Protein Structure

Protein Structure Levels

1° - sequence, 2° - local folding, 3° - fully folded chain (has function), 4° - bringing together more than one chain (subunit)

Conserved Amino Acids

same amino acids between species, usually for important genes because otherwise it is different

Substituted Amino Acids

different amino acids at a given position (either conservative or radical)

Conservative Substitution

substituted with same class/size

Radical Substitution

substituted between classes/sizes

Position Weight Matrix

graph with a sequence of amino acids, with different amino acids shown in different sizes/weights (bigger the size/bigger the weight, the more conserved it is)

1° Structure

sequence of amino acids, can be different in different organisms

2° Structure

local folding of the protein, via partially charged atoms IN THE BACKBONE H-bonding to each other (because partial charges in the backbone need to be stabilize and they cannot H-bond to anything else, since they are in a nonpolar core)

2° Structure Types

a-helix, B-sheets, B-turns

a-helices traits

• backbone groups (partial pos H on Nitrogen)(partial neg O) present that are 4 amino acids away, which can H-bond with other backbone groups | - twisted to the right | - is usually 4-12 amino acids long | - N to C terminus

a-helices interactions

• will interact with other a-helices in opposite directions due to the net dipole that it has

a-helice dipole

bottom (N-terminus) is S+ and top (C-terminus) is S-, because all the S+ hydrogens on N point towards the N-terminus, and all the S- O point towards the C-terminus, and since they are all parallel, it causes them to add together, resulting in a dipole

a-helice representations

cylinders, cylinders with arrows, spiral (arrows point towards C-terminus)

2° Structure Bonus

sidechain interactions (not required but gives more stability)

a-helix sidechains traits

R groups point outwards (interacts in any nonpolar interactions) and those that are 3-4 AA can interact, but no Pro, no Gly

Why can't Pro and Gly interact?

Pro is too rigid (because of ring confined to specific phi si angles that prevent a-helice from forming) and Gly is too flexible

amphipathic a-helix

where in an a-helix, one side is polar and the other is nonpolar, alpha helices do this where one side faces outside and the other, the inside, and you can find interactions (IMFs) between side chains on the wheel

3₁₀ helice

narrowest helice, sidegroup interactions that are 3 amino acid away

pi helice

widest helice, backbone AA interactions 5 away

narrowest to widest

3₁₀, a, pi (no flexibility in number of amino acid interactions because the interactions are specific and rigid)

B-sheets traits

• B-strands combine to make B-sheets | - 2 types of possible B-sheets: antiparallel and parallel because there is no overall dipole | - no Pro or Gly (same reasons) | - 4-10 AA long, 2-15 strands/sheet | - N to C terminus

Which type of B-sheet is most stable?

antiparallel, because the H-bonds are straight on, making them stronger than parallel, whose H-bonds are more bent

B-sheet representations

arrows pointing from N terminus to C-terminus, with little lines

Why are a-helices and B-sheets common in the protein core?

due to lack of water in the protein core forcing the backbone of the protein to make H-bonds with each other to stabilize its partial charges

B turns

used to connect antiparallel B-strands and a-helices, with 4 residues present in the turn (lots of Gly/Pro because they are the right confirmation for B-turns)

2° Structure in Ramachandran Plots

certain secondary structures require certain combinations so they will all have different ramachandran pltos depending on what they are, but they are mostly in allowed regions

Protein Data Bank

protein structures made freely available when published here, given a 4 letter/number code and can look at the structure with a protein visualization program

3° Structure

results from folding of polypeptide chain into closely packed 3D structure

3° Structure traits

amino acids that are far apart in the primary structure may be brought together | - stabilized prim. by hydrophobic effect/water entropy | - has a function of some kind

Protein Structure Hierarchy

Secondary Structure -> Supersecondary Structure (Motifs) -> Folds -> Domains -> Fully Folded Chain or 3° Structure

Supersecondary/Motifs

primary interactions of different secondary structures

Folds

supersecondary structures interact, making even bigger structures

Domains

independently folded, compact units in proteins, which can have a particular function, and in multifunctional enzymes each domain have its own catalytic activity (not all proteins have, so not required to have this for 3° structure)

4° Structure

organization of multiple protein chains (subunits) in a protein (aka a multimer) held together by intermolecular interactions (mostly LDFs)

4° Structure Traits

• can be concentration dependent (so needs more than one subunit to become active) | - subunits have defined stoichiometry and arrangement (homo = same, hetero = different, dimer/trimer/tetramer, etc.)

Mass Spec

used to determine 1° structure of a protein

CD

can monitor the properly folded state of a protein (used to monitor protein unfolding/denaturation)

NMR

used to determine the 3° structure of saller proteins (5%)

X-Ray Crystallography

Used to determine the 3° and 4° structure of any protein

Cryo-Electro Microscopy

USed to determine the 3° and 4° structure of very large protein complexes

Mass Spec Steps

1) cleave with a protease to get different sized pieces | 2) separate fragments | 3) use mass spec to characterize each digest fragment | 4) overlap segments ot build back full sequence

Circular Dichroism (CD)

chiral molecules can rotate plane polarized light, this is circular polarized light, and when passing through proteins (chiral) it shows how much it absorbs, and since each protein has a unique CD spectrum (when native), it ends up indicating 2° structure, so that you can check whether or not the protein is properly folded

NMR (explanation)

is done in solution, 2D NMR sees which hydrogens are close to each in space so you can build the 3° structure, and is good because protein breathes/moves a lot so NMR represents that (more leeway in interactions)

X-Ray Crystallography (exp)

crystal form of a protein, shoot x rays at a it and get a pattern back, from protein you get electron density and can build back the structure (3° & 4° Structure) (kind of like objects in water and their displacement)

X-Ray Crystallography Mistakes

proteins are not solids so may have some additional conformations, which cryo-EM would not have