1. A report submitted to the
CENTRE FOR CELLULAR AND MOLECULAR BIOLOGY
On
NMR Spectroscopy and its application in the
study of Effect of Polar Organic solvents on
6B, a mutant of Bacillus subtilis Lipase
In partial fulfilment of the
Summer Training Program
Submitted by
Shreya Ray
Supervised by
Dr. Mandar V. Deshmukh
2012
1

2. CERTIFICATE
This is to certify that the report titled “NMR Spectroscopy and its application
in the study of Effect of Polar Organic solvents on 6B, a mutant of Bacillus
subtilis Lipase” is submitted by Shreya Ray. The summer training work has
been carried out by her under my supervision at the Centre for Cellular and
Molecular Biology (CCMB), Hyderabad, for a period of two months.
Dr. Mandar V. Deshmukh
Scientist
CCMB
Hyderabad
2

3. DECLARATION
I hereby declare that the report titled “NMR Spectroscopy and its application
in the study of Effect of Polar Organic solvents on 6B, a mutant of Bacillus
subtilis Lipase” was carried out by me under the supervision of Dr. Mandar V.
Deshmukh at the Centre for Cellular and Molecular Biology (CCMB),
Hyderabad, during the period June-July 2012.
Shreya Ray
Place:
Date:
3

4. ACKNOWLEDGEMENT
I would like to thank Dr. Ch. Mohan Rao, Director, CCMB, for providing me
with the opportunity to spend my summer here at CCMB.
I am also grateful to Dr. Ramesh K. Aggarwal, Coordinator, Summer Training
Programme, for ensuring that I had a pleasant stay.
Above all, I would like to express my heartfelt gratitude to Dr. Mandar V.
Deshmukh, he was kind enough to mentor and guide me, and for all the freedom
and space he gave me to learn.
4

5. NMR Spectroscopy and its
application in the study of Effect of
Polar Organic solvents on 6B, a
mutant of Bacillus subtilis Lipase
Nuclear Magnetic Resonance, or NMR, is an extremely valuable tool for the
structural determination of many molecules, particularly proteins, and their
kinetics and their dynamics. NMR gives information about the identity and the
chemical environment of atomic nuclei. This project report on NMR is divided
into two sections. The first section is about the theory of NMR. This includes the
basic principles on which NMR spectroscopy is based, and explains the
fundamental concepts involved. It goes on to discuss about the basic 1D NMR
pulse experiment and the data analysis which follows, finishing off with a small
introduction to 2D NMR. The second section consists of the application of NMR
in a study of the behaviour of the enzyme 6B Lipase in the presence of various
concentrations of different organic solvents; these concentrations were
successively increased (titration). Enzymes generally break down at higher
concentrations of organic solvents. The 6B Lipase, however, has displayed high
stability in such environments and holds promise for catalytic application in new
types of chemical reactions.
5

6. Contents
Part I
1. What in NMR? 08
2. What the Chemist knows 08
chemical shift, line width, line shape, coupling
3. The theory of 1D NMR
a. Nuclear Spin 10
b. The external magnetic field 10
c. The Larmour Frequency 11
d. Spin Populations 12
e. Shimming and Locking 12
f. RF pulse and resonance 13
g. Relaxation and the FID 13
h. Dealing with noise 13
i. Fourier Transformation and Data Processing 14
j. Peak Identification 14
4. Introduction to 2D NMR 15
Part II
1. Background 17
2. Experiment 17
3. Protein Dynamics 18
4. Solvent Binding 20
5. Discussion 26
6

