CHEM 437 Chp 5

What are the critical roles of proteins in living systems?

• Structural

• Catalysts for chemical reactions (enzymes)

• Signal transduction

• Regulatory

• Mobility

• Transport

How are the roles of proteins given?

By their distinctive structures.

How are two amino acids formed?

They can react with the loss of a water molecule to form a covalent bond which is known as a peptide bond.

Describe the primary structure of a protein.

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

• Amino acids

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

• Peptide sequences are written from the N- to C-terminus.

Compare peptides and proteins.

Peptides:

• Short polymer of amino acids.

  • Dipeptide, tripeptide, oligopeptide, polypeptide.

Proteins:

• A molecule compose of one or more polypeptide chains.

  • Monomeric, multimeric proteins.

  • Homormultimer, one kind of chain.

  • Heteromultimer, two or more different types of chains.

What reflects a protein’s function?

• Sequence and composition of the amino acid reflect the function of the protein.

  • Ex: Membrane proteins have more hydrophobic residues, whereas fibrous proteins may have atypical sequences.

Simple Proteins

The proteins that consist of normal amino acids only for their biological function.

Conjugated Proteins

The proteins that utilize accessory molecules or chemical groups to carry out their function.

Prosthetic Group

If the non-protein part is essential to the protein’s function.

Describe how proteins can be grouped into families based on amino acid sequence.

• Proteins can be grouped into protein families based on similarity in their amino acid sequence.

• Proteins that show sequence and structural similarity are homologous as well as proteins performing the same task in the cell.

  • Orthologous: Proteins from different species with similar sequence and function.

  • Paralogous: Proteins from the same species with similar sequence.

What is an example of a conserved protein?

Cytochrome c, which is the electron transport protein found in the mitochondria of all eukaryotic organisms. Its variation in amino acids increases as the phylogenetic difference increases.

What is likely if two proteins have a similar sequence?

They come from a common origin, and may have a related biological function.

Describe the effects of mutations, and their extremes.

• Mutations in the genes that code for proteins give rise to divergent evolution.

  • The same common ancestor evolves and accumulates differences.

• Silent mutation and lethal mutations.

  • Ex: A single amino acid mutation in the hemoglobin-B is responsible for sickle-cell anemia.

How do you do charge calculations for peptides and proteins?

Very similar to individual amino acids, just make sure to include calculations for the N-terminus, side chain, as well as the C-terminus.

How does pI/size of proteins allow them to be purified?

• Can be separated and purified based on size and electrical charge.

  • Tend to be least soluble at their isoelectric point.

• Increase ionic strength first increases protein solubility (salting-in) and then decreases it (salting-out).

What is the breakdown of the purification and analysis of proteins?

• Production

  • Endogenous production

  • Molecular biology

  • Chemical synthesis

• Purification

  • Salting-in and out

  • Chromatography (affinity, ion exchange, hydrophobic interaction, gel filtration, gel permeation, or size exclusion)

• Analysis

  • SDS page

  • Chromatography

  • Sequencing

  • Mass spectrometry

What is salting-in and out of proteins?

• Salting-In: A low concentration of metal salt generally increases protein solubility.

• Salting-Out: High metal salt concentrations reduce protein solubility.

• Proteins are most insoluble at their isoelectric point.

• Different proteins have different solubilities and thus require different concentrations of salt (ammonium sulfate) to precipitate.

What is dialysis of proteins?

• Allows small molecules and ions to pass through a semipermeable membrane.

• The 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.

What is chromatography? What are the different types?

• 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.

  • Ion-exchange Chromatography: Positively or negatively charged molecules.

  • 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.

What is ion-exchange chromatography?

• Depends on the net charge of a protein.

• The protein’s net charge will depend on its pI and the pH of the solution in which it is.

• This considers all ionizable side chains as well as the N and C-terminals.

• When pH > pI, the charge is -ve. When pH < pI, the charge is +ve.

