A report submitted to the   CENTRE FOR CELLULAR AND MOLECULAR BIOLOGY                           OnNMR Spectroscopy and its...
CERTIFICATEThis is to certify that the report titled “NMR Spectroscopy and its applicationin the study of Effect of Polar ...
DECLARATIONI hereby declare that the report titled “NMR Spectroscopy and its applicationin the study of Effect of Polar Or...
ACKNOWLEDGEMENTI would like to thank Dr. Ch. Mohan Rao, Director, CCMB, for providing mewith the opportunity to spend my s...
NMR Spectroscopy and its   application in the study of Effect of     Polar Organic solvents on 6B, a     mutant of Bacillu...
ContentsPart I   1. What in NMR?                                              08   2. What the Chemist knows              ...
Part 1     ………………………..Nuclear Magnetic Resonance       Spectroscopy                             7
1. What is NMR?Nuclear Magnetic Resonance, or NMR, is a powerful technique used to probe the structure,dynamics and chemic...
Based on the position of the peaks in the spectrum, we can guess how many types of hydrogenatoms are there and where each ...
Of course, a peak might split further and further due to coupling with multiple nuclei (multipletstructure), and the resul...
of +ħ/2 or –ħ/2 along the direction of the applied field. These orientations have different energiesand can be viewed as t...
strength of the magnetic field applied along the z-direction. The sign of ω0, if present, indicates thedirection of the pr...
f. RF pulse and resonanceAt this point of time, we apply an RF pulse of a certain frequency. It is just a chain of radiofr...
data sets are continuously added up. This tends to reinforce the signals due to repeated occurrenceand cancels out the noi...
4. Introduction to 2D NMRTwo dimensional NMR experiments use the phenomenon of scalar coupling to extract moreinformation,...
Part 2       ………………………..Effect of Polar Organic Solvents    on the enzyme 6B Lipase                                   16
1. BackgroundProteins that act as enzymes are the molecular catalysts that help perform all the metabolicactivities in any...
3. Protein DynamicsThe first to be designed were the Cleanex experiments. These experiments focus on a particularscale of ...
Another experiment was designed, called HDex, which was used to measure the rate of exchange ofthe amine hydrogen atoms of...
4. Solvent BindingDue to inability in extracting further information from the experiments on protein dynamics, it wasthoug...
40% Methanol                          0.30                          0.252 0.5 = (((N) /23)+(H) )                      ...
40% Acetonitrile                                0.75                                0.70                                0....
40% Isopropanol                                     0.50                                     0.45  2 0.5                  ...
40% Methanol Fig11. Representation of peaks with higher peak shifts in 40% methanol on the surface of the protein in red. ...
10% DMF   Fig14. Representation of peaks with higher peak shifts in 10% DMF on the surface of the protein in red.         ...
5. DiscussionThe results obtained support the claim that 6B Lipase can remain quite stable even in the presenceof polar or...
ResourcesZahid et al, 2011. In Vitro Evolved Non-Aggregating and Thermostable Lipase:Structural and Thermodynamic Investig...