7. Part 1
………………………..
Nuclear Magnetic Resonance
Spectroscopy
7

8. 1. What is NMR?
Nuclear Magnetic Resonance, or NMR, is a powerful technique used to probe the structure,
dynamics and chemical kinetics of many biomolecules. NMR techniques provide an alternative
method for structure determination if a protein cannot be crystallized, or if there is concern that
packing has distorted the true structure in solution. NMR is also useful in probing molecular
interactions, such as solvent binding and ligand binding.
2. What the Chemist knows
Like all Spectroscopy, NMR spectrum is a plot of intensity of absorption/emission against frequency.
The transitions in this case are not electronic, or rotational, or vibrational but involve the quantum
mechanical property called ‘spin’ of the atomic nuclei. NMR spectra are unusual in that they appear
at rather low frequencies (radiofrequency).
In 1D NMR, we generally measure the spectrum of one isotope only in one experiment. This is
because of the design of the experiment. Every isotope needs a different frequency window and we
can produce only one window at a time, usually. The most common NMR experiments involve the
hydrogen window, where we only check the peak shifts of hydrogen atoms in the spectrum. So we
will discuss about the 1H NMR spectroscopy. The principles are very general and can be applied to
spectroscopy experiments involving all other spin-half nuclei. Following is a 1H NMR spectrum of
glucose, just to show how a typical NMR spectrum looks like:
Fig.1. 1D NMR spectrum of glucose
All the peaks correspond to hydrogen but they are different because every hydrogen atom in the
molecule experiences a different kind of environment created by the surrounding atoms and bonds.
8

9. Based on the position of the peaks in the spectrum, we can guess how many types of hydrogen
atoms are there and where each atom belongs.
The positions (frequencies) of these peaks, however, are not absolute; they depend on the strength
of the magnetic field being used, rather proportional to it. To solve this problem, we define a new
kind of scale, called the Chemical Shift Scale. The Chemical Shift of a peak is defined as the ratio of
its distance from a reference peak to the frequency of the reference peak, both peaks being
acquired at the same magnetic field. In this way, the magnetic field dependence cancels out.
The reference peak belongs to a simple reference compound which has been agreed upon by
everyone based on certain properties of the compound, like symmetry, covalent nature, minimum
shielding, etc. For proton (1H) spectroscopy and carbon (13C) spectroscopy, TMS or tetramethyl silane
is the reference compound.
The values of chemical shifts are very small, often of the order of 10-6, because the peak shifts are
very small compared to the value of the frequencies. To make the numbers more convenient, we
widely use the Chemical Shift δ (ppm)-scale, where we multiply the chemical shifts with 106.
The Resolution of two peaks depends on their line widths and their line shapes.
Although the value of line widths are very small as compared to the value of the frequencies we deal
with, they are not so narrow as compared to the width of the frequency window (the spread of
frequencies we deal with). When the separation between two peaks fall below the line width, the
two peaks merge completely and cannot be distinguished, the exact point of merging depending on
the line shape.
The basic line shape is the absorption mode line shape. It is symmetric about the maximum and its
line width is specified by its breadth at half-height. The intensity of this line is proportional to the
number of protons (1H) giving out the signal, represented by the area under the peak. Hence, for the
same number of protons, a broader peak has a lesser height consequently reducing the signal-to-
noise ratio.
Scalar Coupling or J-coupling is a phenomenon where a peak splits into smaller peaks as a result of
‘coupling’ interactions with neighbouring nuclei transmitted through chemical bonds. There are two
kinds of coupling:
Weak coupling occurs when the chemical shift between coupled nuclei is very large as compared to
the coupling constant J. Coupling between two different isotopes, like 1H and 13C, is always weak. In
the simplest case of coupling, two peaks interact and split each other into two, giving rise to four
peaks in total. Two of these peaks belonging to the same nucleus form a ‘doublet’. Each peak of a
doublet has half the intensity of the original peak, and they are placed symmetrically about the
frequency of the original peak. The distance between these peaks in the doublet is twice the
coupling constant J, which is found to be independent of the field strength and hence expressed in
hertz. (Consequently, it will depend on the field strength whenever expressed in the δ (ppm) scale.)
9

