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[Music]

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hello my name is Victoria Lai and I'm

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here to talk about a potential model for

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life on Mars and how we're using

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combination of genomic and mutagenic

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analysis to look more closely at that

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model so first of all what do aliens

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look like pop culture depicts aliens and

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extraterrestrial life like this but we

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suspect it will look more like this

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so this is hallo bacterium NRC one

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isolated from the San Francisco Bay salt

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urns and then this is hallo rubrum lacus

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profund II and it was isolated from deep

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lake Antarctica so these are hallo

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archaea or salt-loving members of

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archaea that their domain of life and

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hailer archaea make good models for

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extraterrestrial life because they're

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thought to be a very ancient form of

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life and are found in extreme

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environments so NRC one and h-blocks

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profundity or HLA have been subjected to

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stratosphere because conditions such as

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pressure change freeze thaw and

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radiation both in the laboratory setting

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and with weather balloons and have been

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shown to survive relatively well and

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this was recently published in the

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International Journal of astrobiology

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they are also able to survive within

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Brien inclusions in salt crystals as you

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can see here in the colored portions

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that is those are living halo archaea so

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these stressors are similar to the kind

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of stresses that would be experienced on

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Mars specifically the extreme

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environment in which HLA is found is

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analogous to the conditions on Mars

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where there's thought to be periods of

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flowing liquid water shown in the

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occurring slope lineae at noon crater so

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this liquid would most likely be brine

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considering that the flows can be

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observed even at sub-zero temperatures

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so there's a number of sodium chloride

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magnesium perchlorates and other salts

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that have been reported to be present on

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Mars that could allow the water to

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remain liquid at such low temperatures

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so hla is not only able to survive

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extremely high salt concentrations but

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also low temperatures it's even been

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recorded to grow as low as negative 1

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degree Celsius

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so because HLA is a cold-adapted pelo

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rakia it's very promising as a potential

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model for life on Mars and by studying

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its evolutionary genetic adaptations

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we're looking for specific ways in which

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it has diverged from its temperate or

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mesophilic counterparts therefore giving

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an indication of what kind of

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evolutionary change results in the

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ability to survive these extremes so HLA

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has a fully sequenced genome that's been

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compared to more mesophilic helú archaea

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in order to find residues that are

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conserved within the mesophyll meso

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files but then diverged in HLA

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suggesting that there's some

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evolutionary advantage to the residues

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in the cold adapted organism so we can

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look more deeply at a single enzyme by

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comparing the protein sequences from HLA

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to homologs in other mesophilic

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halophiles specifically in this study

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were looking at beta galactosidase just

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that one um and and we found six

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residues that were diverged in the cold

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adaptive beta galactosidase so you can

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see there's one in the beta sheets of

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the tim barrel here there's a couple on

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the Alpha helix ease of domain a and

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then two in domain B so they're found

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all throughout there's no real pattern

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there but beta galactosidase is a

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catalyst that facilitates hydrolysis by

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breaking a glycosidic bond in sugars

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such as lactose and that catalytic

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activity at low temperatures is

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generally even less effective than other

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enzymatic activities just in general

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because of the thermodynamic effect so

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we perform mutagenesis on beta

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galactosidase Express eats mutation in a

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halo archaeal expression system and

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purified it to test the effect of

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changing those single divergent residues

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to the ones found in the meso file

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through comparison to the wild-type and

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we use classical biochemical analysis

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through steady-state kinetics to

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quantify those effects so for those of

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you who may not be familiar steady-state

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kinetics uses this formula under

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conditions of substrate excess so you

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use the enzyme s is the substrate and

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then P is the product and es is what is

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these is what the steady-state is

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referring to

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so @k K minus 1 and K 1 you're measuring

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the rate of dissociation of the

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substrate from the enzyme and at K 2

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you're measuring the rate of reaction so

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we can think of this as like a

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lock-and-key situation where you want

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the key or the substrate to fit well

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with the lock the enzyme but still be

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able to remove the key or the product

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after the lock is unlocked and the

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reaction is completed since I'm looking

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at steady-state kinetics with high

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substrate concentrations I can calculate

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the km and kitkat that's here so the km

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is similar to the dissociation constant

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of the substrate from the enzyme and a

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lower km is better as it indicates lost

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association so k NK cat represents the

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rate of the reaction under steady state

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conditions and then k cat over km is the

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ratio of the two and represents the

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efficiency of the enzyme to perform the

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reaction so the higher the k cat over km

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the more efficient the enzyme is is a

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catalyst and then we can look at these

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three constants compared to the wild

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type comparing the wild type to the

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mutated enzyme at temperatures from 0 to

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25 degrees Celsius in two molar KCl

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solution so this graph is showing the

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kms of the mutated enzymes in red

