1 survey of biochemistry proteins and biomolecular stability

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SURVEY OF BIOCHEMISTRY

Proteins and Biomolecular Stability

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Protein Structure

• Primary (1°): amino acid sequence

• Secondary (2°)– Alpha Helix– Beta Sheet

• Tertiary (3°)

• Quaternary (4°)

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Primary and Secondary Structure

Myoglobin - 2V1K Superoxide Dismutase - 1XSO

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Recap

• Structures of 20 amino acids

• pKa and pI

• 1°: Polypeptide Sequence

• 2°: Secondary Structures– Alpha Helices – Beta Sheets

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Recap continued

• Protein Purification Methods– Gel Filtration– Ion Exchange– Affinity

• How to assess purification?– Purity – Yield

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SDS-PAGE

• Electrophoresis: a method for separating molecules based on size and charge when exposed to an electric field.

Name “SDS-PAGE”:

SDS = sodium dodecyl sulfate

PAGE = polyacrylamide gel electrophoresis

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Sodium Dodecyl Sulfate

SDS confers negative charge on proteins and denatures proteins

Sodium Dodecyl Sulfate(Lauryl Sulfate)

CH3(CH2)11OSO3

Amphiphilic

Hydrophilic Hydrophobic

Anionic Detergentin a wide variety of products

Proteins are primarily denatured by boiling them

prior to electrophoresis!

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Buffers maintain pH control Allow gel to fully polymerize

Stacking Gel

0.5M Tris-HCl

pH 6.8

Resolving Gel

1.5M Tris-HCl

pH 8.8

SDS-PAGE Buffers

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Ensure that sample has fully dentured!

Stacking Gel

0.5M Tris-HCl

pH 6.8

Resolving Gel

1.5M Tris-HCl

pH 8.8

In the Gel In the Sample

Laemmli Sample

Buffer

0.5M Tris-HCl,pH 6.8

SDS

Glycerol

Bromophenol Blue

Sample Preparation

Boil Sample for 1-5 min

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Electrophoresis Buffer

H3N-CH2-COOH+

AcidicForm

H3N-CH2-COO+ -

ZwitterionicForm

BasicForm

H2N-CH2-COO -

FullyProtonated

Loss of 1Proton

Loss of 2Protons

Tris Base, Glycine, SDS

Electrophoresis Buffer

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Stacking Gel0.5M Tris-HCl

pH 6.8

Resolving Gel1.5M Tris-HCl

pH 8.8

In the Gel

H3N-CH2-COO

H2N-CH2-COO

+ -

Zwitterion Form

-

Basic Form

SDS-PAGE

Gly lags

Gly leads

Note: Discontinuous SDS-PAGE is depicted here!

12+

-

Migration in an SDS-PAGE Gel

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Migration in an SDS-PAGE Gel

Stop electrophoresis when dye front reaches

bottom of gel

Stain with Coomassie

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Purity

• Purity is a measure of how undefiled a protein sample is.

Lots of impurities

Pure protein

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Yield

% Yield =Amount of protein recovered

Amount of protein initially

Example:

1

2

3

% Yield = (208 / 358.2) x 100 = 58.1%After 2 steps of purification

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Protein Sequencing

• Separate subunits

• Dansyl Chloride Reaction

• Proteolytic Digestion

• Cyanogen Bromide Cleavage

• Edman Degradation

Read on yourown!

Study how each works!

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How to separate subunits?

Dithiothreitol, DTT 2-Mercaptoethanol

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Proteolytic Digestion

Know this!

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Proteolytic Digestion

How many fragments would result from digestion with trypsin?

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Proteolytic Digestion

Trypsin cleaves after Lys (K) and Arg (R):

16 fragments!

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Protein Structure Classifications

• 1°: amino acid sequence

• 2°: local spatial arrangement of a polypeptide backbone without regard for side chains

• 3°: 3D structure of a protein including its side chains

• 4°: spatial arrangements of subunits

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Tertiary Folds

Alpha Beta

Some proteins only have

alpha helices (plus turns and random coils).

Others only have beta

sheets (plus turns and random coils).

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Alpha/Beta Tertiary Folds

Some proteins have a combination of alpha

helices and beta sheets(plus turns and random

coils).

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Biomolecular Stability

Nucleic acids and proteins are stabilized by the same types of intermolecular forces.

Hydrophobic Effect: the tendency of water to minimize its contact with

hydrophobic groups in molecules.

How does the hydrophobic effect impact proteins and nucleic acids?

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Entropy

Entropy measures the spontaneous dispersal of

energy:

how much energy is spread out in a process

-or- how widely spread out it becomes — at a specific

temperaturehttp://www.entropysite.com

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Entropy & the Hydrophobic Effect

How is entropy increased by the hydrophobic effect?

http://www.cryst.bbk.ac.uk/PPS2/projects/day/TDayDiss/Major.html

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How do bond energies compare?

Type of Bond Bond Strength (kJ/mol)Covalent 348 - 460 Ionic Interaction 86Hydrogen Bond 20Dipole-dipole 9.3London Dispersion 0.3

Table 2-1

Relatively speaking, H-bonds are weak,but they are not nearly as weak as one might expect!

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Why Do Base Pairs Stack?

ENTROPY

Hydrophobic effect induces release of

water “binding” to DNA bp’s such that the hydrophobic ring

systems can stack on top of each other to

minimize contact with water.

Consider the magnitude of stacking

energy…Etc.

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Forces Stabilizing Biomolecule Structure

Proteins Nucleic Acids

Hydrophobic Effect Hydrophobic Effect Globular shape Base Stacking

Disulfide Bonds

H-Bonds H-bonds Alpha Helices Base Pairing Beta Sheets

Ionic Interactions Ionic Interactions Salt Bridges Metal Ions