Topic 5d: amino acids and proteins

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The central α carbon holds the AA together.

It is bonded to 4 groups: amino group (-NH2), carboxyl group (-COOH), a hydrogen atom, and a side chain (-R).

The R group of an AA gives it unique properties, influences its size, polarity, charge, and its chemical interactions.

All 20 AAs are chiral, except for glycine, which has a H atom as its R group, which means it has 2 of the same substituents and is thereby achiral.

Although the 19 chiral amino acids have D and L enantiomers, the human body uses only L-amino acids to synthesize proteins.

18 of the 19 chiral amino acids have an S absolute configuration. The exception is L-cysteine, which has R because it is the only one with a higher priority functional group.

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Most amino acids are zwitterions because the ions cancel each other out resulting in net neutral charge UNDER PHYSIOLOGIC CONDITIONS.

The amino group is a base that is typically protonated (+ CHARGE) in the neutral cell environment (pH 7.4).

The C-terminus is a carboxylic acid, which is typically deprotonated (- CHARGE) in the cell environment.

Therefore, both groups are typically ionized under physiological conditions; the only other groups that are ionizable are the acidic and basic amino acids.

acidic residues (aspartic acid and glutamic acid) are deprotonated and have a negative charge UNDER PHYSIOLOGIC CONDITIONS

basic residues (arginine and lysine) are protonated and have a positive charge UNDER PHYSIOLOGIC CONDITIONS.

​​​​​​​histidine is an exception, because it has a basic residue but is deprotonated under physiologic conditions and therefore is neutral

When NOT under physiologic conditions:

In acidic environments, more residues will be protonated, resulting in a net positive charge.

In basic environments, more residues will be deprotonated, resulting in a net negative charge.

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Nonpolar aromatic AAs usually are in the interior of proteins, as they are hydrophobic; also, more likely to be in the hydrophobic interior of cell membranes.

Polar AAs contains atoms capable of H bonding, are very hydrophilic. Typically make up the exterior of proteins and are in the cytosol or bound to the exterior of cell membranes.

Special cases:

Glycine and proline destabilize local alpha helical structure when added into a protein

Serine, threonine, and tyrosine are targets of phosphorylation. Of note, all 3 of these have a hydroxyl functional group.

Aspartic and glutamic acid have structures similar enough to phosphate that they can mimic the presence of a phosphate group, when substituted for S, T, or Y. This is the phosphomimetic effect; when a point mutation is introduced for S, T, or Y, and they are replaced by D or E, that site on an enzyme can become permanently activated (because phosphorylation is often a way to activate enzymes).

Cysteine can produce covalent disulfide bonds with other cysteine residues, which can contribute to the tertiary structure of a protein.

Basic and acidic residues can interact to form salt bridges as a result of their attractive opposite charges; important in protein stability.

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Synthesis of amino acids

Strecker synthesis:

Ammonia (NH3) is added to an aldehyde with a variable R group, to form an imine (R-N=CH-R).

The R group on the aldehyde can be varied, allowing for a variety of AAs to be synthesized.

Cyanide ion nucleophilically attacks imine to form α-aminonitrile.

this ensures that the reaction produces a racemic mixture of products

Aminonitrile is hydrolyzed, resulting in α-amino acid.

Gabriel Synthesis:

Phthalimide salt and diethyl bromomalonate react to form a phthalimide-substituted malonic ester via an Sn2 reaction.

The substituted malonic ester has an acidic hydrogen (arising from stabilization of the anion by 2 nearby ester group).

This hydrogen is deprotonated, and the resultant anion is alkylated with a variable R-group alkyl, allowing for a variety of AAs to be synthesized.

The phthalimide undergoes base catalyzed hydrolysis.

The product is decarboxylated with heat and acid to form a chiral amino acid.

The decarboxylation is non-stereospecific, however the reaction overall still results in racemic mixture.

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Reactions of amino acids:

disulfide bridges between cysteine residues

affect protein folding and stability

these sulfur linkages form under basic (high pH) conditions and can be broken under acidic (low pH) conditions.

BME (beta mercaptoethanol) can be used to break down disulfide bonds in the lab during a western blot

2 AAs are connected by a peptide bond.

Forms when amino group of 1 AA nucleophilically attacks carboxyl group of another AA, in a dehydration synthesis (aka condensation) reaction. The final product is an amide (N-C=O).

This is an endergonic process, ATP provides activation energy.

Hydrolysis of peptide bond:

Exergonic, spontaneous reaction, however high activation energy of this reaction is high enough to delay the reaction from happening.

Enzymes in living organisms both build and break down peptide bonds

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Protein structure:

Primary: starts at N-terminus and ends at C-terminus, linear chain of AAs can be numbered starting from #1 at N terminus

Secondary: AA chain folding in a way that maximizes protein stability and entropy of the system. Small regions locally fold into beta sheets and alpha helices

α-helices:

can act as DNA-binding site

anchor larger proteins (like GPCR) to plasma membrane by acting as a transmembrane sequence.

Stabilized by H-bonds

β-sheets: several regions of a peptide chain loop back along one another to form a sheet-like structure that is held together by H bonds.

tertiary: the 3D structure that is the peptide’s optimal, most energetically favorable conformation that is its native state

depending on the environment in which folding is taking place, hydrophobic or hydrophilic residues may be in the inside or outside of the peptide (typically hydrophobic in the interior and hydrophilic exposed to match aqueous cell environment).

