welcome to lecture number thirty seven today we will going to start one of our last
discussion right which is a single molecular fluorescence now first question which will
come automatically that why we should bother about the single molecule right till now what
we are doing is the fluorescence response of a molecule when it is present in solution
but the concentration is in the order of typically is in the order of micro molar
so we are going to have at least right six into ten to the power seventeen molecules
right in a liter right if it is a one micro molar so that means we are getting a collective
response from all that fluorophores present in the solution now in some case right these
kind of collective response of the molecule present in the solution right which is known
ensemble average fluorescence property of the molecule may be different from the single
molecular response of this molecules present in the solution
it will be clear let me take one example lets for example you have such a molecule
with you is made of some d n a base pair where these base pairs are pairing these are
typically called the molecular beacon so here you have a fluorophore and here you have a
quencher right so this one is your fluorophore lets say it is showing you green colour emission
now once this quencher is present nearby then these quencher can quench the fluorescence
of this [guy] right and the fluorescence intensity is very low let me consider that this is one
of the structure of these molecular beacon present in the solution the other structure
of these molecular beacon which is present in the solution could be in the open form
so this is my closed form and the other one is by open form so here is your fluorophore
and here is your quencher now as the quencher molecule is far apart
from this fluorophore obviously the fluorescence intensity in this case will be much more compare
to the other case compare to the closed form right so this is your fluorophore and this
is your quencher over here and this is your fluorophore and this i referred to as the
open form consider that i know the emission spectra
of this closed form and this open form understandably the fluorescence intensity of this closed
form will be much smaller and the fluorescence intensity of this open form is much stronger
right so if i now draw the fluorescence intensity
of this closed form and open form what you will going to see is lets say this is the
fluorescence spectrum of the closed form lambda emission verses intensity this is the closed
and lets say this is the fluorescence intensity of the open form
now consider you have taken this molecule in a different solvent and what you are getting
experimentally is something like this now my question is this is your measurement
so my question is what is your conclusion
number one you can conclude it like this way that fifty percent of these particular molecule
is present in the closed form and fifty percent of this molecule is present in the open form
and as a result fifty percent will show the low intensity fifty percent will show the
high intensity and all together it is showing the moderately intense fluorescence
so the option one is your fifty percent closed and fifty percent open but there could be
another situation because you are doing this experiment in a different solvent right
so in that particular solvent it is also possible that all of them are having a particular form
which is neither open form nor the close form it is intermediate so that the quenching is
right the quenching of the fluorescence by this quencher is not that much permissible
and you are getting a moderately intense fluorescence intensity
for example what i want to say is that lets say this is the situation right another type
of form so i will name it as partial partially open form is not fully open partially open
form so in this case what you will going to get is moderate intense fluorescence right
because the quencher is not very far not very close here is very close here is far but here
is not very far or very close right so probably these fluorescence intensity we
will going to give you this moderately intense fluorescence intensity so the second option
could be that all these molecules under investigation right you have certain concentration but all
of them are present in a partially open form right so all of them are present in a partially
open form now if i ask you which one is correct of this
this one a a one or two which one is correct if you are just simply doing the ensemble
average fluorescence spectroscopy with this fluorescence spectroscopy it is impossible
for you to tell which one is the correct explanation correct
you may say that ok let me measure the anisotropy even you measure the anisotropy as you know
in this case as it is in the compact structure for the closed form then the steady state
anisotropy will be smaller and in this open form the steady state anisotropy will be larger
but as mixer because in the anisotropy we were looking all the molecules together right
so we cannot distinguish whether it is partially open or fully open is a mixer fully open and
fully close or it is just a all of them are partially open so we cannot do
the only solution is that we will going to we have to look these molecule individually
suppose you are doing this experiment individually how i will come later but consider that you
have the technique so that you can measure the fluorescence intensity of such molecule
one by one now you are measuring for the molecule number one right next you are measuring for
molecule number two next you are measuring for molecule number three and so on and so
on and so on right in that case what you will going to see you
