Chapter 6 Covalent Structures of Proteins

• Proteins, such as rhodopsin in the retina, acquire sensory information that is processed through the action of nerve cell proteins.
• The proteins of the immune system, such as the immunoglobulins, form an essential biological defense system.
• Proteins such as collagen play structural support roles.
• Protein function can only be understood in terms of protein structure.

  1) A protein's primary structure is the amino acid sequence.

  2) A proteins secondary structure is the local spatial arrangement of polypeptide's backbone atoms without regards to the conformation of its side chains.

  3) Tertiary structure refers to the three-dimensional structure of the entire polypeptide.

  1. Primary Structure Determination

• Insulin was sequenced by Sanger in 1953, 51 residues long, > 100 g required.
• b-galactosidase, 1021 residues long 1978

• Steps In Primary Structure Determination
  • 1. Prepare the protein for sequencing
  • 2. Sequence the polypeptide chains
  • 3. Organize the completed structure

   

  A. End Group Analysis: How Many Different Types of Subunits?

• Dansyl Chloride
  • Dansyl Chloride + polypeptide -> Dansylated polypeptide
  • Dansylated polypeptide + acid hydrolysis -> N terminal residue stained yellow

• Edman degradation
  • phenylisothiocyanate + polypeptide -> phenylisothiocarbamyl adduct
  • phenylisothiocarbamyl adduct + trifluoroacetic acid -> released residue

   

• Edman takes the first element of the list off on the N-terminal end, and leaves the rest of the list intact. Edman is therefore a computational protein operator. We can write this as:

  List = First(List) + Rest(List)

  NPolypeptide = N + Polypeptide

  Edman(NPolypeptide) = N + Polypeptide

• Edman degradation can be replied repeatedly until the Polypeptide is completely sequenced, and is thus amenable to automation.
• C-Terminus Identification
• Done Using Exopeptidases.
• Carboxypeptidase A works provided that Rn != Arginine, Lysine, or Proline and Rn-1 != Proline. These relationships are summarized below:

B. Cleavage of Disulfide Bonds

• Permits separation of polypeptide chains if they are connected via Cysteine disulfide bonds, e.g.

T D I Q G C W W H C S

. . . . . |

R Q E I A C D

• To expose internal protein structures to proteolytic (protein-cleaving) agents for further processing.
• Cleave disulfide bonds with performic acid
• This converts S-S bridges (in Cys-Cys) form to cysteic acid
• but messes up Met residues
• A better way is 2-mercaptoethanol, dithiothreitol or dithioerythritol (Cleland's reagent).
• Iodoacetic acid is used to alkylate free sulfhydrl groups to prevent renaturation of disulfide bonds by free oxygen in the air.

C. Separation, Purification and Characterization
of the Polypeptide Chains

• Urea and guanidinium ion are denaturing agents.
  
  

D. Amino Acid Composition

• Before sequencing we determine amino acid composition.
• Amino Acid composition is determined by complete hydrolysis followed by quantitative analysis of the liberated amino acids.
• Endopeptidases are used for hydrolysis.

• Amino Acid Analysis Has Been Automated
• AA's are separated via ion exchange chromatography and visualized using fluorescent adducts.

• The Amino Acid Compositions of Proteins Are Indicative of Their Structures
• The amino acid analysis of a vast number of proteins indicates that they have considerable variation with respect to their amino acid compositions.
• Polar/Nonpolar > 1 for globular proteins
• Nonpolar residues predominate in membrane-bound proteins!

E. Specific Peptide Cleavage Reactions

• Polypeptides that are longer than 40 to 80 residues cannot be directly sequenced.
• Polypeptides of greater length must be cleaved enzymatically or chemically into fragments small enough to be sequenced.
• Trypsin Cleaves Peptide Bonds AFTER positively charged residues N and K if
  Rn != P
• Cyanogen Bromide Specifically Cleaves Peptide Bonds after Met Residues

F. Separation and Purification of Peptide Fragments

• Reverse-Phase Chromatography via HPLC has reduced separation of peptide fragments to routine procedure.

G. Sequence Determination

• Repeated Edman Degradation in a sequenator accomplishes sequencing.
• Usually, a peptide's 40 to 60 N-terminal residues can be identified
  (100 or more with the most advanced systems) before the cumulative effects of incomplete reactions, side reactions and peptide loss make further amino acid identification unreliable.

H. Ordering the Peptide Fragments

• Peptide fragments are ordered by comparing amino acid sequences of one set of peptide fragments with those of a second set whose specific cleavage sites overlap those of the first set.

I. Assignment of Disulfide Bond Positions

• Disulfide Bond Positions can be determined by diagonal electrophoresis.

J. Peptide Sequencing by Mass Spectrometry

• Good for up to 25 residues.
• Fast Atom Bombardment in concert with Tandem Mass Spectrometer
• Good for mixtures of peptides

K. Peptide Mapping

• Related proteins can be sequenced more rapidly once a primary structure has been solved.
• This is called fingerprinting or peptide mapping.