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compared to the wild type in blue so a

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brief overview of our results showed

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that all of the mutated enzymes affected

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temperature activity compared to the

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wild type with all of them in exhibiting

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an increased km at zero degree Celsius

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the degree of difference varying as you

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can see and then the wild type exhibited

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a direct relationship between the km and

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the temperature which would be

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characteristic of a cold adapted enzyme

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where it has the lowest km at the lowest

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temperature and then a higher km at

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where the six mutated enzymes showed

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inverse character compared to the wild

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type with a higher km at low

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temperatures so we that was exactly what

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we were hoping to see in changing it

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from the divergent cold-adapted residue

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to the one found in the Meza file and

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these results confirm the evolutionary

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importance of at least four of the six

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divergent amino acids in the improved

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function of the beta galactosidase

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enzyme at colder temperatures so I'm

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going to go into more detail about one

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of my mutants

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so one mutation in particular was at

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residue 482 which is located in a beta

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sheet in domain B and in all these

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graphs the mutated enzyme will be shown

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in red and the wild-type in blue so this

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mutation showed the highest increase in

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the km at lower temperatures suggesting

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that the mutated enzyme was less

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affected than the wild less effective

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than the wild-type at lower temperatures

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which is in line with the hypothesis

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that changing that single residue will

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negatively impact the cold adaptiveness

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of the enzyme the change in the k-kat

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however is much more subtle than the km

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though you are still seeing a change at

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the higher temperatures where the

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mutated enzyme is more is has a better

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rate of reaction at those higher

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temperatures compared to the wild type

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which again fits with our hypothesis as

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we're changing it from the residue found

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in the cold adaptive enzyme to the one

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found in the Meza file and an increased

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rate of Zac reaction when you're losing

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that cold adaptiveness at higher

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temperatures make sense and then finally

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the enzyme efficiency was decreased for

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the mutated enzyme when compared to the

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wild type over the lower temperatures

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concei and increased at the higher

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temperatures and this trend was mainly

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influenced by the km as you can see

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before it had it was a much more drastic

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difference between the two but the k-kat

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is still taken into account and most

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likely shows its effects at those higher

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temperatures so these three constants

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when compared to the wild-type paint a

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picture of a less cold adapted enzyme

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supporting idea that these diverged

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residues are diverged to increase the

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ability of the beta galactosidase in HLA

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to function in the

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the next step is to examine the enzyme

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metal the enzyme model structurally are

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there patterns that can show us why

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certain residues in certain positions

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can affect the temperature range of the

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enzyme so if you think of proteins or

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enzymes like molecular machines which

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need to flex are moved to be

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catalytically active they need space to

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work effectively and at high

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temperatures the surrounding chemical

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groups and atoms are moving enough that

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there's plenty of space to work with but

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at lower temperatures this as we've

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talked about in the opening talk at

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lower temperatures the surrounding

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molecules are more densely packed and

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frozen like water in a liquid compared

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to in an ice state where the molecules

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are more structured so it stands to

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reason that the enzyme would need to be

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more flexible to function at those lower

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temperatures to counteract those effects

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so we look at the homology model

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comparing valine at position 482 here to

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leucine at that same position in these

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images what you're seeing is the the

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residue in question and then residues

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and side chains which within six

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angstroms of that and the white area

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represents the edges of the Vander Waal

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shells for each side chain each atom so

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you can see with valine you're getting

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this space here

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that's unoccupied and you're getting a

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similar space in leucine here maybe a

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little bit smaller you are losing that

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space here so there's more potential

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there's more potential flexibility here

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and fewer potential interactions with

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these residues up here whereas with the

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leucine there's more potential for

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interaction here a greater chance of

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reduced flexibility and leucine is the

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one that's in the meso file and the

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valine is the original one in the cold

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adapted enzyme and then if we look at it

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from another angle you can see even more

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clearly that this valine here there's

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this additional space that isn't present

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when you have that additional carbon

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when it's a leucine so you're losing

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that space and then you have more

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potential for interactions more

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potential less potential flexibility so

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the studies performed on these six

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different

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Haitians are providing deeper insights

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into how enzymes may function at lower

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limit of temperature in an extreme of

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bio living in Antarctica so in

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conclusion when thinking about the

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potential for life on other planets we

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can first look at life in the extremes

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on earth to give us a better framework

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for how this kind of life may survive in

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our approach we can first look at those

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divergent regions and the proteins

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encoded in the genomes of microorganisms

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adapted to extreme environments and then

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use experimental methods to confirm

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their importance in the ability to

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survive these extremes and in doing so

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we're able to use past evolution as a

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guide pointing us towards novel adaptive

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mechanisms leading to new discoveries

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and expanding our understood ranges of