will going to see two different depending on the two different case right if it is the
case number one right then the fluorescence intensity versus the number of molecule so
here is intensity here the which molecule you are measuring so number number so you
are measuring the molecule now number one molecule then second molecule then third molecule
then forth molecule and fifth so on and so forth right
so number of molecule or i can change this number of molecule to something else lets
say you are measuring from this this is your sample cell right and you are measuring from
here and you let the molecule to come one by one here and let it go out of this so molecule
is coming once it is entering in this region you can see fluorescence because you are measuring
from here and when the molecule will leave from here you will not be able to see the
fluorescence so say number one molecule is coming entering
and it is going to give you the fluorescence and when it will leave you will not going
to see any fluorescence then probably the second one will come it will enter here and
then the you will see the fluorescence and when it will leave from here then you will
not going to see any fluorescence so this number of molecule i can for such kind of
arrangement i can replace with the time right so for the a continuous monitoring process
so here i can also refer this as a time right for a specific observation volume
in this case what we will going to see if it is the case one right you will see the
fluorescence intensity probably well is zero because none of the molecules were present
inside this observation volume then fluorescence intensity will increase right and then fluorescence
intensity again decrease then again probably other molecular will enter fluorescence intensity
increase but when the third one will enter right
when the third one will enter probably the fluorescence intensity is less right because
it is now the close form which is entering um so i should draw it properly
so the first one is entering fluorescence intensity is high then when it will leave
fluorescence intensity will be less when second one is entering lets say it is also the open
form sorry it is also the open form yes then fluorescence intensity is very high and when
it will leave fluorescence intensity is less when the third one will enter right then fluorescence
intensity is too low because it is in the close form the forth one is like this sixth
fifth one is like this sixth one probably like this and so on so you will get such kind
of fluorescence intensity versus time plot for this observation volume so this is for
the case one however in case two what you will going to
see is whenever the molecule will enter this observation volume the fluorescence intensity
which you will get from this observation volume will be fixed right it will not going to defense
molecule to molecule which molecule is entering because all of them are partially open right
it is not like that some are fully open and some are fully closed so in this case
what you will it is not necessary that at the same time you will going to see the fluorescence
burst coming out of the observation volume its not necessary right
so but whenever you will going to see it will be of moderate intensity like this way whenever
it will come clear like this so you see the basic difference so from your experiment
if you look at that how this fluorescence fluctuation is happening you will be able
to tell clearly that ok what my proposition is number on is fifty percent close or fifty
percent open is correct or whether my proposition that all of them are present in a partially
open form is correct so for that what i need to see i need to see the fluorescence characteristic
of a single molecule of this particular bio molecule right
moreover lets say i can also say that somehow i came to know that this is present in the
partially open form partially open form now i can think of that this is the structure
of the partially open form and these structure remain fixed right throughout the time right
what i mean is that there is no fluctuation in the structure but recent studies like this
can be represented as a protein right recent studies suggesting that the protein structure
which we use to see in the text books right the p d b structure the crystallographic structure
of the protein they are not they are not rigid right in solution in solution they undergo
a slow breathing type of motion the time skill is very slow it is in the order of
few micro second to tens or twenties or micro second right but they are undergoing a slow
breathing motion right and those breathing motions can be captured by using such kind
of single molecular technique what i want to say is that the conformational
fluctuation dynamics right may also change the fluorescence property of such kind of
molecule right for example if because of this conformational fluctuation these part this
quencher will come little close or far from this original position when this quencher
will come at this position quenching will be more when this one will go here then quenching
will be less that means there will be some fluorescence intensity fluctuation right within
these molecule which is not much change in the structure right both are are partially
open form but there is small change in the structure right within this partially open
form it is not that it is converting between the close form and open form not that big
fluctuation right small fluctuation but that small fluctuation is good enough to change
the fluorescence characteristics of this molecule right so such kind of slow conformational
fluctuation dynamics can also be observed and in such case if you now do such kind of
