Proteins Flashcards
What interactions are involved in the protein native fold?
Hydrophic, hydrogen bonds, van der waals, and electrostatic interactions
Primary structure
Amino acid sequence. Structure is partially dictated by the properties of the peptide bond.
Phi rotation
Angle around the alpha carbon and amide nitrogen bond.
Psi rotation
Angle around the alpha carbon and carbonyl carbon.
What determines the secondary structure of the protein?
The organization around the peptide bond and the identity of the R groups. Refers to spatial arrangement of the polypeptide backbone.
2 arrangements of secondary structure
Alpha helix and beta sheet
Alpha helix is stabilized by…
Hydrogen bonds between near by residues.
Beta sheet is stabilized by…
Hydrogen bonds between adjacent segments that may not be near by.
Random coil
Irregular arrangement of the polypeptide chain.
What is the helical backbone of a protein held together by?
Hydrogen bonds between the backbone amides of n and n+4 amino acids.
How many residues per turn?
3.6
Outside cell environment is…
Oxidizing, disulfide bonds form.
Inside cell environment is…
Reducing.
Protein with the lowest free energy =
Most stable, one with maximum weak interactions.
How are peptide bonds aligned with the helical axis?
Roughly parallel. So are H-bonds.
How are side chains aligned with the helical axis?
Roughly perpendicular.
Which hydrophobic residues are strong helix formers?
Ones with small hydrophobic residues such as Ala and Leu.
Which amino acids act as helix breakers?
Proline, because rotation around N-C bond is impossible. Glycine because tiny R group supports other conformations.
How is the large dipole moment of the alpha helix enhanced?
By unpaird amides and carbonyls near the ends of the helix.
Where do negatively charged residues occurs in the helix?
Near the positive end of the helix dipole.
How do side chains sit in the beta sheet protein structure?
They stick out from the sheet, alternating up and down direction?
How are beta sheets held together?
By the hydrogen bonding of the amide and carbonyl groups of the peptide bond from opposite strands.
When do beta turns occur?
When beat sheets change direction. The 180 turn is accomplished over 4 amino acids.
How is the beta turn stabilized?
By hydrogen bonds from a carbonyl oxygen to amide proton 3 residues down the sequence.
Type I beta turn
Proline in position 2.
Type II beta turn
Glycine in position 3.
What is the typical connection of beta strands
Beta motif
What stabilizes protein secondary structure?
Numerous weak interactions between amino acid side chains. Mostly hydrophobic and polar interactions, can sometimes be disulfide bonds.
Fibrous proteins
Form of protein teritary structure.
Silk fibroin
Antiparallel beta sheet, small Ala and Gly side chains allow for close packing.
Globular proteins
Arrangement of multiple secondary structures. Composed of different motifs folded together.
Intrinsically disordered proteins
Composed of amino acids whose higher concentration forces less-defined structure (Lys, Arg, Glu and Pro).
Conjugated proteins
Covalently bound to a nonprotein entity
Chromatography
Often used for separation of proteins in which the protein is able to remain fully folded.
Column chromatography
Separation of proteins over a solid phase using a liquid phase to mobilize the proteins. Proteins with a low infinity for the solid phase will wash off first.
Ion exchange chromatography
Separates based on net electric charge at a given pH. Column matrix contains bound charged groups, a proteins affinity to bind with that group is affected by pH.
Cation exchange chromotography
Solid matrix in ion exchange has negative charges. Positive charges move more slowly.
Size exclusion chromotography
Large proteins move faster because they don’t get stuck in the cavities.
Affinity chromotography
Beads in the column have covalently attached ligands, speed at which a protein moves through the column depends on its affinity to bind to that ligand.
SDS
Sodium dodecyl sulfate, a detergent to facilitate protein unfolding so they can be separated by molecular weight. Gives all proteins a uniform negative charge, so rate of movement will only depend on size.
Isoelectric focusing
A pH gradient is established and proteins migrate until they reach the pH that matches its pI.
2D electrophoresis
Combines isoelectric focusing and SDS. Separates identical MW that differ in pI or similar pI that differ in MW.
Horizontal separation in 2D electrophoresis =
Differences in pI.
Vertical separation in 2D electrophoresis =
Differences in MW.
Specific activity ratio
Number of enzyme units / mg of total protein.