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survivability thus giving us a better

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basis for how life may arise even

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outside what we consider life's limits

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so I'd like to thank my lab group for

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supporting me with this in the

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University of Maryland and NASA for

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providing funding

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[Applause]

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uh fans for the nice talk I have a

300
00:11:58,259 --> 00:11:56,440
question about your ki measurements I'm

301
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wondering what is your reporter essay

302
00:12:04,439 --> 00:12:01,180
for studying the km that's my first

303
00:12:08,220 --> 00:12:04,449
question and also are you limiting

304
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yourself to just a single residue

305
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mutagenesis at 482 based on your bowing

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4matic analysis sorry I may not have you

307
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may have covered already but didn't

308
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capture the answer to the first question

309
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I'm using a colorimetric essay using

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onp g at absorbance 420 so I'm doing

311
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like measuring the change in color there

312
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and then using the V zero to calculate

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the km using lineweaver-burk graphing

314
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and the second question I have all of

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those mutations that I talked about the

316
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six different ones there at six

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different residues I just talked about

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482 for the sake of time just going into

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one in specific I do have a poster that

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I'll put up later if you want to see the

321
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other six or the other five questions hi

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I have two questions like so um what are

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the uncertainties and measuring the cake

324
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hat and km values are there any was the

325
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problem with like putting standard error

326
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bars and things like that on these

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graphs is that these values are based

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off of so the way that they're

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calculated is you do those color metric

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assays with different substrate

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concentrations and then based off of the

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v-0 that you calculate from that you

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create a lineweaver-burk graph and then

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based off of the x and y-intercepts of

335
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that lineweaver-burk graph that's where

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you get the km and k-kat so it's a

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little bit difficult to get the

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uncertainties there okay so as an

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astronomer I'll ask you later about what

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half of those words mean okay um are

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were there any mutations in the timbrel

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region there was one so the whole blue

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so the Tim barrel is these beta sheets

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and then the surrounding alpha helixes

345
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with them and the reason why a mutation

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in the Tim barrel would be of interest

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would be because it's that's where you

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would get closest to the active site and

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there was one here a 263 s that was in

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the beta sheets of the Tim barrel that

351
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one didn't have a very strong

352
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temperature dependent effect it just had

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a in general D latias effect so that's

354
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why I didn't go into that one in detail

355
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here but it was interesting okay thank

356
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you hi can you go to the slide with the

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to comparative structures real quick Oh

358
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which one oh yeah one of these so yeah

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you guys did a valine to leucine

360
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mutation what if you did like a valine

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to something smaller like an alanine or

362
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something do you think that it would

363
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show kind of an opposite that's what

364
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we're hoping would happen and that would

365
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that would be a possible next step

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because all of these mutations that we

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did were based on that bioinformatic

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analysis going from the cold-adapted to

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him as a philic but now that we're

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seeing that that does have the effect

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that we were expecting the possibility

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of creating a more cold-adapted enzyme

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exists potentially so that that would be

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a possible next step would be to then

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reverse that go in the opposite

376
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direction of the patterns that we're

377
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seeing take it smaller and see if that

378
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increase in space causes an increase in

379
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flexibility that could then result in an

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enzyme that works better at those colder

381
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thank you for that talk continuing on

382
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that last question I was wondering is

383
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there any advantages to having a less

384
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flexible enzyme at higher temperatures

385
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it seems like it would always be better

386
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to have less kind of steric hindrance

387
00:16:11,319 --> 00:16:08,970
going on no I mean um like was talked

388
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about in the initial talk having that

389
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increased structural aspect in at a

390
00:16:20,180 --> 00:16:17,459
higher temperature you do get that

391
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there's the potential for more stability

392
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and less denaturation at those higher

393
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temperatures so it would potentially

394
00:16:37,600 --> 00:16:36,010
I have a question for you um why did you

395
00:16:39,700 --> 00:16:37,610
guys pick this enzyme and are there

396
00:16:43,510 --> 00:16:39,710
other enzymes that you're thinking about

397
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Joseph enzyme because our hailer Akil