Upcoming SlideShare
Loading in …5
×

6 b lipase nmr

495 views

Published on

0 Comments
1 Like
Statistics
Notes
  • Be the first to comment

No Downloads
Views
Total views
495
On SlideShare
0
From Embeds
0
Number of Embeds
1
Actions
Shares
0
Downloads
10
Comments
0
Likes
1
Embeds 0
No embeds

No notes for slide

6 b lipase nmr

  1. 1. A report submitted to the CENTRE FOR CELLULAR AND MOLECULAR BIOLOGY OnNMR Spectroscopy and its application in thestudy 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. 2. CERTIFICATEThis is to certify that the report titled “NMR Spectroscopy and its applicationin the study of Effect of Polar Organic solvents on 6B, a mutant of Bacillussubtilis Lipase” is submitted by Shreya Ray. The summer training work hasbeen carried out by her under my supervision at the Centre for Cellular andMolecular Biology (CCMB), Hyderabad, for a period of two months. Dr. Mandar V. Deshmukh Scientist CCMB Hyderabad 2
  3. 3. DECLARATIONI hereby declare that the report titled “NMR Spectroscopy and its applicationin the study of Effect of Polar Organic solvents on 6B, a mutant of Bacillussubtilis 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 RayPlace:Date: 3
  4. 4. ACKNOWLEDGEMENTI would like to thank Dr. Ch. Mohan Rao, Director, CCMB, for providing mewith the opportunity to spend my summer here at CCMB.I am also grateful to Dr. Ramesh K. Aggarwal, Coordinator, Summer TrainingProgramme, 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 freedomand space he gave me to learn. 4
  5. 5. NMR Spectroscopy and its application in the study of Effect of Polar Organic solvents on 6B, a mutant of Bacillus subtilis LipaseNuclear Magnetic Resonance, or NMR, is an extremely valuable tool for thestructural determination of many molecules, particularly proteins, and theirkinetics and their dynamics. NMR gives information about the identity and thechemical environment of atomic nuclei. This project report on NMR is dividedinto two sections. The first section is about the theory of NMR. This includes thebasic principles on which NMR spectroscopy is based, and explains thefundamental concepts involved. It goes on to discuss about the basic 1D NMRpulse experiment and the data analysis which follows, finishing off with a smallintroduction to 2D NMR. The second section consists of the application of NMRin a study of the behaviour of the enzyme 6B Lipase in the presence of variousconcentrations of different organic solvents; these concentrations weresuccessively increased (titration). Enzymes generally break down at higherconcentrations of organic solvents. The 6B Lipase, however, has displayed highstability in such environments and holds promise for catalytic application in newtypes of chemical reactions. 5
  6. 6. ContentsPart 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 15Part II 1. Background 17 2. Experiment 17 3. Protein Dynamics 18 4. Solvent Binding 20 5. Discussion 26 6
  7. 7. Part 1 ………………………..Nuclear Magnetic Resonance Spectroscopy 7
  8. 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 alternativemethod for structure determination if a protein cannot be crystallized, or if there is concern thatpacking has distorted the true structure in solution. NMR is also useful in probing molecularinteractions, such as solvent binding and ligand binding.2. What the Chemist knowsLike 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 quantummechanical property called ‘spin’ of the atomic nuclei. NMR spectra are unusual in that they appearat rather low frequencies (radiofrequency).In 1D NMR, we generally measure the spectrum of one isotope only in one experiment. This isbecause of the design of the experiment. Every isotope needs a different frequency window and wecan produce only one window at a time, usually. The most common NMR experiments involve thehydrogen window, where we only check the peak shifts of hydrogen atoms in the spectrum. So wewill discuss about the 1H NMR spectroscopy. The principles are very general and can be applied tospectroscopy experiments involving all other spin-half nuclei. Following is a 1H NMR spectrum ofglucose, just to show how a typical NMR spectrum looks like: Fig.1. 1D NMR spectrum of glucoseAll the peaks correspond to hydrogen but they are different because every hydrogen atom in themolecule experiences a different kind of environment created by the surrounding atoms and bonds. 8
  9. 9. Based on the position of the peaks in the spectrum, we can guess how many types of hydrogenatoms are there and where each atom belongs.The positions (frequencies) of these peaks, however, are not absolute; they depend on the strengthof the magnetic field being used, rather proportional to it. To solve this problem, we define a newkind of scale, called the Chemical Shift Scale. The Chemical Shift of a peak is defined as the ratio ofits distance from a reference peak to the frequency of the reference peak, both peaks beingacquired 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 byeveryone based on certain properties of the compound, like symmetry, covalent nature, minimumshielding, etc. For proton (1H) spectroscopy and carbon (13C) spectroscopy, TMS or tetramethyl silaneis the reference compound.The values of chemical shifts are very small, often of the order of 10-6, because the peak shifts arevery small compared to the value of the frequencies. To make the numbers more convenient, wewidely 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 dealwith, they are not so narrow as compared to the width of the frequency