Biomolecules: Peptides and Proteins Lecture 5, Medical Biochemistry.

Biomolecules:Peptides and Proteins

Lecture 5, Medical Biochemistry

Lecture 5 Outline

• Overview of amino acids, peptides and the peptide bond

• Discuss the levels of protein structure

• Describe techniques used for analysis of proteins

Planar nature of the peptide bond. The partial double bond characteristic prevents free rotation around the C-N bond; keeping it in the same plane with the attached O and H atoms. These planar bonds can pivot around the shared C atom

Levels of Protein Structure

Protein Structure Levels

• PRIMARY: the linear sequence of amino acids linked together by peptide bonds

• SECONDARY: regions within polypeptide chains with regular, recurring, localized structure stabilized by H-bonding between constituent amino acid residues

Protein Structure Levels (cont)• TERTIARY: the overall three-dimensional

conformation of a protein

• QUATERNARY: the three-dimensional conformation of a protein composed of multiple polypeptide subunits

• THE PRIMARY AMINO ACID SEQUENCE IS THE ULTIMATE DETERMINANT OF FINAL PROTEIN STRUCTURE

Ex: INSULIN

Disulfide bonds

Form between two intra- or interchain cysteineresidues, product called cystine- Stabilizes/creates proteinconformation- Prevalent in extracellular/secreted proteins

Stabilizing Forces

1. Electrostatic/ionic 3. Hydrophobic interactions2. Hydrogen bonds 4. Disulfide bonds

2o Structure: -helix

each oxygen of a carbonyl group of a peptide bond forms a H-bond with the hydrogen atom attached to a nitrogen in a peptide bond 4 amino acids further along the chain; very stable structurally; prolines will disrupt helix formation

End-on view of -helix

Parallel

Anti-Parallel

-sheetIn this secondary structure, each amino acid residue is rotated 180o relative to its adjacent residue. Occur most commonly in anti-parallel directions, but can also be found in parallel. H-bonds between adjacent chains aid in stabilizing the conformation.

-bend

Super-secondary structure examples

Super-secondary structurescommonly found in some DNA-binding proteins

Domains, examples:

Saddle-Barrel Bundle

Ex: Tertiary Structure Ex: Quaternary Structure

Myoglobin -subunit Hemoglobin

Structure of Myoglobin and Hemoglobin

• The amino acid sequences of myoglobin and hemoglobin are similar (or, highly conserved) but not identical

• Their polypeptide chains fold in a similar manner

• Myoglobin is found in muscles as a monomeric protein; hemoglobins are found in mature erythrocytes as multi-subunit tetrameric proteins. Both are localized to the cytosol

Sequence Comparison Examples

MyoglobinHb (horse)Hb (horse)Hb (human)Hb (human)Hb (human)Hb (human)

MyoglobinHb (horse)Hb (horse)Hb (human)Hb (human)Hb (human)Hb (human)

(Internal helix)

(Surface helix)

Myoglobin Properties

• At the tertiary level, surface residues prevent one myoglobin from binding complementarily with another myoglobin; thus it only exists as a monomer.

• Each monomer contains a heme prosthetic group: a protoporphryin IX derivative with a bound Fe2+ atom.

• Can only bind one oxygen (O2) per monomer• The normal physiological [O2] at the muscle is high

enough to saturate O2 binding of myoglobin.

Heme-Fe2+ Protein-Heme Complex

with bound oxygen

Heme Structure

Hemoglobin Properties• At the tertiary level, the surface residues of the and

subunits form complementary sites that promote tetramer formation (22), the normal physiological form of hemoglobin.

• Contains 4 heme groups, so up to 4 O2 can be bound• Its physiological role is as a carrier/transporter of oxygen from

the lungs to the rest of the body, therefore its oxygen binding affinity is much lower than that of myoglobin.

• If the Fe2+ becomes oxidized to Fe3+ by chemicals or oxidants, oxygen can no longer bind, called Methemoglobin

Biochemical Methods to Analyze Proteins

• Electrophoresis• Chromatography: Gel filtration, ion exchange,

affinity• Mass Spectrometry, X-ray Crystallography,

NMR• You will not be tested on the sections in your

textbook describing amino acid separations (Ch 4), peptide/protein sequencing and synthesis (Ch 5), and X-ray crystallography/NMR (Ch 6)

Protein Separation by SDS-Polyacrylamide Gel Electrophoresis

Presence of SDS, a detergent, denatures and linearizes a protein (Na and sulfate bind to charged amino acids, the hydrocarbon chain interacts with hydrophobic residues). An applied electric field leads to separation of proteins based on size through a defined gel pore matrix. For electrophoresis in the absence of SDS, separation is based on size,charge and shape of the protein (proteins are not denatured and canpotentially retain function or activity)

SDS-Polyacrylamide Gel (cont)

Separation of proteinsbased on their size islinear in relation to thedistance migrated in thegel. Using protein standards of known mass and staining of the separated proteins with dye, the mass of the proteins in the sample can be determined. This is useful for purification and diagnostic purposes.

Gel filtrationSeparation is based on protein size.Dextran or polyacrylamide beads of uniform diameter are manufactured with different pore sizes. Dependingon the sizes of the proteins to beseparated, they will enter the pore if small enough, or be excluded if theyare too large.

Hydrophobic ChromatographyProteins are separated based on theirnet content of hydrophobic amino acids. A hydrocarbon chain of 4-16carbons is the usual type of resin.

Separation of proteins based onthe net charge of their constituentamino acids. Different salt concentrations can be used to elutethe bound proteins into tubes in a fraction collector. As shown below,resins for binding (+) or (-) chargedproteins can be used

Ion Exchange Chromatography

Affinity Chromatography• Based on the target proteins ability to bind a

specific ligand, only proteins that bind to this ligand will be retained on the column bead. This is especially useful for immunoaffinity purification of proteins using specific antibodies for them.

• Example:

Protein Structure Methods• The sequence of a protein (or peptide) is determined

using sophisticated Mass Spectrometry procedures. The three dimensional structures of proteins are determined using X-ray crystallographic and NMR (nuclear magnetic resonance) spectroscopic methods.

• Protein sequence data banks useful for structural and sequence comparisons

• Please note that the new discipline termed “Proteomics” is evolving to incorporate cross-over analysis of sequence data banks, Mass Spec methodology, and living cells