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BCH 261 Lecture Notes - Lecture 14: Enzyme Kinetics, Linear Form, Zanamivir

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purplecattle498
7 Jun 2018
School
Ryerson University
Department
Biochemistry
Course
BCH 261
Professor
Warren Wakarchuk

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!
1!
BCH 261 – 121 – Biochemistry
Professor Gagan Gupta
April 12, 2018
Lecture: Review
!
Reminders!from!Dr.!Gupta’s!Section!of!the!course.!
!
(See!slide)!
!
DNA!sequence!manipulation!–!question!on!the!midterm!–!how!do!I!design!PCR!primers?!
!
Mutations!–!what’s!happening!at!the!level!of!DNA!and!what!are!the!consequences!of!
different…!
!
Protein!purification!–!pi!in!proteins!and!peptides!–!how!do!we!use!that!information?!
!
Slide:!!
!
How!do!I!design!a!pair!of!PCR!primers?!Many!of!you!had!trouble!with!this.!It!takes!practice.!
!
Generically!speaking,!how!would!we!amplify!this!whole!piece!of!DNA?!First,!think,!if!I!have!a!
single!stranded!piece!of!DNA,!!that’s!got!to!be!5’!and!that!3’.!If!only!given!one!strand,!you!can!
assume!it!goes!from!5!to!3’.!
!
One!is!easy!to!make!and!the!other!requires!translation.!You!start!with!a!double!stranded!
DNA!that!has!this!polarity.!In!order!to!amplify!it!you!need!one!primer!to!bind!to!each!strand.!
Need!to!amplify!strands!independently.!It’s!the!easiest!because!if!you!design!a!primer!that!
contains!sequence,!10!nucleotides,!that!will!be!complimentary!to!the!bottom!strand.!This!
primer!amplifies!the!bottom!strand.!!
!
(See!slide)!
!
Slide:'Proteins'and'Peptides'
!
Slide:''Immunoglobulin’s'and'Functional'regions'
'
You!won’t!have!to!look!at!a!picture!and!interpret!domains!because!I!couldn’t!find!a!good!
printer!to!print,!but!that!doesn’t’!mean!you!don’t!have!to!know!about!domains.!
!
This!is!an!example!of!a!heterotetramer!(spelling).!There!are!four!chains,!two!heavy!and!two!
light.!At!the!end!of!the!chains!we!have!a!small!region!called!the!variable!domain!where!the!
antigen!binding!takes!place.!It’s!possible!to!take!an!antibody!and!cleave!it!into!bits!that!still!
retain!function.!These!Fab!fragments!are!still!functional.!There’s!a!single!binding!site!here…!
!
Slide:'Protein-Ligand…'
!
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!
2!
I!gave!you!serotonin!and!said!if!you!had!to!have!amino!acid!side!chains!to!bind,!where!
would!they!be!and!where!would!they!bind?!All!the!choices!came!from!this!non-polar!
interactions.!Talked!about!ring!stacking.!Could!have!had!the!side!chain!of!losing!stacking!
against!the!chain!–!hydrophobic!interaction.!We!have!the!charge-charge!interactions!and!
the!hydrogen!bonds!is!always!important.!
!
Anything!that!binds!a!ligand!must!have!some!way!to!hold!onto!it.!These!are!the!three!
choices.!
!
Slide:'Binding'to'Proteins:'Graphical'Analysis'
' '
Kd!is!the!dissociation!constant!fort!he!protein!ligand!interaction.!Showed!these!two!plots.!
Talked!about!value!θ,!said!that!Kd!=!.5!θ.!That!means!Kd!is!the!concentration!of!ligand!
where!the!protein!is!half!occupied.!
!
It’s!hard!to!estimate!a!Kd!from!a!plot!where!you’ve!got!the!linear!plot.!Can!take!your!value!of!
.5!and!if!you!use!the!semi-log!plot!it’s!easy!because!you!just!draw!your!line!across!then!come!
down!to!calculate!the!values.!
!
Plots!are!used!to!compare!different!proteins!or!a!single!protein!with!different!kinds!of!
ligand.!
!
Slide:!Myoglobin!versus!Hemoglobin!
!
Spent!quite!a!bit!of!time!talking!about!oxygen!binding.!Two!proteins!do!that,!hemoglobin!
and!myoglobin.!!Myoglobin!is!the!oxygen!molecule!we!need!in!the!tissue!–!hemoglobin!does!
that!–!the!handoff!is!possible!because!those!two!proteins!bind!oxygen!with!very!different!
affinities.!Partly!has!to!do!with!the!structure!of!protein.!Myoglobin!is!a!monomer!and!
hemoglobin!is!a!tetramer.!
!
Slide:'Hemoglobin'Shows…'
!
We!looked!at!this!plot!of!concentration!of!oxygen!binding!to!the!protein!as!a!function!of!….5!
would!be!the!point!where!the!protein!is!50%!saturated.!
!
If!we!look!at!the!concentration!of!oxygen!in!the!lungs,!it’s!relatively!high,!both!proteins!
would!be!completely!saturated…if!we!go!backwards!and!follow!the!curves…the!lung!
concentration!of!oxygen!is!fully!saturated,!the!tissue!concentration!is!almost!completely!
saturated.!Have!to!get!to!really!low!concentration!of!myoglobin!before!it!gives!up!it’s!
oxygen.!!
!
Hemoglobin!is!different.!It’s!our!transporter.!Has!to!take!oxygen!from!lungs!and!transport!to!
the!tissue!where!myoglobin!can!pick!it!up.!Green!line!–!by!the!time!you!get!to!the!
concentration!of!oxygen!in!the!tissue,!hemoglobin!is!only!at!60%!concentration.!Myoglobin!
ready!to!pick!it!up.!
!
Why!do!these!binding!curves!look!different?!Concept!of!cooperativity.!In!hemoglobin!a!
tetramer,!as!soon!as!oxygen!binds…sigmoidal!curve.!At!very!low!oxygen!concentrations!it’s!
sluggish,!then!as!soon!as!we!hit!some!threshold!value,!the!affinity!goes!up!and!it’s!easy!to!
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Document Summary