window (the spread offrequencies we deal with). When the separation between two peaks fall below the line width, thetwo peaks merge completely and cannot be distinguished, the exact point of merging depending onthe line shape.The basic line shape is the absorption mode line shape. It is symmetric about the maximum and itsline width is specified by its breadth at half-height. The intensity of this line is proportional to thenumber of protons (1H) giving out the signal, represented by the area under the peak. Hence, for thesame 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 twokinds of coupling:Weak coupling occurs when the chemical shift between coupled nuclei is very large as compared tothe coupling constant J. Coupling between two different isotopes, like 1H and 13C, is always weak. Inthe simplest case of coupling, two peaks interact and split each other into two, giving rise to fourpeaks in total. Two of these peaks belonging to the same nucleus form a ‘doublet’. Each peak of adoublet has half the intensity of the original peak, and they are placed symmetrically about thefrequency of the original peak. The distance between these peaks in the doublet is twice thecoupling constant J, which is found to be independent of the field strength and hence expressed inhertz. (Consequently, it will depend on the field strength whenever expressed in the δ (ppm) scale.) 9
  10. 10. Of course, a peak might split further and further due to coupling with multiple nuclei (multipletstructure), and the result can be predicted using the knowledge of J-values in case of each split. Thiskind of coupling is very useful in determining which atoms are linked to which ones, and forms animportant part of data analysis. However, in some cases, it might be a nuisance. For example, 13Cspectroscopy of organic spectra would become extremely complex since every carbon atom wouldbe 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 designingthe pulse-sequence experiment.Strong coupling occurs when the chemical shift between coupled nuclei is small, especially in thecase of homo-atom coupling. A doublet resulting from this kind of splitting is asymmetric both interms 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 toobserve this, and hence we can ignore this phenomenon for most of our purposes, where we do notneed so much detail. Therefore, we often say, “Equivalent spins do not split one another”.3. The Theory of 1D NMRThe theory of 1D NMR is illustrated by the most basic single-pulse experiment.a. Nuclear SpinThe theory of NMR involves the quantum mechanical property called ‘spin’ of the atomic nuclei. Thequantum mechanical spin does not have a classical analogue, but on many occasions we candescribe certain of its properties to be very similar to the classical spin. Every fundamental particlehas 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 theexperimental fact that upon applying an external magnetic field to such a particle, the particle willhave an angular momentum of +ħ/2 or –ħ/2 along the direction of the applied field, its totalmagnitude being ħ/2.The nucleus is made up of protons and neutrons. However, in nuclei with an even number of protonsand 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 spinangular momentum. The proportionality constant γ is called the gyromagnetic ratio and it is anintrinsic property of the nucleus in question.b. The external magnetic fieldIt is the presence of the external magnetic field which breaks the spatial homogeneity and gives riseto the breakdown of spin degeneracy. The nucleus is now restricted to have an angular momentum 10
  11. 11. of +ħ/2 or –ħ/2 along the direction of the applied field. These orientations have different energiesand 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 alongthe magnetic field, they never do so because of the quantum restrictions. Their z-componentscannot be changed. At this point, the nuclear spin can be thought of as precessing about themagnetic field because their x and y components continually change while preserving the magnitudeof spin and that of its z-component.As expected, the nuclear state where the direction of the magnetic moment due to nuclear spin is inthe 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 isgiven by:Since the magnetic field is along the z-axis and has the magnitude ‘B’For a spin-half systemOf these, the lower energy state, or the ground state is denoted by α and the higher energy state orthe excited state is denoted by β.The energy of transition from α to β state is given byIn NMR experiments, a strong uniform external magnetic field is provided by powerfulsuperconducting coils, cooled by liquid helium and liquid nitrogen.c. The Larmour FrequencyUsing the relation , where ω is the angular frequency, we can writeω0 is called Larmour Frequency. It is the frequency at which the spin of a nucleus with agyromagnetic ratio γ precesses about a given magnetic field. As we can clearly see, the Larmourfrequency and the energy difference between the two levels are directly proportional to the 11