10. Of course, a peak might split further and further due to coupling with multiple nuclei (multiplet
structure), and the result can be predicted using the knowledge of J-values in case of each split. This
kind of coupling is very useful in determining which atoms are linked to which ones, and forms an
important part of data analysis. However, in some cases, it might be a nuisance. For example, 13C
spectroscopy of organic spectra would become extremely complex since every carbon atom would
be split by many protons; the signal-to-noise ratio would also fall as the peaks become shorter.
Luckily, we can remove the effect of this kind of coupling from the spectra by intelligently designing
the pulse-sequence experiment.
Strong coupling occurs when the chemical shift between coupled nuclei is small, especially in the
case of homo-atom coupling. A doublet resulting from this kind of splitting is asymmetric both in
terms of frequencies as well as intensities. Further splitting gives rise to irresolvable complexities.
Since this can be seen only in the limit of very small chemical shifts, we require a finer scale to
observe this, and hence we can ignore this phenomenon for most of our purposes, where we do not
need so much detail. Therefore, we often say, “Equivalent spins do not split one another”.
3. The Theory of 1D NMR
The theory of 1D NMR is illustrated by the most basic single-pulse experiment.
a. Nuclear Spin
The theory of NMR involves the quantum mechanical property called ‘spin’ of the atomic nuclei. The
quantum mechanical spin does not have a classical analogue, but on many occasions we can
describe certain of its properties to be very similar to the classical spin. Every fundamental particle
has a spin, characterised by a spin quantum number I. This is an intrinsic property of that particle,
like mass or charge. Electrons, neutrons and protons have I =1/2. This can be interpreted as the
experimental fact that upon applying an external magnetic field to such a particle, the particle will
have an angular momentum of +ħ/2 or –ħ/2 along the direction of the applied field, its total
magnitude being ħ/2.
The nucleus is made up of protons and neutrons. However, in nuclei with an even number of protons
and neutrons, the spins always cancel out as this gives stability to the nucleus. The other nuclei,
however, possess spins. The nucleus of our interest is hydrogen, which is just a proton with spin half.
Spin confers a magnetic moment to the particle which is proportional to the value of the spin
angular momentum. The proportionality constant γ is called the gyromagnetic ratio and it is an
intrinsic property of the nucleus in question.
b. The external magnetic field
It is the presence of the external magnetic field which breaks the spatial homogeneity and gives rise
to the breakdown of spin degeneracy. The nucleus is now restricted to have an angular momentum
10

11. of +ħ/2 or –ħ/2 along the direction of the applied field. These orientations have different energies
and can be viewed as two different energy levels. We say that the spins have been ‘polarised’.
A point to note is that although the spins would have lowest energies if they aligned exactly along
the magnetic field, they never do so because of the quantum restrictions. Their z-components
cannot be changed. At this point, the nuclear spin can be thought of as precessing about the
magnetic field because their x and y components continually change while preserving the magnitude
of spin and that of its z-component.
As expected, the nuclear state where the direction of the magnetic moment due to nuclear spin is in
the same direction as the magnetic field is lower in energy as compared to the one opposed to it.
The actual values of energy of the states depend on the strength of the magnetic field being used.
Like the interaction of any other magnetic moment with a magnetic field, energy of the interaction is
given by:
Since the magnetic field is along the z-axis and has the magnitude ‘B’
For a spin-half system
Of these, the lower energy state, or the ground state is denoted by α and the higher energy state or
the excited state is denoted by β.
The energy of transition from α to β state is given by
In NMR experiments, a strong uniform external magnetic field is provided by powerful
superconducting coils, cooled by liquid helium and liquid nitrogen.
c. The Larmour Frequency
Using the relation , where ω is the angular frequency, we can write
ω0 is called Larmour Frequency. It is the frequency at which the spin of a nucleus with a
gyromagnetic ratio γ precesses about a given magnetic field. As we can clearly see, the Larmour
frequency and the energy difference between the two levels are directly proportional to the
11