plot what you will going to see you will going to see such kind of diagram
in all the cases it is similar intensity but within these similar intensity there is some
sort of fluctuation there is some sort of fluctuation sometime it is high sometime it
is low right sometime like this so that can be used further to calculate the conformational
fluctuation dynamics ok so just for your reference i will write here this conformational fluctuation
dynamics ok i have to make it more clear to you i have
this p p t slide for you here you see lets say this is my protein molecule right and
these protein molecule the fluorophore is present in this location and the quencher
is present at this location so when they are far apart i can see a a strong fluorescence
strong pink fluorescence right just i have described like that and when they are closed
by so in this case you see they are closed by thats what i have made this cartoon
so when they are close by the fluorescence intensity is less
so in this case in the solution probably such molecule exist such molecule also exist such
molecule also exist such molecule exist and many many such kind of such molecules will
exist together right when one molecule is unfolding when one molecule is going from
the close to open and many many other form in between right closed one and this is opened
one but many many other form between that is my conformational fluctuation molecules
are doing like this way right in case all the molecules are opening and
closing opening and closing simultaneously in a coherent fashion that means one molecule
when this molecule is doing like this this molecule is also doing like this then when
this molecule is like this this molecule simultaneously like this right
so in that case in the coherent fashion if everything is going to do like this then
ensemble average picture is good enough to see the fluorescence fluctuation of this molecule
because all the molecules are changing its conformation right simultaneously right in
a coherent fashion right but so then i will see the fluorescence intensity will increase
then fluorescence intensity will decrease increase decrease and so on and so forth but
if it is not here in this particular scenario as i am showing here at a particular instance
lets say this is a particular instance in time these molecule you see look here these
molecule showing strong fluorescence this is weak fluorescence this is strong this is
very strong this is weak like here are different so still as i can see by my i in this particular
p p t slide that ok this is strong so this must be open this is strong this must be open
this is close so this is weak so this must be close and so on and so forth but it is
possible for me because i have only one two three four five six seven eight nine ten elven
twelve thirteen fourteen fifteen sixteen seventeen eighteen nineteen twenty twenty only twenty
two such molecules are there consider you have avogadra number lets say
micro molar so ten to the power of seventeen molecules per liter right so in such case
lets see this animation so you have several several several such kind of molecules right
so is a huge number ten to the power seventeen molecules are present in the solution
so in this case what you will going to see from far apart i cannot distinguish this um
different colour of different bio molecules present in my sample that means the information
of intensity fluctuation is now lost right i cannot see so for me that all these colour
are equal right from here to here it looks like a single colour right i cannot distinguish
this so in this case what is the solution my solution
is that i cannot see these molecules in a ensemble average picture right not together
if i am going to see all these bio molecules together then i will lose such kind of
important information that is a fluctuation of the fluorescence intensity right with time
what i have to do i have to see such kind of bio molecule in the individually individually
i have to see this bio molecules and that is my goal how to achieve that is what
we will going to discuss here ok so my aim is that this see individual bio
molecules right as i have shown here so we need to investigate a single protein in bio
molecule with time resolution of the conformational fluctuation right if i am going to see only
this one then i will be able to tell that with time how these bio molecules actually
fluctuating if it is fluctuating that means its fluorescence intensity must fluctuate
because i have a fluorophore over here and i have a quencher present over here so if
it fluctuate its process intensity must fluctuate so if i just sitting over on these particular
bio molecule and see how the fluorescence intensity is changing with time then i should
be able to comment on the conformational fluctuation dynamics of this bio molecule
so for to show this i have this animation with me as you can see here that when this
is in the close form fluorescence intensity is less when it is in the open form fluorescence
intensity is more you can see this as you are seeing only one molecule as i showed earlier
for several molecules such kind of fluctuation information is lost right because many
many molecules are present and because this is non-coherent in nature right
so i showed you the importance of this fluorescence single molecule fluorescence the first
one is that you will be able to say that what kind of structure it is and the second
one you can also able to um determine that with time right how the structure is changing
next i would like to show you another advantage of the single molecule spectroscopy by showing
this type of reaction so here let me have a reaction a substrate level with fluorophore
