D/L isomers?

Amino Acid Abbreviationsamino acid (or

residue in protein)3-letter

abbreviation1-letter

abbreviationMnemonic for 1-

letter abbreviationGlycine Gly G GlycineAlanine Ala A AlanineValine Val V Valine

Leucine Leu L Leucine

Isoleucine Ile I IsoleucineProline Pro P Proline

Methionine Met M MethioninePhenylalanine Phe F Fenylalanine

Tryptophan Trp WtWyptophan (or tWo

rings)Tyrosine Tyr Y tYrosine

Serine Ser S SerineThreonine Thr T ThreonineCysteine Cys C Cysteine

Aspartic Acid Asp** D asparDic acidGlutamic Acid Glu* E gluEtamic acid

Asparagine Asn** N asparagiNeGlutamine Gln* Q Q-tamineHistidine His H Histidine

Lysine Lys K (before L)Arginine Arg R aRginine

* Glx = either acid or amide (when it isn't known which it is)**Asx = either acid or amide (when it isn't known which it is)

Properties of Amino Acid Side Chains

Side chains ("R groups") provide proteins with unique structural and functional properties.Additional C atoms in R groups (after the C) designated by successive Greek

letters: as shown in the structure of the amino acid LYSINE (Nelson & Cox: Lehninger Principles of Biochemistry, 3rd ed., p. 116):

 

Side chain classes

The side chains of the amino acids play an essential role in determining the properties of proteins. 

There is a wide diversity in the chemical properties of amino acid side chains, but they can be grouped into classes, sometimes with overlapping "membership" (e.g., tyrosine is both aromatic and hydroxyl-containing). Other classifications are also possible (for example, the 5 classes in textbook, Fig. 5-5, discussed below).  You are expected to know all 20 amino acid structures and their R group properties, including ionization properties (see table below with "generic" pKa values for groups in peptides and proteins and links to titration curves, and the PDF of proton dissociation reactions).

Side Chain Class Amino Acids

Aliphaticglycine, alanine, valine, leucine, isoleucine

Cyclic proline

Aromaticphenylalanine, tyrosine, tryptophan

Hydroxyl-Containing serine, threonine, tyrosine

Sulfur-Containing cysteine, methionine

Basic histidine, lysine, arginine

Acidic and Their Amidesaspartic acid, glutamic acid, asparagine, glutamine

Structures and classification below are from Nelson & Cox: Lehninger Principles of Biochemistry, 3rd ed., Fig. 5-5. States of ionization are the PREDOMINANT forms found at pH 7.

Nonpolar, aliphatic R groups o Gly: quite water-soluble (as is Pro) o Ala, Val , Leu and Ile: increasing hydrophobicity with

increasing number of C atoms in hydrocarbon chain

o Pro: cyclic (--> unusual properties) shares many properties with the aliphatic group rigidity of ring plays critical role in protein structure (more

about that later) o Met: methyl thioether (S-containing)

quite hydrophobic Met's terminal methyl group important in metabolism

Aromatic R groups o Phe: phenyl group (linked to -CH2, so Phe = alanine with a

phenyl substituent on the methylene C) VERY hydrophobic.

o Trp: indole functional group on C electronegative atom in ring system not as hydrophobic as Phe hydrogen bonding capability (donor? acceptor? how

many hydrogen bonds?) o Tyr: phenylalanine with aromatic OH group (phenolic OH) =

p-hydroxyphenylalanine ionizable (pKa around 10; loss of proton gives phenolate

anion)

hydrogen bonding capability (donor? acceptor? how many hydrogen bonds?)

Tyr R group is the least hydrophobic of the 3 aromatic amino acid side chains.

Polar, uncharged R groups o Ser and Thr : aliphatic OH groups, not ionizable in pH range

1-13 pKa values so high that under any biologically reasonable

pH conditions they're polar but not ionizable. hydrogen bonding capability (donor? acceptor? how

many hydrogen bonds?) o Asn and Gln : amide functional groups

VERY polar, but NOT ionizable hydrogen bonding capability (donor? acceptor? how

many hydrogen bonds?) o Cys : thiol (also called a sulfhydryl group) -- not very polar, and

IS ionizable sulfur atom makes protonated -SH group more

hydrophobic than an aliphatic OH group thiol DOES lose its proton in physiologically relevant pH

range (pKa about 8.5) generates -S- (thiolate anion is quite hydrophilic due to the

charge).