12. strength of the magnetic field applied along the z-direction. The sign of ω0, if present, indicates the
direction of the precession.
The values of γ, however, are very small. And the present methods of obtaining a uniform magnetic
field, which is crucial for our purpose, impose a limit on the strength of the magnetic field that can
be attained. That is why the values of ω0 are very small and lie in the radiofrequency region.
The Larmour frequencies are different for the different types of protons, that is, protons bonded to
different parts of the molecule. This is because each kind of proton experiences a different set of
chemical environment, different electrostatic forces and consequently different magnetic fields.
Movement of electron densities nearby, as a response to the external magnetic field, might shield or
reinforce the original magnetic field around any particular proton, thus changing ω0. This forms the
basis of chemical shift; it is the reason we have different peaks for the different hydrogen atoms in
the compound.
Scalar coupling or J-coupling occurs when the interaction of one spin with the external magnetic
moment polarises that spin, which in turn polarises the bonded electrons, which alters the effective
magnetic field around the nucleus bonded to the former nucleus. Since there can be two possible
polarisations, consequently the magnetic field around the latter nucleus may be either increased or
decreased. The two possible cases give rise to the observed peak split. Since the energy difference
between the spin states is very small, both the polarisations are almost equally likely, hence both
peaks of the doublet have the same intensity. Repeated splitting of peaks produces multiplet
structures.
d. Spin Populations
As a consequence of small γ, the energy difference between the two levels are also very low, hence
both the ground state and the excited state are populated almost equally, with the ground state
having a very slightly larger population. This can be thought of as a result of equilibrium between the
magnetic forces trying to align the spins and the thermal forces trying to disrupt the alignment.
The ratio of populations between the two levels can be predicted by Boltzmann’s relationship:
This kind of population distribution reflects a small longitudinal magnetisation along z-axis.
e. Shimming and Locking
It is very crucial for the magnetic field being used in the NMR experiment to be constant and uniform
throughout the scan and throughout the sample. Shimming is performed to adjust defects in the
existing field uniformity by passing the requisite amount of current through the shim coils. The Field
Frequency Lock is performed to prevent the magnetic field from drifting away from its initial value.
This is achieved using feedback mechanisms from running background scans.
12

13. f. RF pulse and resonance
At this point of time, we apply an RF pulse of a certain frequency. It is just a chain of radiofrequency
photons that we create by passing an oscillating current through a coil whose axis is perpendicular to
the direction of the original magnetic field.
The RF pulse is sent with a well-calculated frequency. For proton NMR, we send a pulse having a
frequency around the middle of the hydrogen window (hydrogen window refers to the range of peak
shifts that a proton can cover). Since the frequencies of the RF pulse and the Larmour frequencies of
the protons are either same or very close, resonance occurs and the photons are absorbed by the
protons. These photons or this electromagnetic wave has an oscillating magnetic field along the axis
of the coil. Although small in magnitude, this magnetic field becomes very powerful due to the effect
of resonance and hence tends to make the spin precess about itself instead of precessing about the
stronger original magnetic field along the z-axis. This phenomenon is known as transverse
magnetisation.
The Larmour frequency about this new oscillating magnetic field is obviously different, and if this
pulse is applied for exactly 1/4th of the time period for this rotation, we will have rotated the original
magnetisation by 90 degrees with respect to the initial direction. This is known as a 900 pulse; it is
the most basic pulse.
g. Relaxation and the FID
When the RF pulse is switched off, the spins start getting back to their previous equilibrium. The
pulse had tilted the entire magnetisation by 900. All the spins now start to relax together, thus
bringing coherence between them. It starts with a precession in the x-y plane about the original
magnetic field along the z-axis. The entire magnetisation precesses with an exact, fixed frequency:
the Larmour frequency. The spins slowly start leaving the x-y plane and dephasing out, the
coherence is slowly lost in this process, known as spin-spin relaxation. Another process that
contributes to this relaxation is the spin-lattice relaxation where the spins try to regain their original
population distribution.
When the RF pulse is switched off, the same coil that was used to generate it now acts as the
receiver coil. The precessing magnetic field projects an oscillating magnetic field in the axis of the
coil at the Larmour frequency. This produces an AC current in the coil which goes to the detector.
This signal is known as FID, or Free Induction Decay.
h. Dealing with noise
In NMR, signals are very weak and they can be easily buried in noise. There are many ways to
improve the signal-to-noise ratio. The most important part of this is signal averaging, where the
pulse sequence is repeatedly sent and FID is repeatedly collected from the sample and successive
13

