chapter 5: primary structure of protein

Amino acids are the building blocks of proteins

• Two amino acids can react with the loss of a water molecule to form a
  covalent bond; the bond joining the two amino acids is called a peptide bond

• Peptides and proteins are unbranched polymers of amino acids linked
  together in a particular sequence (head to tail)

The primary structure of a protein: amino acid sequence

• The primary sequence is encoded in the nucleotide sequence of DNA

• With 20 natural amino acids, the number of possible distinct primary sequences is enormous

• 20^n possibilities for a sequence with n amino acids

• Peptide sequences are written from the N-terminus to the C- terminus (directionality)

Peptides are shorter and proteins are longer polymers of amino acids

The nature of the aa sequence reflects protein's function

the lighter the aa = more frequency

Conjugated proteins contain modified amino acids

• The proteins that consist of normal amino acids only for their biological
  function: simple proteins

• The proteins that utilize accessory molecules or chemical groups to carry out
  their function: conjugated proteins

• These extra molecules can be covalently or non-covalently attached to the
  protein (post-translational modifications)

• If the non-protein part is essential to the protein's function, it is referred to as a
  prosthetic group

Proteins can be grouped into families based on aa sequence

• Proteins that show sequence and structural similarity are homologous

• Proteins performing the same task in the cell are also homologous

• Homologous proteins can be classified as orthologous or paralogous

• Orthologous: proteins from different species with similar sequence and function (common ancestor, for example, hemoglobin in human and cow)

• Paralogous: proteins from the same species with similar sequence (gene duplication, example: alpha and beta subunits of hemoglobin)

Cytochrome c is an example of a conserved protein

• Cytochrome c: the electron transport protein found in the mitochondria of all
  eukaryotic organisms.

• Varies between 103 and 112 amino acids in different species

• Alignment of the amino acid sequences of cytochrome c from 40 different species revealed that 28 positions in the polypeptide chain were IDENTICAL amongst all species

• the variation in amino acids between cytochrome c of different species
  increases as the phylogenetic difference increases

Related proteins share an evolutionary origin

• If two proteins have a similar sequence, it is likely that they came from a common
  origin and may have a related biological function

Mutations in protein sequence can cause fetal diseases

• Mutations in the genes that code for proteins give rise to divergent evolution
  o the same common ancestor evolves and accumulates differences

salting in & salting out

how pH & ions affect protein charge

• Salting in: a low concentration of metal salt generally
  increases protein solubility

• Salting out: when the protein is no longer soluble in the high-salt solution, it
  precipitates

• Proteins are most insoluble at their isoelectric point

Dialysis of proteins

Dialysis allows small molecules and ions to pass through a semipermeable membrane

• macromolecular solution is placed in a semipermeable membrane bag, then immersed in a bathing solution.

• Diffusible solutes in the dialysis bag equilibrate across the membrane.

Chromatographic techniques of protein purification

It takes advantage of relative differences in the physical and chemical characteristics of an amino acid, peptide, or protein

• The molecule of interest flows through a medium with two phases (solid-liquid, liquid-liquid, or gas-liquid) and partitions between them based on its biological and chemical properties

types:

❑ Ion-exchange chromatography: positively or negatively charged molecules.

• depends on the net charge of a protein

• minimally soluble at PI values

• When pH > pI, the protein's overall charge is negative

• When the pH < pI, the overall charge on the protein will be positive

• cation exchange resin (—), so + (cation) can bind

• anion exchange resin (+), so — (anion) can bind

retention: how well/long it stays in column

elution: how slow/quick it goes through column

❑ Hydrophobic interaction chromatography: polarity or hydrophobicity of molecules.
❑ Affinity chromatography: differential affinity of one molecule for other molecules.
❑ Gel filtration, permeation or size exclusion chromatography: molecular size of the molecules.

Gel filtration or size exclusion chromatography

Proteins are separated based on their size

• Column media are composed of beads with varying pore sizes

• The number of times a protein is trapped in the pores will depend on the size of the protein relative to the pores

• Larger molecules are excluded from the gel beads and emerge from the column
  sooner than smaller molecules, whose migration is retarded because they can
  enter the beads

Affinity chromatography

Affinity chromatography is based on the ability of the protein to bind to a ligand.

• Columns are therefore designed to bind the protein being purified specifically

• The protein of interest is then passed through the column, which binds to the target ligands, while other proteins pass through without binding.

• Adding high concentrations of the free ligand or another buffer that dissociates the protein from the ligand can elute the protein.

Protein analysis by SDS-PAGE

The SDS molecules disrupt non-covalent interactions that stabilize the tertiary (or quaternary) structure and denature the protein

• SDS wraps itself around the protein backbone → proteins become rod-like

• Larger proteins bind more SDS than smaller proteins

• The distance of migration is inversely proportional to the size of proteins

• An electric field is applied to the gel to separate proteins

• Negatively charged molecules will go to the positive anode

cations go to cathode

anions go to anode

ion exchange resin stuff

diethylaminomethyl ion exchange = anion exchange resin

carboxymethyl cellulose ion exchange = cation exchange resin

Measurement of peptide and protein concentration

The extinction coefficient for a protein at 280 nm can be calculated as:
𝜺(total) = 5690 × (number of tryptophanes) + 1280 × (number of tyrosines)

Mass Spectrometry (MS) helps analyze peptides and proteins

Mass spectrometers exploit differences in the mass-to-charge ratio (m/z) of ionized atoms or molecules to separate them from each other

• Evaporate and ionize the molecules in a vacuum, separate the ions in space and/or time based on their m/z ratio, measure the number of ions with specific
  m/z ratios

Analyzing peptides and proteins by sequencing methods

Proteins can be sequenced in two ways:

• direct amino acid sequencing

• sequencing the corresponding DNA in the gene

The biochemical strategy for determining the amino acid sequence of a protein involves six steps

1. Separation of the polypeptide chains (if a heteromultimer)

• denaturing

2. Cleavage of disulfide bridges

• Reduction with mercaptoethanol (BME) or dithiothreitol (DTT)

• Followed by treatment with alkylating agents that modify the –SH groups to prevent recombination of disulfides

3. Analysis of N

• Chromatographic techniques are used to identify the PTH-derivative

• phenyl isothiocyanate identifies N terminus

4. Analysis of C terminus

• It involves a series of enzymatic analyses

• Carboxypeptidases cleave the amino acid residues from the C-termini of polypeptides

• The C-terminal residue is removed enzymatically with the carboxypeptidase first, and then reacted with phenyl isothiocyanate to
  produce the PTH derivative (N terminus)

• Iodoacetate is an alkylating agent that reacts with cysteine residues

5. Fragmentation of the polypeptide chain

• Enzymatic fragmentation or chemical fragmentation

• Trypsin cleaves on the carboxy side of Arg and Lys

• Chymotrypsin cleaves on the carboxy side of Phe, Tyr, and Trp

• Generates a set of peptide fragments having either Arg or Lys at their C-termini

• Cyanogen bromide (CNBr) reacts with methionine residues and cleaves
  the peptide bond between the Met and the next amino acid

6. Reconstruction of the amino acid sequence

• Compare the sequences of fragments obtained from two or more cleavage procedures

• the goal here is to find overlapping sequences and align peptides from different
  fragmentations to reveal the overall amino acid sequence

Strength of protein-ligand interactions: dissociation constant (KD)

• Smaller KD indicates better affinity of ligand for protein (typical range – 10-3 M to 10-12 M)

• KD is equal to the concentration of L when half the protein is bound to L