Biochem Midterm 1

weeks 1-3

Carbon covalent bonds

Makes 4 bonds with 4 valence electrons

Rank electronegativities of CHNOPS

O > N > S = C > H = P

How to tell if a molecule is nonpolar

• Made of only one element (e- shared equally)

• Is a hydrocarbon (small electronegativity difference)

• Symmetrical with no lone pairs (same polar bonds evenly distributed around central atom pull equally to cancel out to zero)

How to tell if a molecule is polar

• Asymmetry

• Strong electronegativity difference between bonded atoms (OH groups, O N S P with C H)

• Lone pairs on central atom (often bends the molecule)

Disulfude bonds

Strong covalent cross-links between the sulfur atoms of two cysteine amino acids

Ion dipole interactions

Electrostatic interaction between a full ion (Na+Cl-) and a polar molecule (H2O)

Hydrogen bonding

Strong type of dipole-dipole interaction when a hydrogen atom is covalently bonded to F, O, or N (ex. between waters)

Dipole-dipole interactions

Attractive forces between two polar molecules with permanent dipole moments

London dispersion forces

The weakest type of intermolecular force caused by random constant motion of electrons through temporary induced instantaneous dipoles (type of Van der Waals)

Van der Waals forces

Relatively weak, short-range electrical and attractive interactions between neutral atoms or molecules

Hydrophobic Effect

Entropy-driven process where nonpolar/hydrophobic molecules aggregate in water to minimize unfavorable interactions with polar water molecules and thereby increase the entropy of the water (drives protein folding)

Aggregation decreases the size of the hydrophobic molecule’s hydration shell (ie the water molecules that attract and surround the nonpolar molecule

How does the hydrophobic drive protein folding

Many amino acids are nonpolar from their nonpolar R groups; hydrophobic amino acids pack into hydrophobic cores to increase the entropy of water in the environment; helps drive folding by positioning R groups

Pi-pi stacking

Aromatic interactions, attractive stacking between the electron-rich pi-systems of aromatic rings

Cation-pi interaction

Non-covalent attactive force between a positively charged cation and the electron rich pi-system of an aromatic ring

What does pH mean for an aqueous solution?

pH is the measure of acidity or basicity of an aqueous solution determined by the concentration of hydrogen (H+) ions or hydronium ions (H3O+)

Neutral pH = 7, acidic = pH < 7, basic = pH > 7

What does pKa mean for a molecule?

Measures how tightly a molecule holds onto a proton (H+) with the negative logarithm of the acid dissociation constant (Ka)

Lower pKa = stronger acid (gives up proton easily)

Higher pKa = weaker acid (holds tightly to proton, rarely lets go)

pKa for molecules with O or N

• Protonated O: pKa ~0, very readily gives up proton

• Carboxylic acid O: pKa ~ 5, moderately acidic, stabilized by resonance

• Alcohol or water O: pKa ~15-16, weakly acidic, holds H firmly in normal conditions

• Protonated N: pKa ~10, loses proton in neutral-basic pH

• Neutral amine N (NH2): pKa ~35, strong bases grabbing protons from weaker acids

Henderson Hasselbalch to determine likely protonation state at a given pH

(equation given on sheet)

Plug given pH and pKa into equation. Put everything at base 10 to remove log. Calculate ratio of deprotonated to protonated material to predict how the molecule with react.

Salt bridge

Combination of electrostatic attraction and a hydrogen bond between amino acid side chains

Why don’t peptide bonds rotate freely

Resonance gives them double bond character which prevents them from rotating freely

Why are dihedral angles limited for amino acids?

Steric hindrance between atoms as well as the planar C-N backbone stuck by double bond character

Trans vs cis for peptide bonds, why is one favored?