14. data sets are continuously added up. This tends to reinforce the signals due to repeated occurrence
and cancels out the noise due to random occurrence.
A lot of noise is also cut out by good experimental design and good signal processing after getting
the FID.
i. Fourier Transformation and Data Processing
The FID is recorded in the analogue form and converted into the digital form by sampling the data at
very close intervals. Both real and imaginary parts of the signal are stored; the imaginary parts
contain information about the direction of the precession.
We have information about the FID as a function of time. However, we would like to represent it in
the frequency domain. For this, we perform Fourier transformation.
Before doing so, we multiply the FID with a suitable sine bell function, or any similar function which
decreases the magnitude of the tail of the FID which is predominantly composed of noise and also
decreases the magnitude of the FID at its beginning to control for peak broadening, while increasing
the magnitude of the central part.
Another problem is that many signals may not be in the same phase in which the receiver is. As
hinted earlier, we collect data in the complex form, keeping both the real cosine part and the
imaginary sine part of the signal. Only the real part, however, has got the narrower and better-
resolved Lorentzian peak shape that we want. It is customary to represent only the real part of the
spectrum as the imaginary parts are shorter and broader, decreasing the signal-to-noise ratio.
Whenever the signal and the receiver are not in phase, we get a combination of the real and the
imaginary parts. The solution is obviously to multiply with the phase difference; however there is no
way to know the phase difference.
In the zero-order phase correction, we continuously adjust the phase of the entire spectrum to make
sure that maximum peaks, especially the peaks of interest, are represented in the real form. Also, to
some extent, it has been generally found that the phase difference is proportional to the Larmour
frequency of the peaks. Therefore, in the first order phase correction, we can multiply the entire
spectrum with the corresponding values of phase factors that are a function of frequencies on the x-
axis while continuously adjusting the constant of proportionality.
Lastly, we have got baseline correction. Many a times, the baseline might not be a straight line. It
may be slightly convex or concave or even wiggly. We can correct for these baselines by defining the
baseline which the computer will straighten out for us.
j. Peak Identification
Finally, it remains to convert the scale of the spectrum to the δ(ppm) scale and compare the values
of the Larmour frequencies of the peaks to known values in order to identify the peaks. Which atoms
are linked to which ones can be guessed from the multiplet structure of the peaks.
14

15. 4. Introduction to 2D NMR
Two dimensional NMR experiments use the phenomenon of scalar coupling to extract more
information, as well as increase the resolution by introducing a new dimension.
A nucleus that has been excited in the way described before is made to transfer its magnetisation to
a neighbouring nucleus via coupling interaction. The detector receives a combined FID that contains
relaxation signals from both the nuclei even though only one of them was excited.
The actual experiment consists of a pulse, a first relaxation period, a second pulse or the mixing
period, and a second relaxation period. Since the FID if formed from two time domains, its Fourier
transform generates a matrix with two frequency coordinates. Whenever magnetisation transfer
took place via coupling, we get cross peaks. Or else, we get auto-peaks or self-peaks or diagonal
peaks.
Fig.2. A 2D NMR spectrum
15

