BCHM 312: Exam 2

Factors that can disrupt IMFs and cause unfolding (G>)

• extreme pH levels

• chemical denaturants

• heat

Melting Point of a Protein

50% unfolded

If a protein has a higher melting point

• harder to denature

• more stable tertiary structure

• more negative G folding

Proteases

proteins that hydrolyze protein bonds by catalyzing water-mediated hydrolysis

Insulin

• an alpha and beta chain

• 3 disulfide bonds to stabilize tertiary structure

Conservative Mutations

replaced amino acid with similar biochemical properties

Non-Conservative Mutations

replaced amino acid with different biochemical properties

Protein Purification Steps

1. Lysis of cells —> lysate

2. Fractionation —> separate based on size, charge, and affinity

Size-Exclusion Chromatography

• separate based on size

• largest elutes first

• smallest elutes last

• mech: largest bypasses the pores, smallest travels through the pores

SDS-PAGE

• gel electrophoresis after column chromatography

• approx. MW

• denatures proteins; cannot purify it

• disulfide bonds cleaved by reducing agents

• largest at top; smallest at bottom

Affinity Chromatography

• separate based on affinity for another molecule

• antibodies: seq with most matches to the desired tag will elute last

Cation-Exchange Chromatography

• negative resin attracts the positive ions

• elute by changing: pH and concentration

Isoelectric Point (pI)

pH of a molecule when it is neutral charged

• high pI = positively charged molecule

Isoelectric Focusing

determines the pI of protein

• when towards positive node, it is negative molecule

• when towards negative node, it is positive molecule

2D Electrophoresis

• y-axis represents MW

• x-axis represents pI

2 types of protein interactions

1. alter chemical structure of bound molecule

2. chemical structure is unchanged after binding

Dissociation Constant

[P][L] / [PL] = Kd

Kd trends

• smaller Kd = stronger affinity interaction

Y

• fraction of occupied binding sites

• Y = [L] / [L] + Kd

• when Kd = [L], 50% occupancy

• depends on # of Ligands and Kd strength

Ligand binding is stabilized by

• steric complementarity (fit?)

• IMFs (stick?)

Heme

• prosthetic group for protein folding and function

• 6 coordination bonds to Fe2+

  • 4 N atoms in poryphorin ring

  • 1 O2 on top

  • 1 His from myoglobin at bottom

Myoglobin

1 polypeptide chain with 153 AA

• 1 heme = 1 O2 molecule

• hyperbolic binding curve

Hemoglobin

Tetramer

• 4 heme groups = 4 O2 molecules

• 2 alpha and 2 beta chains in adults

• sigmoidal binding curve

R state

• relaxed (tight curve)

• Presence of O2

• High O2 affinity

• Less 2,3-BPG

• pH increase

• pO2 increase

• pCO2 decrease

• ion pairs broken

T state

• tense (flatter curve)

• Absence of O2

• Low O2 affinity

• More 2,3-BPG

• pH decrease

• pO2 decrease

• pCO2 increase

• ion pairs formed

Ion Pairs

stabilize T state

• His-Lys

• His-Asp

Positive Allosteric Modulators (PAMs)

• improves function

• stabilize R state

  • less O2 to tissues

NAMs

• decreases function

• stabilize T state

• more O2 to tissues

Bohr Effect

pH dec —> more His protonation —> ion pairs more stable (formed) —> T state

CO2 inc —> Neg N.Term from carbamination —> pH dec —> T state bc electrostatics