Biochemistry Exam 1 First Review

Amino Acids, Peptides and Proteins

Proteins: Main Agents of Bio Function

~Catalysis?

• DNA polymerase (in DNA replication)

• RNA polymerase (Transcription)

Proteins: Main Agents of Bio Function

~Transport

• hemoglobin (transports O2 in the blood)

• myoglobin (storage of O2)

• lactose permeate (transports lactose across the cell membrane)

Proteins: Main Agents of Bio Function

~Structure

• collagen (connective tissue)

• keratin (hair, nails, feathers, horns)

Proteins: Main Agents of Bio Function

~Motion

• myosin (muscle tissue)

• actin (muscle tissue, cell motility)

Luciferase

Example: light produced by a firefly

Keratin

fingernail, common contaminants in MS assay

example: rhino horn

General Structure of an Amino Acid

Contains:

• carboxyl group

• hydrogen

• side chain, different in each amino acid

• amino group

• chiral center

What amino acid is an exception for the general structures of AAs?

Proline ; P ; Pro

Amino Acids Classification: Nonpolar, aliphatic R Groups

Glycine (G), Alanine (A), Proline (P), Valine (V), Leucine (L), Isoleucine (i), Methionine (M)

AA Classification: Aromatic R groups

Phenylalanine (F), Tyrosine (Y), Tryptophan (W)

Are the Aromatic R groups non-polar or polar? hydrophobic or hydrophilic? Which two absorb more light than the other?

• Non-polar

• Hydrophobic: normally buried inside the protein core

• Y and W absorb UV light more than F

AAs Classification: Polar uncharged R groups

Serine (S), Threonine (T), Cysteine (C), Asparagine (N), Glutamine (Q)

What is the smallest amino acid?

Glycine (G)

What kind of bonds are formed in polar, uncharged R groups?

Hydrogen bonds

What kind of bonds can Cysteine (C) form?

Disulfide bonds

AAs Classification: Positively charged R groups

Lysine (K), Arginine (R), Histidine (H)

Which AA has an imidazole group attached?

Histidine (H)

What are ampholytes?

Substances that have dual acid and base nature

Example of ampholyte?

Alanine (A)

Formation of Peptides: What are peptides? Are they smaller or larger than proteins?

peptides are small condensation products of amino acids. They are smaller than proteins

Where does numbering and naming peptides start from?

Amino terminus

Practice AA code: Serylglycyltyrosylalanylleucine

SGYAL

What is the average molecular weight of AAs?

138 da

Weight of smaller AAs predominate in proteins?

128 da

Techniques used for protein purification?

• Extraction

• Precipitation: ammonium sulfate

• dialysis

• ultracentrifugation

• size exclusion chromatography

• ion exchange chromatography

• metal binding

• purification of a tagged protein

Techniques used for protein extraction?

• Grinding with or without liquid N

• Grinding with sand

• Bead beater

• sonication

• French press

• buffer with or without mild detergent

Explain what precipitation of ammonium sulfate looks like

What is dialysis used for?

It gets rid of ammonium sulfate

What is SDS? What do its micelles do?

• SDS Is sodium dodecyl sulfate - a detergent -

  • SDS gives all proteins uniformly negative charge

• SDS micelles bind to and unfold all the proteins

What is Electrophoresis? What is it checking in this image?

(SDS-PAGE) Electrophoresis a method to separate proteins by size using an electric field. It is checking each step of the purification process and shows the purification of induced RecA protein getting purer at each stage.

Steps of SDS

• a protein will bind to SDS

• Bound SDS will contribute to negative charge

• unmask intrinsic charge of protein

• >all protein will have similar charge to mass ratio

Levels of Structures In Proteins?

• Primary - amino acid residues - sequence, peptide bond and disulfide bond

• secondary structure - alpha helix - local structure, piece of tertiary structure

• tertiary structure - polypeptide chain (three dimensional)

• quaternary structure - assembled subunits - a protein has two or more polypeptides

The polypeptide is made up of a series of planes linked at _____ carbons.

alpha

X-ray diffraction showed that C-N bond is shorter than regular C-N bond indicating….? Can partial bonds rotate freely

resonance or sharing of two pairs of electrons. partial double bonds cannot rotate freely.

What are secondary structures?

They refer to a local spatial arrangement of the polypeptide backbone.

What two regular secondary structure arrangements are common and what are they stabilized by?

The alpha helix:

• stabilized by hydrogen bonds between nearby residues.

The beta sheet:

• stabilized by hydrogen bonds between adjacent segments that may not be nearby

What is a random coil?

irregular arrangement of the polypeptide chain

What is an alpha helix? Are they right or left handed helices? How are peptide bonds aligned with the helical axis? Are side chains pointing out or in? Are they perpendicular with or alongside the helical axis?

• helical backbone held together by hydrogen bonds between the backbone amides of an n and n+4 AAs.

• right handed

• roughly parallel

• They point out

• They are roughly perpendicular with helical axis

T/F? Sequence affects helix stability

True

Do all polypeptide sequences adopt an alpha helical structure?

No

What small hydrophobic residues AAs are strong helix formers?

Ala (A) and Leu (L)

What two AAs act as helix breakers?

Pro (P) - because rotation around N-C bond is impossible

Gly (G) - because tiny R-groups support other conformations

T/F Parallel or antiparallel orientation of two chains within a sheet are impossible.

False

In parallel B sheets, do the H-bonded strands run in the same or opposite directions? Do they result in bent or linear H-bonds? Are they weaker or stronger?

• same direction

• bent

• weaker

In antiparallel B sheets, do the H-bonded strands run in the same or opposite directions? Do they result in bent or linear H-bonds? Are they weaker or stronger?

• opposite

• linear

• stronger

What happens during permanent waving?

What is fibroin? Does it have antiparallel or parallel beta sheet structure? What AA side chains allow the close packing of sheets? What is the structure stabilized by?

• The main protein in silk from mothers and spiders

• Antiparallel

• A and G

• hydrogen bonding within sheets and London dispersion interactions between sheets

What is spider silk used for? Is it a strong or weak material? Is it flexible? How is it a composite material?

• used for webs, egg sacks, and wrapping prey

• extremely strong material that is stronger than steel and can stretch A LOT

• It is composite with crystalline parts and rubber-like stretchy parts

Steps needed for X-ray Crystallography

• purify protein

• crystallize protein

• collect diffraction data

• calculate electron density

• fit resides into density

Pros and Cons of X-ray Crystallography

Pros:

• no size limits

• well-establish

Cons:

• difficult for membrane proteins

• cannot see hydrogens

What does x-ray crystallography do?

it measures the locations and intensities of spots produced by a be a of X-ray.

Steps of Biomolecular NMR:

• purify protein

• dissolve protein

• collect nmr data

• assign nmr signals

• calculate structure

Pros and Cons of Biomolecular NMR

Pros:

• no need to crystallize protein

• can see many hydrogens

Cons:

• difficult for insoluble proteins

• works best with small proteins

What is NMR (nucleic magnetic resonance)? What happens when static magnetic field is applied?

• manifestation of nuclear spin angular momentum

• nuclear spin generates dipoles

A proteins function depend on its ____.

3D structure

What is denaturation?

Loss of structural integrity with accompanying loss of activity

How can proteins be denatured?

• heat or cold

• pH extremes

• organic solvents

• chaotropic agents: urea and guanidinium

What do chaperones prevent? What do they facilitate?

• Misfolding

• folding

What kind of human diseases are the basis of protein folding?

Amyloidoses: Alzheimer disease, Huntington disease,

Parkinson diseases