16. Part 2
………………………..
Effect of Polar Organic Solvents
on the enzyme 6B Lipase
16

17. 1. Background
Proteins that act as enzymes are the molecular catalysts that help perform all the metabolic
activities in any living cell. Every protein is made up of a long chain of amino acid residues. However,
a protein is not merely a sequence of its residues, which is only the primary structure. In the
presence of water, a protein, first of all, twists and turns to form alpha-helices and beta-sheets,
known as the secondary structure, and finally folds into an overall globular conformation; this is the
tertiary structure of the protein.
In this globular low-energy conformation, the non-polar groups lie in the interior and the polar
groups lie in the exterior, exposed to water. It is this conformation that is responsible for the
functionality of a protein- The ‘hydrophobic interaction’ is necessary. In the absence of these
interactions, in organic solvents, proteins cannot fold in the necessary way and hence cannot
function. Yet, sometimes, when a polar organic solvent is mixed with water, proteins can still
function up to a certain upper limit of the polar organic solvent concentration. As we go on
increasing the amount of this solvent (a process known as ‘titration’), small changes in the protein
continuously take place while still preserving its essential structure, till at some point where the
protein starts breaking down.
This point of breaking down is usually not very high for a protein. Few proteins even break down in a
1% solution of a polar organic solvent, like methanol.
Zahid et al. created a robust variant of a lipase from Bacillus subtilis named "6B" using multiple
rounds of in vitro evolution. The optimum activity temperature of 6B is much higher than that of
wild-type lipase. Most significantly, 6B does not aggregate upon heating. Physical basis of
remarkable thermostability and non-aggregating behaviour of 6B was explored using X-ray
crystallography, NMR and differential scanning calorimetry. Tightening of mobile regions of the
molecule such as loops and helix termini has helped the molecule to attain higher thermostability.
Accordingly, NMR studies suggest a very rigid structure of 6B lipase.* This implies that the enzyme
must not break down or show large perturbations upon titrating with polar organic solvents like
methanol, acetone, etc, up to a certain maximum concentration in water.
Lipases digest lipids. Lipids are non-polar organic compounds popularly known as fats. Since they are
non-polar and consequently immiscible, they form micelles in water. Lipases act on these micelles.
The good thing about having a protein that can withstand high concentrations of polar organic
solvents is that it opens a door to designing many new chemical reactions that were previously
thought to be impossible! It now seems possible to carry out new types of catalytic reactions
mediated by this lipase involving both lipids and polar organic solvents.
2. Experiment
The purpose of the following experiments was to reassert the stability and explore the stability limits
of 6B Lipase in polar organic solvents using NMR techniques. Initially, an attempt was made to do
this using NMR protein dynamics. However, the apparent failure of the initial experiments paved the
way for new experiments that used solvent-binding to establish the same.
17

18. 3. Protein Dynamics
The first to be designed were the Cleanex experiments. These experiments focus on a particular
scale of exchange rate. The faster and the slower peaks wouldn’t even appear in the spectra. It was
expected that Cleanex would yield an entire surface that had intermediate rate of exchange, that is,
a few milliseconds to a few microseconds of exchange period. This surface would be the site of the
protein 6B Lipase that is most affected by polar organic solvents.
However, the first few trials did not give any result- none of the peaks were visible. Upon
modification of the experiment design, finally a few peaks could be seen. Although correct, the
information wasn’t sufficient to make any assertions.
Fig.3. The only peaks visible in the Cleanex experiment.
18

19. Another experiment was designed, called HDex, which was used to measure the rate of exchange of
the amine hydrogen atoms of the residues with deuterium atoms when the protein was dissolved in
100% deuterated water. This experiment produced better results than the previous one, with 50% of
the peaks visible. Here are the results of the experiment.
Table.1. Results of the rate-of-exchange experiments.
Peaks absent Intermediate (Cleanex) Peaks Peaks present
N4, V6, V7, V9, K23, V27, Q29, W31, L36, E2, H3, A38, G145 H10, G11, I12, G13, G14, S15, S16, N18,
Y37, V39, L55, S56, F58, Q60, K61, V62, F19, E20, G21, I22, S24, S28, G30, S32, R33,
L63, D64, E65, V71, D72, I73, V74, A75, D34, K35, D40, F41, W42, D43, K44, T45,
H76, G79, G80, N82, T83, K88, Y89, L90, G46, T47, N48, Y49, N50, N51, G52, V54,
V96, A97, N98, V99, V100, T101, G103, R57, T66, G67, A68, K69, K70, A81, I87,
G104N, N106, Q121, L124, Y125, T126, D91, G92, G93, N94, K95, A105, R107,
S127, S141, A146, R147, V149, L160, Y161, L108, T109, T110, D111, K112, A113, G116,
S162, Y166, I169, K170, E171, G172, L173, T117, D118, N120, K122, I123, V136, R142,
G176 L143, D144, N148, Q150, I151, H152, G153,
V154, G155, H156, M157, G158, L159,
Q164, V165, S167, L168, N174, G175,
G177, Q178, N179, T180
These results are also represented by a colour-coded cartoon of 6B Lipase:
Fig.4. A colour-coded cartoon of 6B Lipase, where red represents residues with fast exchange, blue represents
residues with slow exchange and green represents residues with intermediate exchange. Black is for those residues
whose peaks could not be unambiguously identified in the spectra.
19

