Proteins

Learning Objectives• Understand the importance of the levels of

protein structures• Understand the basis for the stability of protein

structures• Understand how proteins fold into, and unfold

from, their native conformation• Understand the methods employed to analyze

proteins

Biologically Active Peptides

• Aspartame• Glutathione• Vasopressin• Oxytocin• Enkephalins• Insulin

dipeptide

tripeptide

nanopeptide

nanopeptide

pentapeptide

2 polypeptides

Protein classification based on: Composition

• Simple proteins – no other biomolecules present

• Conjugated proteins – presence of metal atom or small organic molecule

Protein classification based on: Solubility

• Globular – water soluble; transport function, immune protection and catalysis

• Fibrous – water insoluble; structural functions collagen, elastin

Protein classification based on: Function

Protein classification based on: Function

Levels of protein structure

• Primary – sequence of amino acids• Secondary – H-bonds bet. backbone C=O and

backbone NH (pleated sheet and helix)

• Tertiary – interactions of secondary structures• Quaternary – association of polypeptide subunits

Levels of protein structure

Levels of protein structure

Non-polar amino acids

Polar, non-charged amino acids

Negatively-charged amino acids

Positively-charged amino acids

The primary structure reveals the amino acid sequence of each protein/peptide.

Levels of protein structure

Secondary structures

• The polar N-H and C=O peptide units in the interior of the protein are held by H-bonds

• Two types which are regular structures in protein

• a-helix and b-pleated sheet

a-helix features• Coil direction – left handed or right handed• L- amino acids favor the right hand coil• One coil has about 3.6 aa residues; there can be several coils

with 650 aa residues(1000Å)• Average length of helix in a globular protein is 15-20Å• H-bonds occur between 1st O of backbone C=O to 13th H atom

of backbone NH• The presence of the ff amino acids do not favor the helix

formation: Pro, adjacent basic or acidic amino acids, Asn, Tyr, Ser, Thr, Ile and Cys

Knowing the Right Hand from the Left

b-pleated sheet

• Two adjacent peptides• Parallel (both NC or CN) • Antiparallel (N to C running in opposite

directions)• Antiparallel more common in the structure of

proteins• Peptides with this structure are rich in alanine

and glycine (silk fiber and spider web)

Supersecondary structuresor structural motifs

• The clusters are held together by favorable non covalent interactions

• Some structural motifs of folded proteins: aa motif; bb motif antiparallel; the Greek key (bbbb) motif; bab motif parallel

Structure of triose phosphate isomerase with several bab motifs combine to form a superbarrel (a) side view (b) top view of the protein

Levels of protein structure

Tertiary structure

• Combination of several motifs of secondary structures into a compact arrangement

• Noncovalent forces bring about the interactions and stability; – H-bonds, – electrostatic, – hydrophobic, – Van Der Waal’s,– pi-pi complexation between R-side chains– Disulfide bonds occur between Cys residues

Tertiary structures are quite varied

Charged/polar R-groups generally map to surfaces on soluble proteins

Non-polar R-groups tend to be buried in the cores of soluble proteins

Myoglobin

Blue = non-polar R-group

Red = Heme

Membrane proteins have adapted to hydrophobic environments

• Water excluded from the hydrophobic interior• Folding of protein occurs after translation in

the presence of molecular chaperones• Heat shock proteins (proteins are highly

expressed when cells are exposed to increase in temperature) – prevent aggregation of heat-denatured polypeptides

• Misfolded proteins aggregate and deposit in certain organs

The diagram shows the role of heat-shock proteins and a chaperonin in protein folding.

As the ribosome moves along the molecule of messenger RNA, a chain of amino acids is built up to form a new protein molecule. The chain is protected against unwanted interactions with other cytoplasmic molecules by heat-shock proteins and a chaperonin molecule until it has successfully completed its folding.

PROTEIN DENATURATION

Levels of protein structure

Quaternary structure of proteins

• Oligomeric –two or more polypeptide chains; subunits

• Homotypic – almost identical subunits• Heterotypic – different subunits • Defines the arrangement and position of each

subunit in an intact protein

Examples of other quaternary structures Tetramer Hexamer Filament

SSB DNA helicase Recombinase Allows coordinated Allows coordinated DNA binding Allows complete DNA binding and ATP hydrolysis coverage of an

extended molecule

How do biochemists determine the sequence of amino acids?

• Sanger technique• Edmann technique• Dansyl chloride technique

Sanger Technique

Edmann Technique

Large Proteins should be sequenced in smaller fragments

Protein isolation

• Ion exchange chrom.–based on charge• Gel filtration chrom- based on molecular size• Affinity chrom- selective binding to a specific

molecule• Gel electrophoresis- Based on charge and molecular

size

Column Chromatography

Ion-exchange Chromatography

Gel/ Size -exclusion

Chromatography

Affinity Chromatography

Components of the mixture have a uniform charge, electrophoretic mobility depends primarily on size

Gel Electrophoresis- generally usedsupport medium is cellulose or thin gels made up of either polyacrylamide or agarose. Polyacrylamide is used assupport medium for low molecular weight biochemicals such as amino acid and carbohydrates whereas agarose for large molecules like proteins

End of lecture