Internally, peptide stabilized by H bonds, ionic bonding/salt bridges, weak intermolecular forces (dipole-dipole interactions, LDFs), disulfide bridges (this is the only covalent bond able to influence tertiary structure), and hydrophobic interactions.

If this shape is affected, protein function is lost or altered unless corrected.

Quaternary: formation of protein when multiple peptide monomers join together, or when peptide subunits are linked to non-protein molecules (like co-factors or prosthetic groups).

Prosthetic groups: non-protein molecules that can be organic or inorganic, bound tightly to peptide and required for protein functionality

Organic: carbs, lipids, vitamins

Inorganic: metal ions

Examples of vernacular:

Homodimer: 2 peptide subunits that are identical

Heterotrimer: 3 peptide subunits that are different from one another

Apoprotein or apoenzyme: a protein w/o its prosthetic group, generally non-functional

Holoprotein: apoprotein coupled to its prosthetic group, generally functional

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Protein stability

Dependent on its environment, its INTER-molecular interactions, and its INTRA-molecular interactions.

Hydrophobic interactions affect stability because they lower a protein’s overall entropy (which means a lower energy conformation which is more stable) while increasing the entropy of the system around it.

This is done by the formation of a solvation layer around the protein, where the water molecules surrounding the protein (in an aqueous environment) have less interactions with hydrophobic residues and more interactions with hydrophilic residues.

That state is preferable and most stable which maximizes the entropy of the system and minimizes the protein’s intrinsic energy

Proteins spontaneously fold into low energy conformations

Chaperone proteins may be necessary during the process; ex: needed to move past the molten globule (transient 3° nonfunctional structure that the protein takes on during the process of folding) state if there is a high energetic barrier

Salinity, temperature, pH all affect folding

Ex: hydrophilic residues on outside vs inside, protonation vs deprotonation of AA residues, disruption of internal ionic interactions at high salinity (which impedes folding)

Optimal conditions vary for each protein (ex taq polymerase works at high temps)

Denaturation occurs when optimal conditions are altered; sometimes this is reversible if the factor causing alteration of conditions is corrected, as long as 1° structure is intact.

Functional ability is affected until optimal conformation is assumed.

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Isoelectric point: the pH at which the net charge on a protein = 0.

At a pH above the isoelectric point (which tends to be basic), proteins are typically negatively charged.

At a pH below the isoelectric point (which tends to be acidic), proteins are typically positively charged.

Higher isoelectric point when there are more basic residues

Lower isoelectric point when there are more acidic residues

Isoelectric focusing: a pH gradient is created on top of a membrane

One end of membrane is connected to cathode (- charge), other end to anode (+ charge)

The baseline charge of the proteins on the membrane determines which end the protein migrates to.

The protein is stationary when it reaches the location along the pH gradient that is = to its isoelectric point, because the protein is neutral at that point.

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Analysis and separation techniques:

SDS page and native page

Western blot: proteins are transferred from separation gel to blotting membrane, after which specific radioactive antibodies can be added to visualize and/or detect whether a particular protein is present on the gel.

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Proteins bind certain molecules based on their affinity and specificity.

The affinity of a molecule determines how readily it will bind to the protein, while specificity determines which molecule will bind the protein.

Non-enzymatic binding proteins:

Receptors: the affinity of a ligand to its receptor is measured by dissociation constant, Kd (the [ligand] required for 50% binding).

Lower Kd indicates stronger affinity. Helpful for when ligand is scarce

Ion channels: the aqueous pore becomes accessible to ions only after a conformational change is triggered (i.e. binding of ligand)

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Antibodies: aka immunoglobulins, bind to specific antigens or pathogens, coat extracellular pathogens, and neutralize them by blocking key sites on the pathogen.

Neutralization prevents infection of host cells as the pathogen cannot enter them.

Neutralized pathogens are then filtered by the spleen and eliminated in waste.

Antibodies with higher affinity for their antigen would be expected to be a stronger defense against the associated pathogen.

Each tip of an antibody contains a paratrope specific for 1 particular epitope on an antigen, which allows precise binding. This also allows tagging of microbes or infected cells

All antibodies have a similar baseline structures:

Y shaped molecule consisting of 4 polypeptide chains ---> 2 identical heavy chains and 2 identical light chains

The heavy and light chains are connected by disulfide bonds and are held together by beta sheets

Each chain is made of immunoglobulin domains, which have constant and variable regions

The constant region determines Ig class (A, M, D, G, and E)

The variable region is hypervariable allowing for millions of possible antibodies to exist, as antibodies are highly specific and each binds a specific antigen, and there are millions of possible antigens. This is a key feature of the adaptive immune system

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Motor proteins: molecular motors that can move along the cytoplasm of animal cells

Use ATP hydrolysis to obtain energy for conformational change that results in movement of associated biomolecules

Myosin: helps in movement of muscles

Myosin filaments are bound to actin filaments. Myosin changes shape using ATP hydrolysis which moves the actin bound to it.

Kinesin: involved in chromosome movement and vesicular movement; ANTEROGRADE

Dynein: movement of cilia and flagella RETROGRADE