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CMU BSC 03231 - Lecture

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1Biochemistry I Fall Term, 2004September 15, 2004Lecture 7: Protein Tertiary & Quaternary StructureAssigned reading in Campbell: Chapter 4.4-4.5.Key Terms:Denaturation & refoldingDisulfide bondsElectrostatic interactionsHydrogen bondsvan der Waals forces"Hydrophobic effect"Conformational statesHeme groupX-ray crystallographyNMR spectroscopyDimer, trimer & tetramerOligomer(Topics relating to O2 binding by myoglobin and hemoglobin will be covered later in the course.)Links:(I) Review Quiz on Lecture 7 concepts(I) The CMU Chime Tutorial, covered previously in cluster sesssions, can also be accessedthis week. Be sure you understand the operation of this visualization tool.(I) The Protein Architecture Tutorial introduces the basic principles of protein 3-D structureusing Chime images of Protein G.(S) The "Alpha_bet Helix" shown in lecture today is an example of an ideal α-helix.4.4 Tertiary Structure of ProteinsDisulfide BondsThe formation of a covalent disufide bond between two Cys residues can contribute to the stabilityof protein tertiary structure. The "S-S" bond covalently crosslinks two regions of the structure thatmay be distant in sequence, but nearby in the folded state.Disulfide bonds are only found in proteins that function outside of the cell, e.g. extracellularenzymes, antibodies, plasma proteins, etc. The cysteine residues in intracellular proteins are kept intheir reduced, -SH, state by an active enzyme pathway using glutathione as the reducing agent.Noncovalent Forces in Protein Structure and the Hydrophobic EffectNoncovalent energies are 2 to 3 orders of magnitude smaller than covalent bonds; they act at short-range; and they are exceedingly numerous. A key feature of protein structure is that the stabilitydepends on the simultaneous presence of all of the noncovalent interactions of the native state.Thus, the interactions described below cooperate to produce the native structure.21. Electrostatic InteractionsThe free energy of bringing two charges together is:∆G = z1z2*e2/(ε*r)where z1 and z2 are the ionic valences, e is the unit of charge, r is the distance between the ionsconsidered as point charges, and ε is the dielectric constant of the medium between them. Forcharges of +1 and -1; in water (ε = 80); at a distance, r = 4Å; we calculate ∆G = -4 kJ/mol. In thehydrophobic core of a protein, the value of ε is estimated to be on the order of 2 - 4 (e.g. benzene).Thus, if a charge is buried in the core of a protein, there is a large energetic advantage in burying agroup of opposite charge nearby (or a corresponding penalty for leaving it unpaired). On thesurface of a protein, the advantage and the penalty are both much smaller because of the presenceof water, which shields both charges.2. Hydrogen Bonds are due primarily to partial electrostatic charges. The energetics aredetermined, in part, by the values of the partial charges. The groups of interest in proteins includeamide (N-H) and carbonyl (C=O) groups. Typical partial charge assignments for these groups are-0.3e and +0.3e for N and H, respectively; and -0.4e and +0.4e for O and C, respectively. Typical∆G's are 1 - 4 kJ/mol per H-bond. H-bond donors in the core of a protein are (nearly) alwayspaired with an acceptor. H-bond donors and acceptors on the surface of a protein can also H-bondwith other residues, but are more frequently H-bonded to water.3. van der Waals Forces a) Forces between atoms are attractive and occur between any pair of atoms at distances of 4-6 Å.Typical ∆G's are 0.5 - 1.0 kJ/mol per pair of atoms. b) The repulsion of the electronic shells occur at distances < the sum of the van der Waals radii.The unfavorable ∆G's rise rapidly as two nonbonded atoms are forced to occupy the same space(referred to as "steric repulsion").4. Metal Ion CoordinationThis is another electrostatic interaction. The separate category is justified by the fact that certainproteins have very specific requirements for metal ions. For example, Zn2+ is required to stabilizethe tertiary folding of the zinc finger domain in many DNA-binding proteins.3The Hydrophobic EffectDuring protein folding, the transition from the countless unfolded states to a single native state isaccompanied by the burial of solvated nonpolar side chains (and polar peptide units) into thenonsolvated core of the protein.The "hydophobic effect" or "hydophobic interaction" in protein structure is derived from thecombined properties of H-bonds in water and van der Waals forces applied to amino acid residueswith nonpolar side chains:A nonpolar side chain in water makes less favorable van der Waals interactions than if it weredissolved in an apolar solvent. In addition, the solvating water molecules cannot satisfy their fourpotential H-bonds while they surround the apolar solute.In contrast, a nonpolar side chain in the apolar core of a protein has gained favorable van der Waalsinteractions and has rid itself of the dissatisfied solvating water.The interior of folded proteins is tightly packed. Proteins have very few cavities on the order of thesize of a water molecule. The rule, in a phrase coined by Francis Crick, is that "Knobs fit intoholes." In other words, each core side chain fits into a complementary space created by several(from 5-8) of the other core side chains.Entropy disfavors the folded structure of proteins.The most significant energetic effect opposing all of the favorable interactions described above isthe unfavorable entropy of forming a unique 3-D structure. If it were not in its native state, theprotein could assume a huge number of conformations. To illustrate this point, consider thenumber of conformational states in peptides containing only alanine residues:1. Ala-Ala-Ala: The tripeptide has two rigid peptide bonds that remain fixed. However, the two Ψand two Φ angles can each rotate to three positions. Thus, the number of different conformationalstates is 32*32 or 34. This result (81) while large, is not too large to comprehend.2. (Ala)25 forms a helix in solution. Under denaturing conditions, it is a flexible polymer; the totalnumber of conformational states results from 32 states for each peptide unit or 32*24. This resultis comparable to Avogadro's number.A typical small protein (say, 100 residues) will have many amino acids with more side chaindegrees of rotational freeedom than do alanine peptides. On average there will be roughly 35conformational states for each peptide unit. Viewed as a flexible chain,


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