  12. 12. strength of the magnetic field applied along the z-direction. The sign of ω0, if present, indicates thedirection of the precession.The values of γ, however, are very small. And the present methods of obtaining a uniform magneticfield, which is crucial for our purpose, impose a limit on the strength of the magnetic field that canbe 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 todifferent parts of the molecule. This is because each kind of proton experiences a different set ofchemical environment, different electrostatic forces and consequently different magnetic fields.Movement of electron densities nearby, as a response to the external magnetic field, might shield orreinforce the original magnetic field around any particular proton, thus changing ω0. This forms thebasis of chemical shift; it is the reason we have different peaks for the different hydrogen atoms inthe compound.Scalar coupling or J-coupling occurs when the interaction of one spin with the external magneticmoment polarises that spin, which in turn polarises the bonded electrons, which alters the effectivemagnetic field around the nucleus bonded to the former nucleus. Since there can be two possiblepolarisations, consequently the magnetic field around the latter nucleus may be either increased ordecreased. The two possible cases give rise to the observed peak split. Since the energy differencebetween the spin states is very small, both the polarisations are almost equally likely, hence bothpeaks of the doublet have the same intensity. Repeated splitting of peaks produces multipletstructures.d. Spin PopulationsAs a consequence of small γ, the energy difference between the two levels are also very low, henceboth the ground state and the excited state are populated almost equally, with the ground statehaving a very slightly larger population. This can be thought of as a result of equilibrium between themagnetic 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 LockingIt is very crucial for the magnetic field being used in the NMR experiment to be constant and uniformthroughout the scan and throughout the sample. Shimming is performed to adjust defects in theexisting field uniformity by passing the requisite amount of current through the shim coils. The FieldFrequency 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. 13. f. RF pulse and resonanceAt this point of time, we apply an RF pulse of a certain frequency. It is just a chain of radiofrequencyphotons that we create by passing an oscillating current through a coil whose axis is perpendicular tothe direction of the original magnetic field.The RF pulse is sent with a well-calculated frequency. For proton NMR, we send a pulse having afrequency around the middle of the hydrogen window (hydrogen window refers to the range of peakshifts that a proton can cover). Since the frequencies of the RF pulse and the Larmour frequencies ofthe protons are either same or very close, resonance occurs and the photons are absorbed by theprotons. These photons or this electromagnetic wave has an oscillating magnetic field along the axisof the coil. Although small in magnitude, this magnetic field becomes very powerful due to the effectof resonance and hence tends to make the spin precess about itself instead of precessing about thestronger original magnetic field along the z-axis. This phenomenon is known as transversemagnetisation.The Larmour frequency about this new oscillating magnetic field is obviously different, and if thispulse is applied for exactly 1/4th of the time period for this rotation, we will have rotated the originalmagnetisation by 90 degrees with respect to the initial direction. This is known as a 900 pulse; it isthe most basic pulse.g. Relaxation and the FIDWhen the RF pulse is switched off, the spins start getting back to their previous equilibrium. Thepulse had tilted the entire magnetisation by 900. All the spins now start to relax together, thusbringing coherence between them. It starts with a precession in the x-y plane about the originalmagnetic 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, thecoherence is slowly lost in this process, known as spin-spin relaxation. Another process thatcontributes to this relaxation is the spin-lattice relaxation where the spins try to regain their originalpopulation distribution.When the RF pulse is switched off, the same coil that was used to generate it now acts as thereceiver coil. The precessing magnetic field projects an oscillating magnetic field in the axis of thecoil 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 noiseIn NMR, signals are very weak and they can be easily buried in noise. There are many ways toimprove the signal-to-noise ratio. The most important part of this is signal averaging, where thepulse sequence is repeatedly sent and FID is repeatedly collected from the sample and successive 13
  14. 14. data sets are continuously added up. This tends to reinforce the signals due to repeated occurrenceand 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 gettingthe FID.i. Fourier Transformation and Data ProcessingThe FID is recorded in the analogue form and converted into the digital form by sampling the data atvery close intervals. Both real and imaginary parts of the signal are stored; the imaginary partscontain 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 inthe 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 whichdecreases the magnitude of the tail of the FID which is predominantly composed of noise and alsodecreases the magnitude of the FID at its beginning to control for peak broadening, while increasingthe magnitude of the central part.Another problem is that many signals may not be in the same phase in which the receiver is. Ashinted earlier, we collect data in the complex form, keeping both the real cosine part and theimaginary 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 thespectrum 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 theimaginary parts. The solution is