A. Methods to determine 3-dimensional protein structures
1. X-ray crystallography -- Fig. 6-22,23
- x-rays have short wavelength (ca. 1.5 Å) required to measure small distances between atoms
- no lenses for X-rays ==> focus diffraction patterns mathematically
- yields three-dimensional electron density intowhich atoms are positioned in building a model
2. 2-dimensional nmr spectroscopy (and recently 3D and 4D) gives distances between specific atom pairs Box 6-3
- NOESY spectroscopy -- distances through space
- COESY spectroscopy -- distances through bonds
1. globular proteins contain common secondary structural elements
- first structure know is sperm whale myoglobin which is almost all a-helix
- other proteins contain both a-helix and b-sheet
- other proteins contain only b-sheet
2. amino acid sidechains are distributed by polarity
- Nonpolar residues in the interior (Val, Leu, Ile, Met, & Phe)
- Charged polar residues (Arg, His, Lys, Asp, & Glu) are on the exterio
- Uncharged polar amino acid residues (Ser, Thr, Asn, Gln, Tyr, and Trp) usually on the surface, but also in the interior; in the latter case they make H-bonds to other groups to neutralize their polarity
3. Core of globular proteins is quite compact with very little space
4. Large proteins have domains which are structurally independent but may interact
- each domain has at least 2 layers of secondary structure to seal the hydrophobic core
- each domain may have a specific function
C. Large Polypeptides Form Domains:
1. Each Domain consists of 100-200 amino acids
2. Domains contain two or more layers of secondary structure required to form a hydrophobic core separated from aqueous environment.
3. Domains are often structurally independent units with the characteristics of a small protein and a specific function, e.g. substrate binding site.
4. Neighboring domains are often connected by only one or two polypeptide segments.
5. small molecule binding sites may be between two domains providing flexibility
D. Protein Stability--thermodynamic measurements indicate that native proteins are only marginally stable. DG ~ 0.4 kJ/mole aa ==> 40 kJ for 100 residu protein (10 kcal/mole or ~2 H-bonds)
1. Hydrophobic Forces-The most important determinant of tertiary structure.
- DG for transfer of hydrophobic groups from H2O to nonpolar solvent is < 0, but DH > 0 for aliphatics and ~0 for aromatics!! ==> driving force is entropy (rmember DG = DH - TDS) Table 2-2
- Entropy changes are from ordering of H2O molecules hydrophobic groups to maximize their H-bonds Fig. 2-8
- Sidechain hydropathies are good predictors of which portions of polypeptide are inside the protein. Table 6-2
2. Hydrogen Bonds: -D-H - - - A- : The most important stabilizing force in secondary structure
predominantly electrostatic with energy of 12-30 kJ/mole and strong directionality. Nearly all possible H-bonds are made within a protein ==> is a major influence in determining protein structure, but provides little stabilization energy because H-bonding groups can bond with H2O in denatured form. Fig. 2-2,7
3. Electrostatic Forces
- Ionic Interactions - Salt Bridge (bond): strong interactions but contribute little to stability because charged groups can be solvated by water in denatured form
- Dipole-Dipole Interactions - van der Waals forces: weak but large number contributes significantly to stability Fig. 2-5
4. Disulfide Bonds--not present in cytoplasm but form in extracellular secreted proteins to stabilize them
5. Metal Ions may stabilize proteins by cros-linking sidechains, e.g. zinc finger motif in Fig. 6-35
E. Protein Denaturation--small stabilization energy means protein structure is readily disrupted ==> denatured. This occurs coopertively (all at once). Conditions which can denature are
1. Temperature--most proteins denature with TM < 100°C
2. pH - extremes of pH change the ionization state (and consequently the charge) of many amino acid sidechains.
3. detergents (e.g. SDS) - bind to the hydrophobic sidechains disrupting hydrophobic interactions.
4. chaotropic agents (guanidinium ion and urea) at high concentration (5-10 M) denature proteins by disrupting hydrophobic interactions
F. Protein Renaturation
1. Many denatured proteins can be renatured by returning the conditions to those in which the protein is inherently stable (e.g. removing urea or detergents, returning pH to near 7.0, lowering the temperature). Fig. 6-36
2. This suggested that the sequence of amino acids is the sole determinant of protein tertiary structure, and newly synthesized proteins fold spontaneously into the correct structure.
G. Molecular Chaperones: When synthesized inside cells, proteins begin folding immediately.
1. Since there is a very high concentration of protein in the cytosol, newly synthesized proteins can form incorrect associations with other proteins and becom misfolded
2. Molecular Chaperone proteins prevent misfolding of proteins providing a sheltered environment where they can fold properly before being released; this process required energy from ATP Fig. 6-40
E. Conformational Flexibility or "Breathing": Protein tertiary structure is specific but not rigid, many parts of the protein, particularly N- and C- termini and Lys sidechains, can move up to 2 Å Fig. 6-41
E. Quaternary Structure--many proteins contain more than one polypeptide (or subunit) which associate in a specific way) ==> make large assemblies out of smaller replacable units; also enzymes with many subunits provides a way to regulate them
1. Subunit interactions-contact regions are complementary surfaces held together by
- hydrophobic bonds
- hydrogen bonds
- van der waals interactions
- salt bonds
- occasionally disulfice bonds
2. Symmetry-in majority of multisubunit proteins the subunits are arranged with geometrically equivalent positions Fig. 6-32,33