Positively charged R groups (sometimes called "basic" R groups) o Arg: guanidino group

VERY high pKa (~12+), so a very weak acid (stronger base)

carries + charge all across physiological pH range resonance forms of guanidino group stabilize protonated

form (charge is delocalized) hydrogen bonding capability (donor? acceptor? how

many hydrogen bonds?) o Lys: -amino group (a primary amine)

pKa about 10 protonated form (predominates at physiological pH)

carries + charge hydrogen bonding capability (donor? acceptor? how

many hydrogen bonds?) o His: imidazole functional group (has 2 N atoms in 5-membered

unsaturated ring) pKa about 6-6.5

protonated form carries + charge, but at pH 7 predominant form is neutral (despite textbook's categorization as "positively charged")

very important player in catalytic activity of many enzymes hydrogen bonding capability, and also proton

donor/acceptor

Negatively charged R groups (sometimes called "acidic" R groups) o Asp and Glu: side chain carboxyl groups

pKa values around 4 predominant form at physiological pH = carboxylate

anion hydrogen bonding capability (donor? acceptor? how

many hydrogen bonds?)

Relative hydrophobicity/hydrophilicity of amino acid R groups

Table 12.2 (Berg, Timoczko and Stryer, Biochemistry, 5th ed.): Polarity scale for amino acid residues based on free energy changes for moving a residue from a hydrophobic environment (dielectric constant = 2) into H2O.

Similar trends for relative hydrophobicities in text Table 5-1 (diff. numerical scale, and not arranged in order of relative polarity)

Depending on how transfer experiments are done, different absolute numbers can be obtained, but the general trends of relative polarity are clear

o Phe, Met, Ile, Leu, Val are very hydrophobic o Arg, Asp, Lys, Glu, Asn, Gln, and His are quite hydrophilic o The rest are in between -- neither very polar nor very hydrophobic

Fig. 5-7: Reversible oxidation of 2 cysteine side chain thiols to form cystine, or re-reduction to 2 thiols

o disulfide bonds between 2 Cys residues in a (usually extracellular) protein

o often a critical structural feature in extracellular proteins (stabilize folded structures, in interior of protein structure)

o When found in intracellular proteins, usually have a functional role.

Ionization Properties of Amino Acid Functional Groups (in PEPTIDES AND PROTEINS)

weak conjugate acid/base groups in peptides and proteins crucial to functions

o only one -amino and one -carboxyl group on a peptide or proteins (at the termini of the chain) because the rest of the -amino and -carboxyl groups are tied up in amide bonds holding monomers together in polymer (more later)

o side chain ionizable groups (only 7 of the 20 amino acids) PDF of the acid dissociation reactions for functional groups of amino

acid residues in peptides and proteins ionization states of side chain weak acid groups control charges on

protein Note: local environment in peptide or protein determines actual pKa of

that specific group, so the ranges shown below (and the rather arbitrary "generic" values, rounded off for simplicity) are only the usual expected ranges for pKa values for the functional groups in peptides and proteins; the pKa of a specific group in a specific protein can lie significantly outside the expected range if the local environment is unusual.

links in table below are to titration curves for that amino acid or functional group

Groupusual pKa range, in peptides & proteins

(approx."generic"pKa )

-Carboxyl (terminal group of peptide or protein)

~3.0 - 4.0 (generic 3.0)

Asp, Glu (side chain carboxyl) ~4.0 - 4.5 (generic 4.0)

His (imidazole) ~6.0 - 7.4 (generic 6.5)

Cys (thiol, SH) ~8.5 - 9.0 (generic 8.5)

Tyr (phenolic OH) ~9.5 - 10.5 (generic 10.0)

-Amino (terminal group of peptide or protein)

~8.0 - 9.0 (generic 8.0)

Lys (-amino) ~9.8 - 10.4 (generic 10.0)

Arg (guanidino) ~12.0 - 12.5 (generic 12.0)

 

Isoelectric point (pI)

pI = "isoelectric pH" = "isoelectric point" = pH at which the NET charge on a molecule is ZERO. 

o If pH < pI, net charge is positive (more + than - charges) o If pH > pI, net charge is negative (more - than + charges)

pI = the pH exactly halfway between the two pKa values surrounding the zero net charge equivalence point on the titration curve (examples to be analyzed in class: Gly and His)

Fig. 5-10. Titration curve of glycine (Nelson & Cox: Lehninger Principles of Biochemistry, 3rd ed.)

Fig. 5-12b. Titration curve of histidine (Nelson & Cox: Lehninger Principles of Biochemistry, 3rd ed.)

Molecular separations based on charge properties (paper electrophoresis of amino acids as an example)

paper strip soaked in buffer, in contact with 2 reservoirs with electrodes connected to a power supply

Buffer reservoir #1

+(anode;

anions move toward it)

O

Buffer reservoir #2

_

(cathode; cations move

toward it)

^origin (sample of an amino acid applied)

When a voltage is applied, in which direction will the amino acid move? What do you need to know to answer that question?

o ________________________ o ________________________

e.g., Histidine: (sample dissolved in buffer, applied at origin above, and voltage applied)

buffer pH net charge direction of migration

1

3.9

7.6>

11

Ultraviolet absorbance of amino acid side chains

Aromatic amino acids (Trp, Tyr, Phe) absorb light in the near ultraviolet region of the spectrum (250-300 nm). 