obviously to multiply with the phase difference; however there is noway to know the phase difference.In the zero-order phase correction, we continuously adjust the phase of the entire spectrum to makesure that maximum peaks, especially the peaks of interest, are represented in the real form. Also, tosome extent, it has been generally found that the phase difference is proportional to the Larmourfrequency of the peaks. Therefore, in the first order phase correction, we can multiply the entirespectrum 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. Itmay be slightly convex or concave or even wiggly. We can correct for these baselines by defining thebaseline which the computer will straighten out for us.j. Peak IdentificationFinally, it remains to convert the scale of the spectrum to the δ(ppm) scale and compare the valuesof the Larmour frequencies of the peaks to known values in order to identify the peaks. Which atomsare linked to which ones can be guessed from the multiplet structure of the peaks. 14
  15. 15. 4. Introduction to 2D NMRTwo dimensional NMR experiments use the phenomenon of scalar coupling to extract moreinformation, 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 toa neighbouring nucleus via coupling interaction. The detector receives a combined FID that containsrelaxation 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 mixingperiod, and a second relaxation period. Since the FID if formed from two time domains, its Fouriertransform generates a matrix with two frequency coordinates. Whenever magnetisation transfertook place via coupling, we get cross peaks. Or else, we get auto-peaks or self-peaks or diagonalpeaks. Fig.2. A 2D NMR spectrum 15
  16. 16. Part 2 ………………………..Effect of Polar Organic Solvents on the enzyme 6B Lipase 16
  17. 17. 1. BackgroundProteins that act as enzymes are the molecular catalysts that help perform all the metabolicactivities 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 thepresence 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 thetertiary structure of the protein.In this globular low-energy conformation, the non-polar groups lie in the interior and the polargroups lie in the exterior, exposed to water. It is this conformation that is responsible for thefunctionality of a protein- The ‘hydrophobic interaction’ is necessary. In the absence of theseinteractions, in organic solvents, proteins cannot fold in the necessary way and hence cannotfunction. Yet, sometimes, when a polar organic solvent is mixed with water, proteins can stillfunction up to a certain upper limit of the polar organic solvent concentration. As we go onincreasing the amount of this solvent (a process known as ‘titration’), small changes in the proteincontinuously take place while still preserving its essential structure, till at some point where theprotein starts breaking down.This point of breaking down is usually not very high for a protein. Few proteins even break down in a1% solution of a polar organic solvent, like methanol.Zahid et al. created a robust variant of a lipase from Bacillus subtilis named "6B" using multiplerounds of in vitro evolution. The optimum activity temperature of 6B is much higher than that ofwild-type lipase. Most significantly, 6B does not aggregate upon heating. Physical basis ofremarkable thermostability and non-aggregating behaviour of 6B was explored using X-raycrystallography, NMR and differential scanning calorimetry. Tightening of mobile regions of themolecule 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 enzymemust not break down or show large perturbations upon titrating with polar organic solvents likemethanol, 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 arenon-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 organicsolvents is that it opens a door to designing many new chemical reactions that were previouslythought to be impossible! It now seems possible to carry out new types of catalytic reactionsmediated by this lipase involving both lipids and polar organic solvents.2. ExperimentThe purpose of the following experiments was to reassert the stability and explore the stability limitsof 6B Lipase in polar organic solvents using NMR techniques. Initially, an attempt was made to dothis using NMR protein dynamics. However, the apparent failure of the initial experiments paved theway for new experiments that used solvent-binding to establish the same. 17
  18. 18. 3. Protein DynamicsThe first to be designed were the Cleanex experiments. These experiments focus on a particularscale of exchange rate. The faster and the slower peaks wouldn’t even appear in the spectra. It wasexpected 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 theprotein 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. Uponmodification of the experiment design, finally a few peaks could be seen. Although correct, theinformation wasn’t sufficient to make any assertions. Fig.3. The only peaks visible in the Cleanex experiment. 18
  19. 19. Another experiment was designed, called HDex, which was used to measure the rate of exchange ofthe amine hydrogen atoms of the residues with deuterium atoms when the protein was dissolved in100% deuterated water. This experiment produced better results than the previous one, with 50% ofthe peaks visible. Here are the results of the experiment.Table.1. Results of the rate-of-exchange experiments.Peaks absent Intermediate (Cleanex) Peaks Peaks presentN4, 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, T180These 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 representsresidues with slow exchange and green represents residues with intermediate exchange. Black is for those residueswhose peaks could not be unambiguously identified in the spectra. 19
