Protein- DLA Primary Structure

Question 1

What are the 4 levels of protein organization?

Answer 1

Primary

Secondary

Tertiary

Quarternary

Question 2

Describe the primary structure of proteins

Answer 2

• sometimes called “beads on a string”

- sequence of amino acids=primary structure

Question 3

How are primary protein structures related to genetic diseases?

Answer 3

Many genetic disorders result from proteins that contain abnormal sequences

   - Recall the central dogma of cellular biology
   - DNA-> RNA-> protein

Question 4

What’s the purpose of insulin?

Answer 4

Lowers blood sugar by glucose absorption

Question 5

How are peptide/amide bonds formed?

Answer 5

Formed by ribosome, dehydration synthesis

Question 6

Describe the possible breakage of the peptide bond

Answer 6

• Not broken when proteins are denatured
• Prolonged exposure to acid or base at high temperatures is necessary to break bonds (hydrolysis)
• Nature has many enzymes to cleave peptide bonds
  • these are proteases
  • More on this later

Question 7

In what direction are amino acids named?

Answer 7

From the amino terminus to the carboxy terminus

Question 8

How are peptides named?

Answer 8

We always read from left to right

• Nature creates N-> C so we read the same way
• Left(an-terminus aa) is written first with C is next

Question 9

What are component amino acids in peptides?

Answer 9

Moieties or residues

Question 10

How are polypeptides named?

Answer 10

Except for the C-terminal amino acid, all moieties have the suffix changed to -yl

• the -ine, -ate, or doc is dropped

E.g. valylglycylleucine

Question 11

What reaction forms a peptide/amide bond?

Answer 11

A peptide bond is formed from a condensation reaction (dehydration) involving two amino acids as a molecule of H2O is eliminated

Question 12

What kind of configuration is formed by R groups when a peptide bond is Formed ?

Answer 12

-Ribosome forms amino acids with a trans configuration with respect to side chains

-

Question 13

What are the characteristics of a peptide bond?

Answer 13

Lack of rotation around the peptide bonds

• this partial double bond is rigid and planar between the carbonyl oxygen and amide nitrogen due to to the partial pi bond to cross/break it is to great, it cannot rotate
• the bond between the alpha-Carbon and alpha-amino is rotatable
• The bond between the alpha-C and alpha-CO group is also rotatable

Question 14

Why is trans configuration good for peptide bonds?

Answer 14

It minimizes steric hindrance, trans-good cis- bad

Steric hindrance due to the side chains

Question 15

What are the Chemical Characteristics of the peptide bond?

Answer 15

• peptide bind is uncharged but polar:
• In amide linkages, the N or O atoms in a peptide bond do accept protons betwee pH 2-12(no acid base chemistry)
• Polypeptides have a limited ability for ionized groups to form (COO-, NH3+)
• the bond itself is polar but due to O and N atoms, hydrogen bonds may form

Question 16

What cases the partial double bond character of peptide bonds?(comeback for structures)

Answer 16

It is due to resonance

Question 17

How can rotation be affected by the kind of protein that is made from its amino acids?

Answer 17

Rotation and location of R groups might have limitations based on what the amino acids are

Question 18

The ____________________ of the peptide bond is restricted

Answer 18

Stereochemistry

Question 19

Why is the peptide bond uncharged yet polar?

Answer 19

There is a partial separation of charge which forms a permanent dipole at peptide bonds

Question 20

Peptide bonds are…

Answer 20

Rigid and planar

Question 21

Explain the determination of amino acid composition of a polypeptide by acid hydrolysis

Answer 21

• First, identify and quantify constituent amino acids
• pure sample must be used, contamination gives errors

Acid hydrolysis:

• Hydrolyzed by strong acid at 110 degrees Celsius for 24 hours
• Peptide binds cleaved
• Gln and Asn-> Glu and Asp; Trp mostly destroyed
• gives composition but nit sequence

Question 22

List methods to determine amino acid composition

Answer 22

1. Acid hydrolysis
2. Chromatography
3. Quantitative analysis
4. Sequencing from N-terminal(phenylisothiocyanate-Edman’s reagent)- gives sequence

Question 23

How can Chromatography be used to determine amino acid composition?

Answer 23

Individual amino acids separated by ion exchange chromatography

     - cation-exchange for negatively charged amino acids
     - anion-exchange resin for negatively charged amino acids

Elated from column by buffers of increasing ionic strength and pH

Amino acids separated at different ionic strength and pH

Question 24

How can amino acids be separated and identified by quantitative analysis?

Answer 24

• quantified with ninhydrin-> purple compound with amino acids, NH3 and amines(yellow color with imino group of Pro
• intensity of color measured with spectrophotometer
• Area under curve proportional to amount of amino acid
• From this, if MW of protein is known, then can calculate the number of residues of each amino acid in the protein
• Done in amino acid analyzer

Question 25

How can amino acids be sequenced from the N-terminus?

Answer 25

• Phenylisothiocyanate-Edman’s reagents used to label N-terminal residue under mildly alkaline conditions -> phenythiohydanton(PTH)
• This covalent modification the N-terminal residue peptide bond weak, it can then be cleaned without breaking the other peptide bonds and be identified
• Above process occurs in a repetitive cycle to sequence peptide using “sequenator”

Question 26

What is the limit of amino acids in a protein which can be used in N terminus sequencing ?

Answer 26

For polypeptides of up to 100 amino acids

Some is lost each time, so sample runs out