20. 4. Solvent Binding
Due to inability in extracting further information from the experiments on protein dynamics, it was
thought to be a good alternative to try and see if solvent binding could give a better picture of the
effect of polar organic solvents on 6B Lipase.
In the following experiments, a solution of 6B Lipase was titrated with various polar organic solvents,
most of the peaks being recognisable till up to 40% concentration of the organic solvent. Solvent
binding would perturb almost every peak by some amount. Some peaks would be much more
perturbed than others. It was hoped that the experiments on solvent binding would generate
coherent results about the more active side/surface of the protein.
Peak shifts, of course, could be due to direct solvent binding, or due to solvent binding on
neighbouring residues, as well as due to actual physical movement of the residues. We cannot
comment on the actual phenomena conclusively but we can certainly identify the side of the protein
that is affected the most in polar organic solvents. And depending on the increased or decreased
protein activity in these solvents, we can guess how much the perturbed side and the binding site
overlap.
The residues in the core of the protein are much more inaccessible than those on the surface. It will
be hard for a solvent molecule to penetrate such a solid structure and bind to an interior residue. In
spite of this, if such a binding is still taking place, represented by a significant chemical shift in the
peak of a core residue in the NMR spectrum, we can be sure that this was accomplished by ‘opening
up’ of the protein at some place. This kind of ‘opening up’ corresponds to structural instability of a
protein. Rigid proteins don’t open up easily.
First of all, the peaks were assigned in all the spectra. After that, a plot of the residues versus their
peak shifts was created for the highest concentration used in the experiment, which is usually 40%.
The nitrogen dimension was normalised to the hydrogen dimension while calculating the distances.
The next step of analysis was a pictorial representation of the more active sites, identified by virtue
of greater peak shifts. To do this, a baseline was defined first, below which all the residue peak shifts
were so low that they could be treated equivalently. Baselines were qualitatively chosen from the
plot of peak shifts by making sure that a very large majority of the residues shift by at least the
baseline.
After this, the part of the graph below the baseline was cut, setting the value of all the peaks that
are below the baseline or just touching the baseline to zero. The remaining part of the graph was
normalized to a scale of 0-50, 50 being the label for the most shifted peak. This information, known
as b-factor, was fed to a PyMol file of 6B Lipase. A convenient range of colours was chosen, say
yellow to red, such that yellow corresponds to a zero peak shift and red corresponds to maximum
normalised peak shift (that is, 50). All the intermediate values are represented by the appropriate
shade.
20

21. 40% Methanol
0.30
0.25
2 0.5
 = (((N) /23)+(H) )
0.20
0.15
2
0.10
0.05
0.00
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180
Residue number
Fig.5. A plot of peak shifts versus residue number for6B Lipase dissolved in 40% methanol
40% Acetone
0.60
0.55
0.50
2 0.5
 = (((N) /23)+(H) )
0.45
0.40
0.35
0.30
0.25
2
0.20
0.15
0.10
0.05
0.00
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180
Residue number
Fig.6. A plot of peak shifts versus residue number for 6B Lipase dissolved in 40% acetone
21

