Protein Biochemistry, Structure, and Folding

1) Biochemistry

a) Bonds/Interactions

i) Hydrogen bonding of the R-side chain

ii) Covalent bonds – Disulfide bonding between cysteines

iii) Metal Ion interactions or even other valent ions like Ca²⁺

iv) Electrostatic interactions of the R-side chains

v) Helical structure

vi) Sheet structure

2) Structure

a) The peptide bond is rigid and planar; the six atoms of a peptide group lie in a single plane, thus limiting the range of conformations that can be assumed by a polypeptide chain.

i) Conformation can be defined by the bond angles represented in a Ramachandran plot.

b) Secondary structure- local conformation of some part of the polypeptide.

i) Folding patterns

(1) a-helix – simplest arrangement the polypeptide chain could assume with its rigid peptide bonds. Polypeptide is tightly wound around a longitudinal axis traversing the middle of the helix, with the R-side chains pointing outward from the backbone.

(a) Stability factors:

(i) Electrostatic repulsion (or attraction) between successive amino acid residues with charged R groups.

(ii) Bulkiness of adjacent R groups.

(iii) Interactions between amino acid side chains spaced 3-4 residues apart.

(iv) Occurrence of proline and glycine residues that destabilize the helix.

(v) Interaction between amino acid residues at the ends of the helical segment and the electric dipole inherent to the helix.

(2) b-sheet – more extended conformation of the polypeptide chains into a zigzag structure.

(a) Hydrogen bonds are formed between adjacent polypeptide chains.

(b) R groups of adjacent amino acids protrude from the sheet in opposite directions creating an alternating pattern.

(i) Parallel – same amino-to-carboxyl orientations.

(ii) Anti-parallel – opposite amino-to-carboxyl orientations.

(iii) Layering of sheets requires the R groups of the touching surfaces must be small.

(3) b-turns – connect the ends of two adjacent segments of an antiparallel b-sheet.

(a) Type I can tolerate any amino acid in any position except proline in position 3.

(b) Type II always has a glycine at position 3.

(i) Proline and glycine are the residues most common in turns because glycine is small and flexible and proline will readily convert the imino nitrogen to its cis conformation.

ii) Determining Secondary Structure

(1) Circular dichroism is very sensitive to the secondary structure of polypeptides and proteins. It uses a form of light absorption spectroscopy that measures the difference in absorbance of right- and left-circularly polarized light (rather than the commonly used absorbance of isotropic light) by a substance. It has been shown that CD spectra between 260 and approximately 180 nm can be analyzed for the different secondary structural types: alpha helix, parallel and antiparallel beta sheet, turn, and other.

iii) Determining 3-D structure of secondary structure

(1) Angle Plots – Ramachandran plots

(2) Hydrogen bonding – NMR

3) Tertiary Structure – overall 3-D arrangement of all atoms in a protein; amino acids that are far apart in the polypeptide sequence and that reside in different secondary structures may interact within the completely folded structure.

a) Hydrophobic and H-bonding are the primary forces holding a structure together but also electrostatic interactions, in some cases metal or other ions interact to stabilize the overall structure.

b) Determining 3-D tertiary structure by x-ray diffraction/crystallography – determine the spacing of atoms in a crystal lattice by measuring the locations and intensities of spots produced on photographic film by a beam of x-rays of given wavelength after the beam has been diffracted by the electrons of the atoms. Now the amount of information obtained depends on the degree of structural order in the sample.

c) NMR for H, C, F, N, and P can be used to determine structure to complement crystallography.

4) Post-translational modifications

a) N-Terminal modifications

i) N-formylmethionine in bacteria is lost

ii) N-Terminal Met in eukaryotes is lost and the new N-Terminus can be acetylated.

b) Loss of signal sequence – N-terminal

c) Modification of individual amino acids

i) Hydroxyl groups can become phosphorylated

ii) Extra carboxyl groups may be added to Glu

iii) Monomethyl- and dimethyllysine residues occur

d) Attachment of carbohydrate side chains

i) N-linked oligosaccharides on Asn

ii) O-linked oligosaccharides on Thr

e) Addition of isoprenyl groups – can aid in anchoring of proteins to membranes

f) Addition of a prosthetic group – heme group of cytochome c

g) Proteolytic processing – insulin must be trimmed by proteases like trypsin or chymotrypsin before becoming active

h) Formation of disulfide cross-links – common in eukaryotic cells for proteins to be exported; helps protect the native conformation from the denaturative effects of the extracellular environment which is generally oxidizing.

5) Quatrernary Structure – 2 or more separate polypeptides or subunits which are the same or different that arrange to form complexes. 2 or more proteins joined together.