Protein Structure

Protein Structure I Primary Structure

Figure 5-1 Primary Structure Insulin Signal sequenceChain B MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLV C Peptide CGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEG Chain A SLQKRGIVEQCCTSICSLYQLENYCN Bovine: Insulin Human: ProInsulin

Figure 5-1 Primary Structure Insulin Signal sequenceChain B MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLV C Peptide CGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEG Chain A SLQKRGIVEQCCTSICSLYQLENYCN Bovine: Insulin Human: ProInsulin

Value of Primary Structure Information Primary sequence information is –prerequisite for determining three-dimensional structure –essential in understanding molecular mechanism of action Sequence comparisons among analogous proteins –provide insights into protein function –reveal evolutionary relationships Sequence of proteins whose mutations result in inherited diseases –assist in development of diagnostic tests –assist in development of effective therapies

Primary Structure Determination

Strategy Purification of protein to homogeneity Prepare protein for sequencing Sequence polypeptide chains Organize completed structure Alternative: Nucleic Acid Sequencing

Figure 5-12 Sequencing Strategy Summary

Figure 5-12 Sequencing Strategy I

Figure 5-12 Sequencing Strategy II

Figure 5-12 Sequencing Strategy III

Purification of Protein to Homogeneity

Prepare Protein for Sequencing End Group Analysis: How many different subunits Cleavage of disulfide bonds Separation and purification of the polypeptide chains Amino acid composition

End Group Analysis (How Many Different Subunits?) N-Terminal Identification

Sanger’s Reagent

Dansyl Chloride

End Group Analysis (How Many Different Subunits?) C-Terminal Identification

Reduction

Hydrazinolysis

Cleavage of Disulfide Bonds

Oxidative Cleavage

Problem (Oxidation of Methionine to Methionine Sulfone)

Reduction and Alkylation

Problem

Solution

Separation and Purification of Polypeptide Chains

Sequence Polypeptide Chains Specific peptide cleavage reactions Separation and purification of peptide fragments Sequence determination

Hydrolysis Polypeptide Amino Acids Hydrolysis

Acid Hydrolysis

Mechanism

Problems Complete destruction of Trp Partial destruction of Ser, Thr, and Tyr Deamination of Asn and Gln

Base Hydrolysis (Many Amino Acids Destroyed) (Racemization)

Enzymatic Hydrolysis Mild Conditions Many proteases and peptidases Specific and non-specific Problem: contribution of amino acids from hydrolysis of proteases

Amino Acid Analysis (Automated) Ion-exchange chromatography High performance liquid chromatography Colorimetric Analysis

Specific Peptide Cleavage Reactions

Proteolytic Enzymes Cleave peptide bonds Specificity: R 1

Table 5-3 Specificity of Endopeptidases

Chemical Cleavage (Cyanogen Bromide)

Separation and Purification of Peptide Fragments

Sequence Determination: -Edman degradation -Mass Spectrometry

Edman Degradation I

Edman Degradation II

Edman Degradation III

Figure 5-16a part 1 Electrospray Ionization Mass Spectrometry (ESI)

Figure 5-16a part 2 Electrospray Ionization Mass Spectrometry (ESI)

Figure 5-16b Electrospray Ionization Mass Spectrometry (ESI)

Figure 5-17 Tandem Mass Spectrometry

Organize Completed Structure Ordering peptide fragments Assignment of disulfide bond positions Determine position of amides

Ordering Peptide Fragments

Figure 5-18 Generating Overlapping Fragments

Ordering Peptide Fragments

Assignment of Disulfide Bond Positions Hydrolyze without breaking disulfides Reduce, alkylate, and identify linked fragments (disulfides)

Assignment of Amide Positions Hydrolyze without breaking amides Hydrolyze fragments and measure NH 3 (need fragments having a single Asn or Gln)

Protein Evolution Evolution by Natural Selection Mutations

Table 5-5 part 1 Cytochrome c All look like this

Sequence Comparisons Provide Information on Protein Structure and Function Homologous proteins: evolutionarily related proteins –Invariant residues –Conservative substitutions –Hypervariable positions Neutral drift

Figure 5-21 Phylogenetic Trees Depict Evolutionary History

Proteins Evolve by the Duplication of Genes or Gene Segments

Protein Families Can Arise through Gene Duplication Orthologous proteins: homologous proteins with the same function in different species Paralogous proteins: independently evolving proteins derived by duplication of a gene (globin family) Pseudogenes

Figure 5-22 Globin Family

Figure 5-23 The Rate of Sequence Divergence Varies