so f is my fluorophore and it is getting bonded with this enzyme to form some enzyme substrate
complex which is denoted like this e q and ultimately this is going to give you the product
s f and this enzyme back right so if you want to study the kinetics of this
reaction what you should do you should i am talking about our normal fluorometric
assay of this enzymatic reaction so in that case what you should do you should take
your s f or s e q in the q head right let me take
e q in the q head and then if you add this s f initially right if you follow as a function
of time the fluorescence intensity initially the fluorescence intensity must be zero right
let me change the colour initially the fluorescence intensity must be zero because e q right does
not contain any fluorophore so fluorescence intensity should be zero over here
once you add this s f in the mixer right then the e q will react with s f to form this free
fluorophore right so with time the fluorescence intensity of this will increase and when the
reaction will complete all the s f will be converted to s plus f and then fluorescence
intensity will be maximum so from here you will be able to say what
is the [rate] constant of this reaction and you can now change the temperature you can
change the environment and see how the enzymatic reaction is actually depending on those cases
but now you consider that when you are mixing s f with a q obviously you need some time
right you need some time so whether you you will be able to monitor this enzymatic reaction
right that will be guided by the date time of mixing right this is the drawback of our
this setup right so one of the drawback
is dead time of mixing and it will also not going to tell me right whether there is a
formation of any intermediate state for such kind of experiment would not reveal any intermediate
states in the reaction right so what is the solution right
solution is lets say if i have solution is single molecule level spectroscopy single
molecular fluorescence consider this situation that you have s f sitting over here and somehow
you are just monitoring this s f right so e q will come and interact and will go right
so now if i plot so this is my single molecule right you are monitoring single molecule fluorescence
in this case so in this case if you just see it and look at these s f molecule as a function
of time what you will going to see right s f this f is present but it is attach to a
s so let me define this intensity as its fluorescent
but weak weak fluorescence and this f is strong fluorescence when it is free from s
right so in this case what you will going to see is that there is some fluorescence
intensity right so the fluorescence intensity is like this way lets say but when this e
q sorry sorry sorry sorry sorry sorry i should write this weak fluorescence not here but
here but when it is this form obviously the q is nearby so that means very weak very weak
fluorescence so this one is very weak fluorescence this one ok
so the fluorescence intensity of s f you are looking here right is like this but when this
e q binds to s f the fluorescence intensity will decrease so you will going to see a decrease
in the fluorescence intensity and that fluorescence intensity in the lower low fluorescence intensity
region will stay for some time and then when the reaction will takes place when the reaction
will finish right then there will be a formation of s and f the free f so the free f fluorescence
intensity will be there if you are still looking this f right if this f will go away then obviously
will not be able to see but consider that you are still looking this thing then the
fluorescence intensity will increase like this fluorescence intensity will increase
right that means i have both my s f and e q in the
solution i am just only looking at the s f all all of them are there so if s f is
alone right then i see a moderately fluorescence intensity when a q come and interacted with
s f then fluorescence will decrease and that reaction will take its own time and then when
the reaction is over it is forming s plus f at the fluorescence intensity again will
increase so if i do this thing several several several
times then i will get a clear idea about what is the rate of this enzymatic reaction here
the plus point is that there is no dead time of mixing right because all the substances
are present in the solution beforehand only what you are doing you are monitoring the
s f right e q is already present so unlike this right if lets say the reaction
will takes place in one second and your dead time you are mixing very slowly like it took
ten second then by the time you mix shake reaction is over right you wont be able to
see such kind of rise but in this case you will be able to see that such kind of reaction
because all the molecules are present reaction mixer
another important information what you will going to see is that whether there is formation
of any intermediate state lets say the fluorescence intensity of this reaction is not like
what i have shown in this green but it is like this this one and then decreased and
then is like this and then it increased and remain like these and then again increase
and it become like this so clearly i can tell that here is my s f
here is my s plus f this for sure in between that s f must have interacted with e q that's
why fluorescence intensity is less and in this intermediate one the structure must be
s f little bit of different type of interaction with e q that means you will be able to see
this intermediate state very easily if you monitor such kind of single molecular level
fluorescence ok let me finish here and we will continue our discussion in the next lecture
thank you very much