What is gel filtration or size exclusion chromatography?

• Proteins are separated based on their size.

• Column media are composed of bead 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 emerge sooner than smaller molecules.

What is affinity chromatography?

• Based on the ability of the protein to bind to a ligand.

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

• A small molecules is immobilized on a matrix, and when the protein of interest passes through, it binds to the target ligands, whereas other proteins pass through.

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

What is protein analysis by SDS-page?

• The SDS molecules disrupt non-covalent interactions that stabilize the tertiary structure and denature the protein.

• SDS wraps itself around the protein backbone, and proteins become rod-like.

• The proteins will have a uniform charge density because SDS binds at a constant weight ratio.

• Larger proteins bind more SDS, and the distance of migration is inversely proportional to the size of proteins, so smaller go farther than bigger.

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

• Negatively charged molecules will go to the positive anode.

• Coomassie blue dye is used to stain the proteins.

At what absorbance can proteins and peptides be found?

• 215 nm for the peptide backbone.

• 280 nm for the aromatic side chains.

How can the concentration of a protein be calculated?

A = Ecl

How can the extinction coefficient for a protein at 280 nm be calculated?

Etotal= 5690 (number of tryptophanes W) + 1280 (number of tyrosines Y)

What is mass spectrometry?

• Exploit differences in the mass-to-charge ratio 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.

• Commonly used as electrospray ionization and tandem mass spectrometry.

How can proteins be sequences?

• Direct amino acid sequencing.

• Sequencing the corresponding DNA in the gene.

What is the biochemical strategy for determining the amino acid sequence of a protein?

• Separation of the polypeptide chains.

• Cleavage of disulfide bridges.

• Analysis of N and C-terminals.

• Fragmentation of the polypeptide chain.

• Reconstruction of the amino acid sequence.

Explain the separation of the polypeptide chains.

• Subunit interactions depend on weak forces.

• Polypeptide separation is achieved with 8 M Urea, 6 M guanidinium, HCl, extreme pH or high salt concentration.

• If the protein is a heteromultimer, chromatographic methods can then separate the different chains for individual sequencing.

Explain the breakage of the disulfide bonds.

• Can be done by performic acid oxidation or reduction with mercaptoethanol (BME) or dithiothreitol (DTT).

• Then, treatment with alkylating agents that modify the –SH groups to prevent recombination of disulfides.

Explain the analysis of the N-terminus.

• Edman degradation allows sequential identification of a series of residues starting at the N-terminus.

• Chromatographic techniques are used to identify the PTH-derivative.

Explain the analysis of the C-terminus.

• Carboxypeptidases cleave the amino acid residue from the C-terminus.

  • A: All residues except Pro, Arg, Asp, Glu, and Lys.

  • B: Arg and Lys residues only.

  • Y: Any residue.

• The C-terminal residue is removed enzymatically with the carboxypeptidase and then reacted with phenyl isothiocyanate to produce the PTH derivative.

Explain the fragmentation of the polypeptide.

• Enzymatic fragmentation is done with trypsin (carboxy side of Arg and Lys), chymotrypsin (carboxy side of Phe, Tyr, and Trp), chlostripain, staphylococcal proteases.

• Chemical fragmentation is done with cyanogen bromide (cleaves peptide bond between Met and the next amino acid).

Explain the construction of the complete sequence.

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

procedures.

• Sequence all the peptides produced (usually by Edman degradation).

• The goal here is to find overlapping sequences.

• Align peptides from different fragmentations to reveal the overall amino acid sequence.

What do proteins interact with?

• Other proteins

• Nucleic acids

• Small molecules (metabolites)

• Metal ions

What is the formula for the dissociation constant of proteins? And what does it indicate?

KD = [P][L]/[PL]

v= [L]/(Kd+ [L])

When v=0.5, KD= [L].

Smaller KD indicates better affinity of ligand for protein.