398
00:16:48,280 --> 00:16:45,140
expression system doesn't have a beta

399
00:16:50,320 --> 00:16:48,290
galactosidase and there's a very easy

400
00:16:52,870 --> 00:16:50,330
colorimetric assay as a sort of proof of

401
00:16:59,200 --> 00:16:52,880
principle but we could do it with other

402
00:17:00,610 --> 00:16:59,210
ones it's definitely possible okay we

403
00:17:09,020 --> 00:17:00,620
have time for more questions if there

404
00:17:15,650 --> 00:17:13,340
have you used any control enzyme to for

405
00:17:17,360 --> 00:17:15,660
your experiment related to enzyme

406
00:17:21,620 --> 00:17:17,370
activity means you compare your results

407
00:17:24,199 --> 00:17:21,630
with mesophilic enzymes and cyclic

408
00:17:28,850 --> 00:17:24,209
enzymes oh the problem is there aren't a

409
00:17:31,900 --> 00:17:28,860
lot of enzymes that have been looked at

410
00:17:34,760 --> 00:17:31,910
enough to be a control at least in these

411
00:17:37,160 --> 00:17:34,770
in these specific conditions because

412
00:17:39,230 --> 00:17:37,170
like I said I had to use a 2 molar

413
00:17:40,880 --> 00:17:39,240
potassium chloride solution in order to

414
00:17:43,090 --> 00:17:40,890
do these reactions and generally you

415
00:17:46,850 --> 00:17:43,100
wouldn't use that high of a salt for

416
00:17:48,020 --> 00:17:46,860
enzymatic activity assays and I wouldn't

417
00:17:49,340 --> 00:17:48,030
want to necessarily compare it to

418
00:17:50,750 --> 00:17:49,350
something that doesn't have that high of

419
00:17:52,280 --> 00:17:50,760
salt because you don't know what kind of

420
00:17:54,799 --> 00:17:52,290
confounding variables you're adding in

421
00:17:58,310 --> 00:17:54,809
at that point and which method you use

422
00:18:00,830 --> 00:17:58,320
for to study the data to change in

423
00:18:03,590 --> 00:18:00,840
protein conformation as well as amino

424
00:18:06,560 --> 00:18:03,600
acid sequence so this is homology

425
00:18:07,880 --> 00:18:06,570
modeling using Swiss PDB viewer so

426
00:18:10,760 --> 00:18:07,890
that's a free software that's available

427
00:18:12,890 --> 00:18:10,770
online and so we made a homology model

428
00:18:15,230 --> 00:18:12,900
of our enzyme using thermus thermophilus

429
00:18:18,049 --> 00:18:15,240
because it has a characterized and

430
00:18:20,840 --> 00:18:18,059
crystallized structure of its beta

431
00:18:22,790 --> 00:18:20,850
galactosidase so we use that to create

432
00:18:25,370 --> 00:18:22,800
apology model and like so this is all

433
00:18:29,540 --> 00:18:25,380
very theoretical we we haven't been able

434
00:18:33,350 --> 00:18:29,550
to get our enzyme crystallized so this

435
00:18:35,510 --> 00:18:33,360
is all super theoretical this is just

436
00:18:39,410 --> 00:18:35,520
the most likely one given the sequence

437
00:18:41,419 --> 00:18:39,420
that we've put into the system and like

438
00:18:42,890 --> 00:18:41,429
obviously that would be a really good

439
00:18:43,790 --> 00:18:42,900
next step is to get it crystallized so

440
00:18:45,169 --> 00:18:43,800
if anyone knows any good

441
00:18:47,390 --> 00:18:45,179
crystallographers that they want to

442
00:18:52,910 --> 00:18:47,400
refer to me i'd be happy to take the

443
00:18:58,550 --> 00:18:55,700
what about cryo-em have you done create

444
00:19:01,970 --> 00:18:58,560
cryo Yemen I have tried to but nobody

445
00:19:03,680 --> 00:19:01,980
will respond to my emails I really want

446
00:19:14,060 --> 00:19:03,690
to get this crystallized you guys trust

447
00:19:15,890 --> 00:19:14,070
me hi just a super quick question so I'm

448
00:19:17,270 --> 00:19:15,900
a geologist at work in XRD what are the

449
00:19:21,020 --> 00:19:17,280
obstacles of crystallizing your amino

450
00:19:22,400 --> 00:19:21,030
acids I think it's the salt I got talked

451
00:19:23,510 --> 00:19:22,410
to a lot of crystallographers and

452
00:19:25,910 --> 00:19:23,520
whenever I bring up the fact that

453
00:19:28,130 --> 00:19:25,920
there's a lot of salt in it they see

454
00:19:31,550 --> 00:19:28,140
they get kind of excited they think that

455
00:19:33,110 --> 00:19:31,560
it's good but then in practice there

456
00:19:34,610 --> 00:19:33,120
seems to be some sort of hindrance I

457
00:19:36,770 --> 00:19:34,620
don't know a ton about coastal ography

458
00:19:39,530 --> 00:19:36,780
so I don't know what's holding it back

459
00:19:41,000 --> 00:19:39,540
maybe it's the size I'm not sure but I

460
00:19:43,670 --> 00:19:41,010
mean we've crystallized things that are

461
00:19:44,600 --> 00:19:43,680
smaller and bigger than this so I the

462
00:19:53,730 --> 00:19:44,610
only thing that I could think of would