What is an oligopeptide?
General term for a series of amino acids joined via peptide bonds (often synthetic peptides)
What is an polypeptide?
general term for long chains of amino acids (generally natural products of translation)
What is a protein?
functional structures that may contain one or more polypeptides and may also contain prosthetic groups (i.e. carbohydrates/lipids/metal ions/heme)
Most polypeptides are how long?
What is the issue with long proteins?
100-1000 amino acids long, average of 350 residues
longer proteins may be error prone
The average mass of one amino acid residue in a protein is 110 Da
What is the approximate protein mass formula? Why 110?
Approximate protein mass = number of amino acids x 110 Da
a free amino acid = 128 Da
each peptide bond formation releases one H20 (18 Da)
128-18 = 110 Da per residue
Match the conjugated structure for this name: lipoprotein
lipids
Match the conjugated structure for this name: glycoproteins
carbohydrates
Match the conjugated structure for this name: heme
heme
Match the conjugated structure for this name: flavoproteins
flavin nucleotides (FMN/FAD)
Match the conjugated structure for this name: metalloproteins
metal ions (zinc, iron, calcium)
Match the conjugated structure for this name: phosphoproteins
phosphate groups
What does the BLOSUM matrix look at?
The BLOSUM matrix is used to score alignments between protein sequences.
High score → substitution is common / conservative
Low or negative score → substitution is rare / disruptive
“How acceptable is this amino acid swap in evolution?”
What are gaps in protein structure?
Two sections of amino acid sequences may align well, but be different lengths apart.
Gaps represent insertions or deletions at the genetic level.
In protein structure, gaps typically correlate to loops on the surface.
When comparing sequence, we look for the best match. What are the three types of residues?
Invariant residues (same amino acid in all sequences, usually essential for function)
conservative substitutions (replaced by amino acids with similar properties, often tolerated)
non-conservative substitutions (replaced by very different amino acids, often alter function or occur in less critical regions)
Best alignments maximize what type of residues
invariant and conservative substitutions
What are homologs, orthologs, and paralogs?
homologs: two proteins which share a common ancestor (one single gene)
orthologs (homologs which arise out of speciation events, same function in diff organisms)
paralogs (arise out of gene duplication events, may have same of different functions)
What is activity?
How is it indicated?
ability to convert substrate into product, usually indicated in units, which is an amount
1 unit = 1 μmol substrate converted per minute
What is specific activity?
How is it indicated?
activity as a fraction of total protein, which is purity of the enzyme
Specific activity = units/mg total protein
As you purify an enzyme preparation, what do you
expect (realistically) to happen to the specific activity and total activity?
Total activity will drop (because some enzyme is lost at every step, during binding, washing, elution…) and specific activity should rise (you’re removing contaminating proteins, so a higher fraction of the remaining protein is the enzyme of interest)
Before and after a step (or a series of steps) purification can be determined.
What is fold purification?
Change in specific activity associated with a purification process.
Before and after a step (or a series of steps) purification can be determined.
What is percent recovery?
Number of total units after a process compared to the number before that process.
How do you find specific activity?
find total activity, in units
find total protein, in mg
specific activity = units/mg
What is the colour and the chemical that causes the colour of human and other vertebrate blood
blood, haemoglobin
What is the colour and the chemical that causes the colour of spiders, crustaceans, some mollusces, octopuses, and squid?
blue, haemocyanin
