Biochem 1

What determines an amino acid’s hydrophobicity or hydrophilicity?

Amino acid side chain (R group)

Name six hydrophobic amino acids.

Alanine, Isoleucine, Leucine, Phenylalanine, Proline, Valine

Name six hydrophilic amino acids.

Arginine, Asparagine, Aspartic acid, Glutamic acid, Histidine, Serine

What is a peptide bond?

A covalent bond between amino acids formed by condensation (loss of water)

What is the directionality of a protein sequence?

From the N-terminus to the C-terminus

What differentiates a peptide, polypeptide, and a protein?

Peptides: <20 residues

What is a heteromultimer?

A protein composed of different polypeptide chains

What are the four levels of protein structure?

Primary (sequence), Secondary (helices/sheets), Tertiary (3D fold), Quaternary (subunit assembly)

What is the average molecular weight of an amino acid in proteins?

Approximately 110 Da (after accounting for water loss per peptide bond)

What's the difference between preparative and analytical purification?

Preparative yields usable protein

Which properties are used to purify proteins?

Size, charge, binding affinity, hydrophobicity, solubility

What does SDS-PAGE separate proteins by?

Size (molecular weight)

What effect does SDS have on proteins?

Denatures them and gives uniform negative charge

What does native PAGE preserve?

Protein shape, size, and charge

How does differential centrifugation separate components?

Based on size and density

When are proteins least soluble?

At their isoelectric point (pI)

What is "salting out"?

Precipitating proteins by adding high salt to outcompete water for solvation

What is ion exchange chromatography (IEX) based on?

Charge differences between proteins

How does hydrophobic interaction chromatography (HIC) work?

Binds hydrophobic residues in high salt

How does affinity chromatography achieve high specificity?

Uses a ligand that selectively binds the target protein

What does size exclusion chromatography (SEC) separate by?

Molecular size

Who first sequenced a protein and which one?

Fred Sanger

What are the main steps in protein sequencing?

Chain separation → disulfide reduction → cleavage → peptide sequencing → sequence assembly

What does trypsin cleave after?

Arg and Lys (basic residues)

What does cyanogen bromide (CNBr) cleave after?

Methionine

What does Edman degradation do?

Sequentially removes and identifies the N-terminal amino acid

What reagent is used in Edman degradation?

Phenylisothiocyanate (PITC)

What does MS (Mass Spectrometry) measure?

Mass-to-charge ratio (m/z) of ionized molecules

What is ESI in mass spectrometry?

Electrospray Ionization—produces ions from solution

What is MALDI used for?

Producing ions using a laser and matrix—good for intact proteins

What does TOF stand for in mass spectrometry?

Time-of-Flight—measures ion velocity to determine mass

What is peptide mass fingerprinting?

Identifying proteins by comparing tryptic peptide masses to database predictions

What happens in tandem mass spectrometry (MS/MS)?

Peptides are fragmented, and the resulting ion series reveals sequence information

What are b- and y-ions in MS/MS?

b-ions: N-terminal fragments