Cis structure causes steric clash between the C-alphas and R groups

Special properties of glycine

Smallest amino acid means high flexibility because virtually no steric hindrance

Often helps facilitate beta-turns because of flexible geometry

Special properties of proline

Cyclic secondary amine, the R group covalently bonds to its own backbone N atom, causing geometric constraints from restricted rotation of N-alpha C bond

Acts as a “helix breaker” in alpha helices because it can’t coil properly, initiates beta-turns

Ramachandran plots

Describe “allowed conformations” of amino acids

x-axis measures angle for N and C

y-axis measures angle for alpha-C and carbonyl C

Primary protein structure

Amino acid sequence

Secondary protein structure

Local spatial arrangement of the polypeptide backbone with regards to the conformations of the R groups

Tertiary protein structure

3D structure of the entire protein including R groups

Quaternary protein structure

How different “chains” or “subunits” of polypeptide chains within a protein are arranged

Alpha helices

Stabilized by hydrogen bonding of peptide backbone

R group points out of helix

Right-handed coil (thumb can be carbonyl group pointing to H-bond with NH group of amino acid four residues later)1

Beta sheets

Form from hydrogen bonds between neighboring polypeptide chains

All backbone hydrogen bonding opportunities taken (backbone)

Can be antiparallel (C and N termini opposite) or parallel (matching termini, longer bond distance)

Motifs

AKA folds, recognizable folding patterns involving 2 or more secondary structure elements (ex.ß-barrels, zinc finger)

Intrinsically disordered proteins

Many proteins have intrinsically disordered regions with no alpha helices or beta sheets, yet they still have functions in the cellpr

Protein native structure

Fully folded biologically active 3D shape of a polypeptide chain under physiological conditions

Chaperones

Aid misfolded or intermediate protein states before they become too unstable and dangerous

GroEL

Works as a chaperone to protein folding, provides a safe folding environment in its “folding cavity”

Has large hydrophobic regions exposed to interact favorably with improperly folded residues and bind to it

When substrate is bound, GroEL gains cap to close cavity and make hydrophilic environment to force proper folding

Creates safe pocket for folding without possible aggregates or degradation

Chromatography

Purifies proteins by separating molecules of interest from a mixture with a stationary and mobile phase

Stationary phase: fixed phase (solid matrix or gel)

Mobile phase: freely moving phase in which molecule is dissolved

First must lyse cells to release protein of interest and bind it to the stationary phase, then wash, and then elute the protein

Cation chromatography

Separates molecules according to charge

Cation exchangers (often resin) = negatively charged and bind to cations

The binding of a protein to the exchange resin depends on the salt ion concentration in the mobile phase buffer and the pH of the mobile phase buffer (bc of charge states of amino acid R groups + salt ions compete with protein for resin binding)

His tag purification

Affinity purification strategy, several His residues added to C or N terminus of a protein and act as the affinity handle.tag

NTA forms basis of affinity resin; charged with nickel to make Ni-NTA that can bind to histidine R groups (His tag velcro)

To release, imidazole (R group of His) outcompetes protein for nickel bonding and molecule is released

Unit of protein molecular weight

kilodaltons (kDa) where 1 Da = 1 g/mol, so 1 kDa = 1 kg/mol = 1000 g/mol

SDS-PAGE

Technique used to separate proteins by size and then visualize them like bands on a gel

Run vertically, higher up = larger, lower = smaller

Wash steps should remove all but the desired molecule if buffer salts and pH are correct so that elution brings out what you want

Metal chelator

Molecules that bind to metals very tightly, forming a cage ring-like structure (ex EDTA or NTA)

NMR

Process: need very pure, NMR spectroscopy, assign NMR peaks

Sample: very small, very pure, flexibility OK, radioisotopes = hard

Best for: Determining dynamics of sample structure (conformational changes, interactions between molecules)

X-ray crystallography

Process: purify protein, get crystals, bring crystal to x-ray, determine diffraction patter, interpret results

Sample: must be extremely pure, can’t be too flexible, smaller = better, has to crystallize (sometimes hard)

Best for: Small, well-ordered samples + situations where need very good resolution (ex protein-drug interactions)

Cryo-electron microscopy

Process: purify proteins in good grid conditions, collect large microscopy datasets, process microscope images to build model

Sample: pure but not as pure, flexibility OK, bigger = better

Best for: large complex structures, samples can be lower concentration and purity than for other methods