Reminders from dr. gupta"s section of the course. (see slide) Mutations what"s happening at the level of dna and what are the consequences of different . First, think, if i have a single stranded piece of dna, that"s got to be 5" and that 3". If only given one strand, you can assume it goes from 5 to 3". One is easy to make and the other requires translation. In order to amplify it you need one primer to bind to each strand. It"s the easiest because if you design a primer that contains sequence, 10 nucleotides, that will be complimentary to the bottom strand. This primer amplifies the bottom strand. (see slide) You won"t have to look at a picture and interpret domains because i couldn"t find a good printer to print, but that doesn"t" mean you don"t have to know about domains. This is an example of a heterotetramer (spelling).

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Related Questions

Answered most of the questions. Can you tell me if my answers are correct? Last question of the month. Thank you. I appreciate it.

4. You perform electrophoresis on the product of a PCR and see that instead of the 300-base band you expected that there are five different bands.

What controls the specific sequence to which a PCR primer binds? Complementary sequence and annealing temperature control the specific sequence to which a PCR primer binds.

What are two factors that contribute to the annealing temperature of the primer? Two factors that contribute to the annealing temperature of the primer is primer length and the Guanine and Cytosine content.

What part of your thermal cycle protocol could you change, and how would you change it, to try and get rid of the extra bands?

To get rid of the extra band, we have to increase the annealing temperature.

If, after performing electrophoresis, there were no bands on the gel, what part of your thermal cycle protocol would you change, and how would you change it?

After performing electrophoresis, if there were no bands on the gel then we have to decrease the annealing temperature of the primer

5. If you are using a DNA sequence from a related species to design primers for your species of interest, why is it better to design your primers to complement an exon?

In related species there are similarities but their genetic makeup will not be the same. It is better to design your primers to complement an exon because ??

6. Why is it better to use a 25-base primer than a 10-base primer? Why do you have to have a lower annealing temperature for a 10-base primer?

It is better to use a 25-base primer than a 10-base primer because a 10-base primer is not long enough and may pair with a wrong portion of DNA, which would give wrong results. You have a lower annealing temperature for a 10-base primer because annealing temperature increases when the number of base pairs increase. If the temperature is too high the primer might not bind.

Identify the three steps in PCR and tell the function of each step.

Denaturation - when the double-stranded DNA is heated to 92-95℃ for about one minute, to separate it into two single strands.

Annealing - temperature of the reaction is lowered to a temperature that is between 45℃ and 65℃ to allow the DNA primers to attach to the template DNA.

Extension - temperature of the reaction is raised to a temperature between 45℃ and 65℃ and the new strand of DNA is made by adding nucleotides to the ends of the primers.

