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

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

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:

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.

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.

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

7

Part 1

………………………..

Nuclear Magnetic Resonance

Spectroscopy

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.

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.)

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

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

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.

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

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.

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

16

Part 2

………………………..

Effect of Polar Organic Solvents

on the enzyme 6B Lipase

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.

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.

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, Y37, V39, L55, S56, F58, Q60, K61, V62, L63, D64, E65, V71, D72, I73, V74, A75, H76, G79, G80, N82, T83, K88, Y89, L90, V96, A97, N98, V99, V100, T101, G103, G104N, N106, Q121, L124, Y125, T126, S127, S141, A146, R147, V149, L160, Y161, S162, Y166, I169, K170, E171, G172, L173, G176

E2, H3, A38, G145

H10, G11, I12, G13, G14, S15, S16, N18, F19, E20, G21, I22, S24, S28, G30, S32, R33, D34, K35, D40, F41, W42, D43, K44, T45, G46, T47, N48, Y49, N50, N51, G52, V54, R57, T66, G67, A68, K69, K70, A81, I87, D91, G92, G93, N94, K95, A105, R107, L108, T109, T110, D111, K112, A113, G116, T117, D118, N120, K122, I123, V136, R142, 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.

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.

21

40% Methanol

Fig.5. A plot of peak shifts versus residue number for6B Lipase dissolved in 40% methanol

40% Acetone

Fig.6. A plot of peak shifts versus residue number for 6B Lipase dissolved in 40% acetone

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

0.00

0.05

0.10

0.15

0.20

0.25

0.30

=

(((

N)2

/23)+

(H

)2)0

.5

Residue number

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

=

(((

N)2

/23)+

(H

)2)0

.5

Residue number

22

40% Acetonitrile

Fig.7. A plot of peak shifts versus residue number for 6B Lipase dissolved in 40% acetonitrile

10% DMF

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.

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

0.000.050.100.150.200.250.300.350.400.450.500.550.600.650.700.75

=

(((

N)2

/23)+

(H

)2)0

.5

Residue number

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

0.00

0.05

0.10

0.15

0.20

0.25

=

(((

N)2

/23)+

(H

)2)0

.5

Residue number

23

40% Isopropanol

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

Fig10. A plot of peak shifts versus residue number for 6B Lipase dissolved in 20% isopropanol

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

=

(((

N)2

/23

)+(

H)2

)0.5

Residue number

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

= (

((

N)2

/23

)+(

H)2

)0.5

Residue number

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.

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.

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

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.