Unit 12 - Protein Structure Determination

What does protein composition analysis reveal?

The types and quantities of amino acids, but not their sequence.

What chemical is used to hydrolyze all amide bonds in composition analysis?

6 N HCl at 100–110°C for 12–36 hours.

What amino acids are degraded under hydrolysis conditions?

Serine, threonine, tyrosine, tryptophan.

Which residues are converted during hydrolysis and can’t be detected as-is?

Glutamine → glutamate, asparagine → aspartate.

What reagent is used for N-terminal analysis?

Dansyl chloride.

What does dansyl chloride react with?

The α-amino group (and lysine side chains).

How are dansyl-labeled amino acids identified?

By standards and chromatography after hydrolysis.

What are exopeptidases?

Enzymes that remove amino acids from the ends of peptides.

What are endopeptidases?

Enzymes that cleave within the peptide chain.

What do carboxypeptidases do?

Cleave amino acids one-by-one from the C-terminus.

What do aminopeptidases do?

Cleave amino acids from the N-terminus.

Why is time-course analysis used with carboxypeptidases?

To monitor the sequential release of amino acids.

What is the limitation of carboxypeptidase analysis?

It usually reveals only the last few residues.

How can disulfide bonds interfere with sequencing?

They prevent chain separation and interfere with cleavage.

How are disulfide bonds reduced for sequencing?

With β-mercaptoethanol.

Why is iodoacetate used after reduction of disulfides?

To prevent disulfide bonds from reforming by alkylation.

What sequencing method starts at the N-terminus?

Edman degradation.

What reagent is used in Edman sequencing?

Phenylisothiocyanate (PITC).

What does Edman degradation release?

One amino acid at a time as a stable derivative for identification.

How many residues can Edman sequencing accurately identify?

Typically 30–50.

Why can’t Edman sequencing proceed through disulfide bonds?

Because they link chains and block N-terminal access.

What enzymes are used to cleave proteins into fragments for sequencing?

Trypsin and chymotrypsin.

Where does trypsin cleave?

After Lys or Arg (unless followed by Pro).

Where does chymotrypsin cleave?

After Phe, Tyr, or Trp (unless followed by Pro).

What chemical cleaves peptides at methionine residues?

Cyanogen bromide (CNBr).

What bond does CNBr cleave?

After methionine, forming a lactone.

What are the typical steps in sequencing a protein?

Purify, reduce/alkylate, digest with proteases, separate fragments, sequence via Edman.

What’s the purpose of chromatography after digestion?

To separate peptide fragments for individual analysis.

How can peptide fragments be aligned into the full sequence?

By overlapping sequence data from different cleavage methods.

What is an example of overlap strategy?

Trypsin and CNBr fragments aligned to reconstruct full sequence.

Why sequence both ends of the protein?

To determine directionality (N- to C-terminal order).

What method can be used instead of Edman for short peptides?

Mass spectrometry.

What does MS measure to identify peptides?

Mass-to-charge ratio (m/z).

What does MALDI-TOF stand for?

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight.

What is electrospray ionization (ESI)?

A soft ionization method used in mass spectrometry for peptides.

What is a typical output of mass spectrometry?

A spectrum of m/z values representing fragment ions.

What facility equipment is used in WATSPEC?

Micromass Q-TOF, JEOL HX110, Agilent GC/MS, Bruker MALDI-TOF.

Why compare a new protein’s sequence to known databases?

To infer function from homology.

What is the benefit of finding a related protein with a crystal structure?

It can help model your protein's 3D structure.

How can expression conditions help identify proteins?

By exposing cells to treatments and analyzing overexpressed proteins.

What’s the value of DNA sequencing in protein studies?

It can be translated into the protein’s amino acid sequence.

What is the main challenge of sequencing long proteins?

Edman sequencing isn’t effective past 50 residues.

What is a practical solution to sequence long proteins?

Sequence DNA instead or use overlapping peptide fragments.

What did the Lecture 12 example involve?

Sequencing an antibacterial octapeptide from a soil microorganism.

What were the peptide’s components in the example?

Ala, Lys, 2Gly, Ser, Met, Tyr, Thr.

Which techniques were used to analyze the peptide?

CNBr, trypsin, chymotrypsin cleavage, dansylation, carboxypeptidase.

What was the final sequence of the octapeptide?

Gly–Tyr–Ser–Met–Thr–Lys–Ala–Gly.

How did CNBr help in the example?

It cleaved after methionine, allowing fragment ordering.

What does a dansyl chloride reaction identify?

The N-terminal amino acid.

What does carboxypeptidase analysis identify?

The first amino acid released from the C-terminus.