Tom is a molecular biology graduate student who needs to isolate a gene from the pathogenic bacteria Salmonella typhi, which he is studying. He wiselydecides to use PCR to amplify the gene before trying to purify it. The gene has the general structure shown below:

~1000 Base Pairs ! ! 5’-ATGGCTTACGTG-----------CCATAGGGCTAA-3’ ! ! 3’-TACCGAATGCAC-----------GGTATCCCGATT-5’

The gene is about 1000 nucleotide base pairs long, but only the sequences at the ends of the double strand are shown. Use the information from the video, what you learned about base pairing in class, and the fact that PCR always proceeds along the parent DNA strand the 3’ to 5’ direction. Design two, 6-nucleotide long, PCR primers for Tom. When you have these primers designed, proceed to the “Exercises and videos” link. You will be prompted for important information in drop-down menus. Answer each question and click continue to see a first-person performance of a PCR reaction.

Question #1 (These will be multiple choice and are automatically graded) a) What different components will do you want to add to your PCR?

b) Which primers will you use?

c) How many PCR cycles should you set the thermocycler for to obtain 128 TOTAL strands of DNA at the end of the reaction (this includes the template strands that you started with)?

d) To analyze the PCR you need to make a DNA gel. What do you want to add to make the gel?

2

a) You also wish to synthesize a degenerate oligonucleotide primer that could be used eventually to clone DNA that encodes your mutant protein. Design this degenerate oligo, keeping in mind the rules we talked about in class. More information about designing degenerate primers and primers in general are located on the following page.

Write out the sequence of this oligo here

b) If you synthesized the above oligonucleotide primer, how many different oligos would be present in the mixture?

RULES TO FOLLOW for creating primers (Modified from the Thermo Fisher web site):

Designing degenerate primers:

Write out your amino acid sequence, label amino and carboxy termini

For each amino acid, use the genetic code to predict the various nucleic acid codons that can encode this amino acid. For all but Met, you should have redundancy present.

Now you need think about 5’ and 3’ considerations, and keep in mind that DNA within genes is double-stranded. It is helpful to keep in mind that the 5’ end of a gene corresponds to the amino terminus of a protein. Using 5’ and 3’ labels write out a single strand DNA sequence corresponding to the your amino seq of interest. You may choose to start with only possible codon for each amino acid, or you can do all possibilities using the notation I showed you in class to account for the wobble position of codons.

Now make your DNA double stranded by filling in the opposite DNA strand; label the 5’ and 3’ ends of this second strand and make sure you have an anti-parallel arrangement of DNA strands.

If you haven’t already, now consider the redundancy present on both strands, and make sure you have indicated sequences that account for all possible redundancies.

Realize that when a researched synthesizes a degenerate oligo that the final synthesis contains oligos with every substitution possible. The amount of degeneracy is defined by the number of different primer combinations in the mix. You can determine the degree of degeneracy by multiplying the number of changes present at each position together. If a position has no degeneracy then you multiple by 1 for that position.

Keep in mind that the trade-off between primer specificity and efficiency can be modified by altering the degeneracy of the primer. For example, the more degenerate the primers, the less specific annealing will be; however, decreased degeneracy will allow more potential to identify unknown variants. Anything over 1024 fold degeneracy is at the upper limit of potential usefulness.

Designing Primers:

Good primer design is essential for a successful PCR reaction. There are many factors to take into account when designing the optimal primers for your gene of interest. This information is taken from the Fisher Scientific website and are used when actually designing an experiment. Here are some tips to consider when designing primers.

In general, a length of 18 nucleotides is the minimum necessary for efficient primer binding.

Try to make the melting temperature (Tmm) of the primers between 65°C and 75°C, and within 5°C of each other.

If the Tmm of your primer is very low, try to find a sequence with more GC content, or extend the length of the primer a little.

Aim for the GC content to be between 40 and 60%, with the 3' of a primer ending in C or G to promote binding. This is called a GC clamp.

Typically, 3 to 4 nucleotides are added 5’ of the restriction enzyme site in the primer to allow for efficient cutting. Restriction enzymes are ENDO nucleases, so they cut better when their recognition site is not on the end of DNA.

Try to avoid regions of secondary structure, and have a balanced distribution of GC-rich and AT-rich domains.

Avoid intra-primer homology (more than 3 bases that complement within the primer) or inter-primer homology (forward and reverse primers having complementary sequences). These circumstances can lead to self-dimers or primer-dimers instead of annealing to the desired DNA sequences.