22. 40% Acetonitrile
0.75
0.70
0.65
2 0.5
0.60
 = (((N) /23)+(H) )
0.55
0.50
0.45
0.40
0.35
0.30
2
0.25
0.20
0.15
0.10
0.05
0.00
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180
Residue number
Fig.7. A plot of peak shifts versus residue number for 6B Lipase dissolved in 40% acetonitrile
10% DMF
0.25
2 0.5
0.20
 = (((N) /23)+(H) )
0.15
0.10
2
0.05
0.00
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180
Residue number
Fig.8. A plot of peak shifts versus residue number for 6B Lipase dissolved in 10% DMF. The spectrum for 10% DMF itself
was too noisy, so we did not go for higher concentrations.
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23. 40% Isopropanol
0.50
0.45
2 0.5
0.40
 = (((N) /23)+(H) )
0.35
0.30
0.25
0.20
2
0.15
0.10
0.05
0.00
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180
Residue number
Fig.9. A plot of peak shifts versus residue number for 6B Lipase dissolved in 40% isopropanol. However, since this
spectrum was excessively noisy, I am not very confident about this plot. Hence, I’ve also plotted the spectrum for 20%
isopropanol, which was very clear.
20% Isopropanol
0.40
0.35
2 0.5
 = (((N) /23)+(H) )
0.30
0.25
0.20
2
0.15
0.10
0.05
0.00
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180
Residue number
Fig10. A plot of peak shifts versus residue number for 6B Lipase dissolved in 20% isopropanol
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24. 40% Methanol
Fig11. Representation of peaks with higher peak shifts in 40% methanol on the surface of the protein in red.
40% Acetone
Fig12. Representation of peaks with higher peak shifts in 40% acetone on the surface of the protein in red.
40% Acetonitrile
Fig13. Representation of peaks with higher peak shifts in 40% acetonitrile on the surface of the protein in red.
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25. 10% DMF
Fig14. Representation of peaks with higher peak shifts in 10% DMF on the surface of the protein in red.
40% Isopropanol
Fig15. Representation of peaks with higher peak shifts in 40%isopropanol on the surface of the protein in red.
20% isopropanol
Fig16. Representation of peaks with higher peak shifts in 20% isopropanol on the surface of the protein in red.
25

26. 5. Discussion
The results obtained support the claim that 6B Lipase can remain quite stable even in the presence
of polar organic solvents with concentrations as high as 40%. Proteins are usually expected to unfold
and get denatured even in much lower concentrations of polar organic solvents. This would be
reflected in the spectra by a drastic change in the landscape and disappearance and appearance of
new unidentifiable peaks. With 6B Lipase, however, the peaks, although shifted, could still be
recognised without much ambiguity. This means that the solvent binding very slightly distorts the
protein.
From the spectra we also got information on the solvent-binding sites for various polar organic
solvents. Thus we know now which surface of the protein will be perturbed the most in a given polar
organic solvent.
From the figures of protein surface in which the solvent-binding sites are represented by a red-to-
yellow gradient of colours, we can see that there is a large overlap between the surface areas that
are active in the protein when solvated by different polar organic solvents. We suspect that this
overlapping region that is highly perturbed in all the solvents consists of more non-polar residues.
Since these are less polar than other residues, the polar organic solvent which is much less polar
than water preferentially binds to these regions and consequently produce larger perturbations
here. This assertion remains to be confirmed.
*The 6B protein has 181 residues and 180 peptide linkages. Its sequence is as follows:
AEHNPVVMVHGIGGSSSNFEGIKSYLVSQGWSRDKLYAVDFWDKTGTNYNNGPVLSRFVQKVLDETGAKKVDIV
AHSMGGANTLYYIKYLDGGNKVANVVTLGGANRLTTDKAPPGTDPNQKILYTSIYSSDDEIVPNYLSRLDGARNVQI
HGVGHMGLLYSPQVYSLIKEGLNGGGQNTN
… Alpha-helix … Beta-sheet
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27. Resources
Zahid et al, 2011. In Vitro Evolved Non-Aggregating and Thermostable Lipase:
Structural and Thermodynamic Investigation.
James Keeler, Understanding NMR Spectroscopy.
Malcolm Levitt, Spin Dynamics: Basics of Nuclear Magnetic Resonance.
Gordon S. Rule and T. Kevin Hitchens, Fundamentals of Protein NMR
Spectroscopy
All the 2D NMR spectra were analysed in Topspin and Sparky.
All the figures of protein structure have been generated in PyMol.
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