Trp has highest molar absorptivity, followed by Tyr, with Phe making only a small contribution.

Disulfide bonds (between Cys residues in proteins) also absorb in the uv range, but much less than the aromatics.

Fig. 5-6 (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed.): Absorbance of ultraviolet light by aromatic amino acids

Posttranslational modifications of amino acid side chains

chemical modifications AFTER biosynthesis of proteins occur for a few amino acid residues in some proteins Some examples (see also Fig. 5-8, Nelson & Cox: Lehninger Principles

of Biochemistry, 3rd ed.)):

O-Phosphoserine 4-

Hydroxyproline 5-Hydroxylysine

-carboxyglutamate

reversible phosphorylation and dephosphorylation of Ser, Thr, and Tyr residues very important in covalent regulation of activity of some enzymes and many biosignalling proteins, including some hormone receptors and transcription factors

4-hydroxyproline & 5-hydroxylysine important in structure of collagen (fibrous protein in connective tissue)

-carboxyglutamate important in a number of proteins whose function involves Ca2+ binding, including several proteins involved in blood clotting

Chemical Reactions of Amino Acids

All amino acids have at least two reactive groups: the amino and -carboxyl groups and these groups can react with a variety of reagents. Here are two examples:

 

A particularly interesting example is the green fluorescent protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria, which has generated intense interest as a marker for gene expression and localization of gene products.  The chromophore, which results from the spontaneous cyclization and oxidation of the sequence -Ser65-Tyr66-Gly67- , is unusual because it does not involve a non-protein chromophore, as is usually the case for colored proteins. The chromophore is buried in the interior of GFP.

The Peptide Bond

Peptides and proteins:polymers of amino acids joined bypeptide bonds amide linkages from condensation of -carboxyl group of one amino

acid with -amino group of another amino acid

process repeated many times --> linear chain of amino acids, a polypeptide chain

convention: sequence written from left to right starting with residue with free -amino group (the N-terminal or amino terminal amino acid residue) and ending with the residue containing the free -carboxyl group (the C-terminal or carboxyl terminal residue), e.g., NH2-Glu-Gly-Ala-Lys-COOH = EGAK

average residue mass ~110 (average Mr of the 20 amino acids minus Mr of H2O)

a polypeptide chain with 100 amino acid residues would have a Mr of about 11,000)

small peptides (a "few" amino acid residues) = oligopeptides

Peptide bond formation endergonic (Go' ~21 kJ/mol)

(How would a cell make the reaction go in the direction of condensation in an aqueous environment? no details needed here for biochemical mechanism -- that's covered in BIOC 411)

peptide bonds metastable in aqueous environment -- equilibrium lies far in direction of hydrolysis, but RATE of hydrolysis very slow in absence of catalyst

Enzymes that catalyze peptide bond hydrolysis = peptidases or proteases, e.g., (specific examples of proteases) your digestive proteases like trypsin and pepsin

Ionization properties of peptides

analyzed the same way as for free amino acids one -amino group (pKa approx. 8) and one -carboxyl group (pKa

approx. 3), plus any ionizable side chains on residues in the peptide To figure out approximate net charge of a peptide at a given pH:

o make yourself notes on the sequence to keep track of what you're doing

o add up charges on all the ionizable groups

Example: Fig. 5-14 (Nelson & Cox: Lehninger Principles of Biochemistry, 3rd ed.): pentapeptide SGYAL = Ser-Gly-Tyr-Ala-Leu = Serylglycyltyrosylalanylleucine

Amino Acid Analysis

Sequence of amino acids in a protein is dictated by the sequence of nucleotides in the gene encoding that protein:

(from Berg, Tymoczko & Stryer, Biochemistry, 5th ed., p. 28)

Each protein (unique sequence) has unique amino acid composition. Can chemically hydrolyze (hot 6N HCl) a pure protein to generate the

free amino acids and determine its amino acid composition chromatographically

Because side chains of the amino acids have different properties, can separate and quantitate all 20 amino acids using a variety of chromatographic techniques, as illustrated below.

 

Peptide bond has resonance structures --> partial double bond character Due to the partial double bond character of the peptide bond, the O, C, N

and H atoms are nearly planar and there is no rotation about the peptide bond (peptide).  As we shall see later, the planarity of the these elements has important consequences for the three dimensional structure of proteins. 

Generally, the two C groups are in a trans configuration, which minimizes steric interaction (cis/trans).