Protein scondary and tertiary structure

What is the secondary structure?

It is defined as the local conformation of its backbone. Which is defined in a couple of ways like Helicies, pleated sheets, and turns.

cis vs trans

Cis are close and cause problems while trans are further away and dont have steric clashing

Ψ

Alpha carbon - carbon bond

180 degrees!

ɸ

Alpha carbon - nitrogen bond

180 degrees!

What does the Ramachandran diagram tell us?

It says most bond conformations are not possible, but there are around 3 possbile conformations

Where are beta sheets?

Top left!

Where are alpha helixes?

Bottom left and top right!

Why is glycine important?

It lacks an R group which means steric clashes do not really affect it as much

Helical structures

It twists! it is characterized by the number of peptide units per helical turn, and by distance the helix rises along its axis per turn.

What is the stregnth of the hydrogen bond in an alpha helix?

2.8Å

What is the only observed alpha helix in nature?

The right handed helix

β pleated sheet

This utilizes the full hydrogen bonding capacity of the polypeptide backbone. Bonding rarely occurs between neighboring polypeptide chains rather than within itself as in the ɑ helix

Anitparallel β pleated sheet

Has hydrogen bonds and the two polypeptide chains run in differnt directions

Parallel β pleated sheets

The hydrogen bonded chain extends in the same direction

Globular proteins

compact shape like a ball with irregular faces, includes enzymes!

Fibrous proteins

Usually span a long distance in the cell, 3D structure is usually long and rod shaped

Zinc Finger

Tertirary structure, coordinates one or more zinc ions in order to stabilize the fold. Two β-strands and one ɑ helix.

Catalytic triad

The part of a protein where chemistry takes place

Base polarizes

Acidic stabalizes

Nucelophilic residue attack

Energetics of folding

ΔG = ΔH - TΔS

What is required for a spontaneous process?

ΔG must be negative

What is the deciding factor of if something folds?

The folding MUST be entropy driven

Levinthal’s paradox

Solved, but the idea that there are a lot of conformations an AA can take and asking how does it decide

Molten Globule

Slightly larger than native conformation, signifcant amount of secondary structure formed with side chains still not ordered or packed

Structure flucuation is much larger

What is the net stability of a protein?

It is defined as the difference in free energy between the native and denatured state

Energy difference between the U and N states

What are some methods that can distinguish between U and N

Urea and Guanidinium hydrochloride

What is the trade-off of between stability and activity?

Something with an active site is going to be inheritly unstable, along with that how stable something is can be altered by what temperature it wants to work best at.

X-ray crystallography

Generates a good protein crystal

Detector sees a patern of spots called reflections from X-ray beam. EACH atom makes a contribution to each spot!

Massive calculations to produce an electron density map. Nuclei have greatest density

Yields map of structure

NMR

Only certain atoms (H, C, N, F, and P) give rise to an NMR signal. Used to ID nuclei and their immediate chemical environment. Also use NOE signals provide info about the distance between atoms

Protein motions

Motions occur on multiple time scales

“Fast” motions require spin relaxation measurements

Slow motions require relaxation dispersion measurements

NMR and x-ray crystallography give us a more complete understanding of proteins

How do I get Exogenous expression of a protein?

With mammalian cell culture, Baculovirus expression in insect cells, bacterial expression

T7 based E. Coli expression

Fast

Cheap

Easy typically

Disadvantages: no post-translational mods, deficient in certian tRNAS, oxidized and secreted protiens more difficult, certain protiens can not fold and form inclusion bodies

How does one deal with inclusion bodies?

Slow down production of a protein, co expression of chaperones, and purift from inclusion bodies

Anion Exchange

You get positive amino acids after this!

Cation exchange

You get negatively changed amino acids

Size exclusion chromatography

Can seperate out particles based off of size, small or large!