Hemoglobin in Birds paper

-red= derived amino acid (non-ancestral)

-black aa= ancestral aa seen in pair bird

-the pair of birds has same aa at this location

what are red aa vs black aa?

blank box?

blue-high altitude bird

white- low altitude pairing

(pairs most closely related to each other)

what does blue vs white shade mean

paralogs

-both share the beta subunit but have their own alpha subunits

HbD vs HbA

amino acid numbered position in globulin

what do the numbers at the top mean

parallel amino acid changes

-convergent amino acid changes not due to common ancestry

THAT LEAD TO INCREASED O2 AFFINITY (near the heme group)

what does box around aa pair mean?

about 140 aa long

how long is the HbA and HbD

-aa positions under strong selection and functionally important

key residues

-proteins the lab made from inferred ancestral sequence info

resurrected ancestral genes

-derived but not associate with the active or heme binding sites

-were associated with high altitude survival just not the topic of this paper

apomorphic allosteric mutations

same species with a high and low altitude grouping

conspecific species pairs

-560 total

-only 49 in HbA and 55 in HbD (not a lot)

-lots of negative selection (do not change)

how many aa out of Hb had derived changes

They isolated the HbA and HbD and were able to measure the affinity of each

How did they get the p50 of each Hb

-this is line where the points would be if there was no difference in affinty of the HbA or HbD between species pairs

what does the line in the center mean

species pair

what does one point mean

-the high altitude taxon has lower p50 and therefor higher O2 affinity

what does it mean if the points are below the line

-the concentration of a ligand (o2 in this case) that it takes to saturate 50% of the available Hb

-low- high affinity

-high= low affinity

what is p50

HbD

which affinity is lower HbA or HbD

-lab technique to mimics the inside of an animal

-hopefully makes the Hb act as it would inside RBCs

why was KCl and IHP added

-reconstruct ancestral Hb and add in the mutations that were parallel aa changes

-want to see if this will cause decreased p50 in anc too

how was protein engineering used in this paper

position 83 of the beta subunit

-often mutated to S

position 67

-often mutated to alanine

what residue was test the most

-83 to S had reduction and so did 67 to Ala

-but together they had a larger decrease than both alone (points to additive reductions

what happened when they measured p50 of mutations in non ancestral

Lowers p50, but the further back you go the less the mutations has an affect (or it didn't have an affect at all)

-genomic background matters and allosteric affects important

what were their findings when they looked at G83S induced mutations in ancestor proteins

allostery

protein interactions not at active site that can change P50