  20. 20. 4. Solvent BindingDue to inability in extracting further information from the experiments on protein dynamics, it wasthought to be a good alternative to try and see if solvent binding could give a better picture of theeffect 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. Solventbinding would perturb almost every peak by some amount. Some peaks would be much moreperturbed than others. It was hoped that the experiments on solvent binding would generatecoherent 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 onneighbouring residues, as well as due to actual physical movement of the residues. We cannotcomment on the actual phenomena conclusively but we can certainly identify the side of the proteinthat is affected the most in polar organic solvents. And depending on the increased or decreasedprotein activity in these solvents, we can guess how much the perturbed side and the binding siteoverlap.The residues in the core of the protein are much more inaccessible than those on the surface. It willbe hard for a solvent molecule to penetrate such a solid structure and bind to an interior residue. Inspite of this, if such a binding is still taking place, represented by a significant chemical shift in thepeak of a core residue in the NMR spectrum, we can be sure that this was accomplished by ‘openingup’ of the protein at some place. This kind of ‘opening up’ corresponds to structural instability of aprotein. 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 theirpeak 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 virtueof greater peak shifts. To do this, a baseline was defined first, below which all the residue peak shiftswere so low that they could be treated equivalently. Baselines were qualitatively chosen from theplot of peak shifts by making sure that a very large majority of the residues shift by at least thebaseline.After this, the part of the graph below the baseline was cut, setting the value of all the peaks thatare below the baseline or just touching the baseline to zero. The remaining part of the graph wasnormalized to a scale of 0-50, 50 being the label for the most shifted peak. This information, knownas b-factor, was fed to a PyMol file of 6B Lipase. A convenient range of colours was chosen, sayyellow to red, such that yellow corresponds to a zero peak shift and red corresponds to maximumnormalised peak shift (that is, 50). All the intermediate values are represented by the appropriateshade. 20
  21. 21. 40% Methanol 0.30 0.252 0.5 = (((N) /23)+(H) ) 0.20 0.152 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 0.502 0.5 = (((N) /23)+(H) ) 0.45 0.40 0.35 0.30 0.252 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. 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 numberFig.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. 22
  23. 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 numberFig.9. A plot of peak shifts versus residue number for 6B Lipase dissolved in 40% isopropanol. However, since thisspectrum 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 23
  24. 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% AcetonitrileFig13. Representation of peaks with higher peak shifts in 40% acetonitrile on the surface of the protein in red. 24
  25. 25. 10% DMF Fig14. Representation of peaks with higher peak shifts in 10% DMF on the surface of the protein in red. 40% IsopropanolFig15. Representation of peaks with higher peak shifts in 40%isopropanol on the surface of the protein in red. 20% isopropanolFig16. Representation of peaks with higher peak shifts in 20% isopropanol on the surface of the protein in red. 25
  26. 26. 5. DiscussionThe results obtained support the claim that 6B Lipase can remain quite stable even in the presenceof polar organic solvents with concentrations as high as 40%. Proteins are usually expected to unfoldand get denatured even in much lower concentrations of polar organic solvents. This would bereflected in the spectra by a drastic change in the landscape and disappearance and appearance ofnew unidentifiable peaks. With 6B Lipase, however, the peaks, although shifted, could still berecognised without much ambiguity. This means that the solvent binding very slightly distorts theprotein.From the spectra we also got information on the solvent-binding sites for various polar organicsolvents. Thus we know now which surface of the protein will be perturbed the most in a given polarorganic 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 thatare active in the protein when solvated by different polar organic solvents. We suspect that thisoverlapping 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 polarthan water preferentially binds to these regions and consequently produce larger perturbationshere. This assertion remains to be confirmed.*The 6B protein has 181 residues and 180 peptide linkages. Its sequence is as follows:AEHNPVVMVHGIGGSSSNFEGIKSYLVSQGWSRDKLYAVDFWDKTGTNYNNGPVLSRFVQKVLDETGAKKVDIVAHSMGGANTLYYIKYLDGGNKVANVVTLGGANRLTTDKAPPGTDPNQKILYTSIYSSDDEIVPNYLSRLDGARNVQIHGVGHMGLLYSPQVYSLIKEGLNGGGQNTN… Alpha-helix … Beta-sheet 26
  27. 27. ResourcesZahid 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 NMRSpectroscopyAll the 2D NMR spectra were analysed in Topspin and Sparky.All the figures of protein structure have been generated in PyMol. 27

×