Protein- DLA Primary Structure Flashcards
What are the 4 levels of protein organization?
Primary
Secondary
Tertiary
Quarternary
Describe the primary structure of proteins
- sometimes called “beads on a string”
- sequence of amino acids=primary structure
How are primary protein structures related to genetic diseases?
Many genetic disorders result from proteins that contain abnormal sequences
- Recall the central dogma of cellular biology - DNA-> RNA-> protein
What’s the purpose of insulin?
Lowers blood sugar by glucose absorption
How are peptide/amide bonds formed?
Formed by ribosome, dehydration synthesis
Describe the possible breakage of the peptide bond
- 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
In what direction are amino acids named?
From the amino terminus to the carboxy terminus
How are peptides named?
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
What are component amino acids in peptides?
Moieties or residues
How are polypeptides named?
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
What reaction forms a peptide/amide bond?
A peptide bond is formed from a condensation reaction (dehydration) involving two amino acids as a molecule of H2O is eliminated
What kind of configuration is formed by R groups when a peptide bond is Formed ?
-Ribosome forms amino acids with a trans configuration with respect to side chains
-
What are the characteristics of a peptide bond?
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
Why is trans configuration good for peptide bonds?
It minimizes steric hindrance, trans-good cis- bad
Steric hindrance due to the side chains
What are the Chemical Characteristics of the peptide bond?
- 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
What cases the partial double bond character of peptide bonds?(comeback for structures)
It is due to resonance
How can rotation be affected by the kind of protein that is made from its amino acids?
Rotation and location of R groups might have limitations based on what the amino acids are
The ____________________ of the peptide bond is restricted
Stereochemistry
Why is the peptide bond uncharged yet polar?
There is a partial separation of charge which forms a permanent dipole at peptide bonds
Peptide bonds are…
Rigid and planar
Explain the determination of amino acid composition of a polypeptide by acid hydrolysis
- 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
List methods to determine amino acid composition
- Acid hydrolysis
- Chromatography
- Quantitative analysis
- Sequencing from N-terminal(phenylisothiocyanate-Edman’s reagent)- gives sequence
How can Chromatography be used to determine amino acid composition?
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
How can amino acids be separated and identified by quantitative analysis?
- 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
How can amino acids be sequenced from the N-terminus?
- 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”
What is the limit of amino acids in a protein which can be used in N terminus sequencing ?
For polypeptides of up to 100 amino acids
Some is lost each time, so sample runs out
