Chapter 6 - Globular Proteins - PowerPointA. 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
B. Tertiary Structure--3dimensional arrangement or folding of secondary
structure elements including the stribution of sidechains1. 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
| All alpha helix - Myoglobin
| Alpha helix & beta sheet - Flavodoxin
| All beta sheet - Superoxide Dismutase
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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
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C. Large Polypeptides Form Domains: 1. Each
Domain consists of 100-200 amino acids2. 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. 6-362. 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 misfolded2. 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
Which amino acids are found in the interior of a globular protein?
So, the amino acids present on exterior of globular protein are aspartic acid and lysine while amino acids present on inside of globular protein are valine and phenylalanine.
Which amino acid would most likely be found in the interior of a globular protein group of answer choices?
amino acids with hydrophobic side chains like valine, alanine, isoleucine, methionine, and leucine will tend to be inside the protein. The amino acids with hydrophilic side chains like lysine, arginine and aspartate will tend to be found on the outside of the protein.
Why are hydrophobic amino acids located in the interior of globular proteins?
During biosynthesis, a globular protein folds into a tight particle with an interior core that is shielded from the surrounding solvent. The hydrophobic effect is thought to play a key role in mediating this process: nonpolar residues expelled from water engender a molecular interior where they can be buried.
Which of these amino acids is most likely to be found near the interior of a membrane?
Glycine and alanine have the highest occurrence among the buried amino acids in membrane proteins, whereas leucine and alanine are the most common buried residue in soluble proteins.
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