L. Nucleic Acid Sequencing

• It is easier to sequence DNA than proteins BUT:
• Disulfide bonds can be located only by protein sequencing.
• Many proteins are modified after biosynthesis
• It is sometimes difficult to locate the nucleic acid that encodes the protein of interest. (that will change with complete human genome project)
• A common DNA sequencing error is the insertion or deletion of a single CTAG.
• The standard genetic code is not universal.

  2. Protein Modification

• A common strategy for identifying proteins is to use side chain specific reagents.
• There is a big table of these in the book.

  3. Chemical Evolution

• A mutation in a protein must increase the probability of survival.

A. Sickle Cell Anemia: The Influence of Natural Selection

• Hemoglobin is a protein for oxygen transport.
• a2b2 tetramer
• two identical alpha chains and two identical beta chains.
• erythrocytes carry the hemoglobin
• Sickle-Cell Anemia is a Molecular Disease
• In 1945 Linus Pauling hypothesized that sickle-cell anemia was the result of a mutant hemoglobin.
• Later they showed that normal hemoglobin HbA had an anionic charge around tow units more negative than HbS.
• Glu in the sixth position of each beta chain was changed to Valine.
• This mutation caused HbS to aggregate into filaments of sufficient size and stiffness to deform erythrocytes.
• The Sickle-Cell Trait Confers Resistance to Malaria
• Individuals Heterozygous for HbS are resistant to malaria.
• Sickle-Cell Anemia provides a classic Darwinian example of a single mutation's adaptive consequences in the ongoing biological competition among organisms for the same resources. (dogma alert)

B. Species Variations in Homologous Proteins:
The Effects of Neutral Drift

• A protein that is well adapted to its function still continues evolving.
• Comparisons of the primary structures of homologous proteins (evolutionarily related proteins) indicates which of the residues are essential to function and which have lesser significance.
• Cytochrome c Is a Well-Adapted Protein.
• It is used in mitochondria as part of an electron transport chain.
• It transfers electrons between cytochrome c reductase and cytochrome c oxidase.
• Protein Sequence Comparisons Yield Taxonometric Insights
• Proteins Evolve at Characteristic Rates
• The rate that mutations are accepted into a protein depends on the extent that amino acid changes affect its function.
• Mutational Rates are Constant in Time
• Existence proof, insects versus plants versus animals.
• Plant characteristic time frames are different than animals or insects!
• Point mutations in DNA accumulate at a constant rate with time via random chemical change rather than resulting from errors in replication.
• Protein Evolution Is Not the Basis of Organismal Evolution
• The rapid divergence of human and chimpanzee stems from relatively few mutational changes in the segments of DNA that control gene expression, that is how much proteins will be made, where and when.

C. Evolution through Gene Duplication

• A Gene duplication is an efficient mode of evolution because one of the duplicated genes can evolve a new functionality through natural selection while its counterpart continues to direct the synthesis of presumably essential ancestral protein.
• The sequences of hemoglobin's alpha and beta subunits and myoglobin are quite similar.
• 1) The primordial globin probably function simply as an oxygen storage protein.
• 2) Duplication of a globin gene ~1.1 billion years ago permitted the genes to evolve separately by a series of point mutations.
• 3) Hemoglobin's tetrameric character is a structural feature that greatly increases its ability to transport oxygen efficiently.
• 4) In fetal mammals, oxygen is obtained from the maternal circulation.
• 5) Human embryos in the first 8 weeks post conception make gene-duplicated alpha and beta variant hemoglobins.
• 6) In primates, the beta chain has undergone relatively recent duplication to form a delta chain. The alpha2delta2 hemoglobin which is about 1% of the normal adult hemoglobin has no known unique function.
• The human genome contains relics of globin genes that are no longer expressed.
• The three dimensional structure of a protein and hence its function is dictated by its amino acid sequence.

  4. Polypeptide Synthesis

• The ability to manufacture polypeptides not available in nature has enormous biochemical potential.
• 1) To investigate the properties of poly peptides by systematically varying their side chains.
• 2) To obtain polypeptides with unique properties, with nonstandard side chains or with isotopic labels.
• 3) To manufacture pharmacologically active polypeptides that are biologically scarce or nonexistent. For example synthetic vaccines.
• Consider the homopolypeptides, polyglycine, polyserine and polylysine.
• Oxytocin was the first nona-peptide (9 peptide) synthesized.

A. Synthetic Procedures

• Polypeptides are chemically synthesized by covalently linking amino acids one at a time.
• The amino acid being protected must have its own alpha-amino group protected, then unprotected in a series of coupling and deblocking steps.
• Amino Acid synthesis is easier if you anchor the C terminus to an insoluble solid support such as beads of polystyrene resin. This is called solid phase synthesis.
• Anchoring the chain to an inert support
• Coupling the amino acids
• Releasing the polypeptide from the resin is done via liquid HF.
• Yield of these steps is low 0.98^2n where n is the number of peptide bonds to be created