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What are the 4 levels of protein structure?
- Primary
- Secondary
- Tertiary
- Quaternary
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What is the primary structure of proteins?
- Amino acid residues
- Amino acids linked by peptide bonds to form peptides or proteins
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What is the secondary structure of proteins?
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What is the tertiary structure of proteins?
Polypeptide chain
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What is the quaternary structure of proteins?
Assembled subunits
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Amino acids are linked to other amino acids by what kind of bonds?
Peptide bonds
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Describe peptide bonds
- Very stable
- Half-life is approximately 70 years at neutral pH
- Peptide bond hydrolysis requires acid or base, plus heat
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What is the average molecular weight of an amino acid in a peptide or protein?
Considered to be 110
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Proteins with different function vary greatly in what way?
Vary in composition
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What makes a protein polymorphic?
Difference in sequence when comparing across different organisms from the same species
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What are the proteins called that also include non-amino acid components?
Conjugated proteins
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Why was the sequencing of insulin significant?
- It was thought to be impossible
- Also the year Watson and Crick figured out the DNA structure
- The relationship between proteins and DNA soon became clear
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Why is it important to know the sequence of the protein we are studying?
Essential to further biochemical analysis
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The actual sequence of a protein is generally determined from what?
DNA sequence
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What are the two methods of studying the primary structure of proteins/peptides?
- Edman Degradation (Classical method)
- Mass Spectrometry (Modern method)
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What is the classical method of studying the primary structure of proteins/peptides?
Edman Degradation
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What is the modern method of studying the primary structure of proteins/peptides?
Mass Spectometry
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What is Edman Degradation?
Successive rounds of N-terminal modification, cleavage, and identification
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What is Edman Degradation used for?
Can be used to identify protein without known sequence
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What is mass spectrometry?
MALDI MS and ESI MS can precisely identify the mass of a peptide, and thus the amino acid sequence
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What is mass spectrometry used for?
Can be used to determine post-translational modifications
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What How did Sanger sequence insulin?
- Amino terminal amino-acid residue is first labeled with a compound like 1-fluoro-2,4-dinitrobenzene (FDNB), that won't denature the protein
- Acid is then used to hydrolyze the peptide bond (acid hydrolysis)
- Partial acid hydrolysis will produce peptides of specific length, which can be separated
- This process can be repeated
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What is Edman's Degradation?
- Peptide bond nearest the amino terminus of the protein or polypeptide is cleaved in two steps
- Two steps are carried out under very different reaction conditions
- This allows one step to proceed to completion before the second is initiated
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What are the conditions for the two steps in Edman's Degradation?
- Basic conditions in step 1
- Acidic conditions in step 2
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What what point does Edman's degradation become inefficient?
For peptides w/ >50 AAs
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How can disulfide bonds be broken?
Through oxidation or reduction
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What can be used in the reduction process of breaking disulfide bonds?
Dithiotheitol (DTT) or β-mercaptoethanol
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Why is the second reaction in the reduction process of breaking disulfide bonds necessary?
To prevent the reformation of the disulfide bond
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What do proteases do?
Catalyse the hydrolytic cleavage of peptide bonds
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What are the only kinds of peptide bonds that most proteases cut?
Bonds that are adjacent to a particular amino acid residue
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Why do many proteases only cut a peptide bond that is adjacent to a particular amino acid residue?
They fracgment the peptide in a reproducible and predictable manner
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Where does trypsin cleave?
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Where does chymotrypsin cleave?
Arg (C)
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Where does V8 protease cleave?
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Where does pepsin cleave?
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Where does cyanogen bromide cleave?
Met (C)
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Currently, what processes have yielded millions of protein sequences?
Genetic sequencing and translation using DNA code
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What does mass spectrometry measure?
Provides a highly accurate measure of the molecular weight of a protein
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What does mass spectrometry provide?
- A highly accurate measure of the molecular weight of a protein
- Specific sequences of short polypeptide segments
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Previously, why couldn't mass spectrometry be applied to macromolecules like proteins and nucleic acids?
Because they would be destroyed when heated to the gas phase
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What were some new methods of mass spectrometry that could be applied to macromolecules?
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What is MALDI MS?
- Matrix-associated laser desorption
- Proteins are placed in a light absorbing matrix
- A short pulse of laser light, the proteins are ionized and desorbed from the matrix into the vacuum system
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What is ESI MS?
Electrospray ionization mas spec
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