Nuclear Magnetic Resonance spectroscopy (NMR) is a one of the powerful and theoretically complex analytical technique. It is a spectroscopy where an ensemble of nuclei of atoms that possesses "magnetic moments" and "angular momentum" is subjected to an external magnetic field. As the nuclear angular momentum is quantized, these nuclei align either parallel or anti-parallel to the applied magnet field. Depending upon the nature of nuclei, one of these aligned states (in most cases, it is parallel) is more energetically favorable; that gives rise to net bulk magnetization. An external radiofrequency pulse is applied to the bulk magnetization to establish a resonance condition which in turn realigns bulk magnetization in the transverse plane of the external magnetic field. Ensemble of spins then precesses at a characteristic frequency and depending upon their chemical environment these precessional frequencies can be different for different chemical entity (Chmical shift). These ensemble of spin precessing in the magnetic field generates current which is a time decaying function as the spins restore their equilibrium states. Time domain signals are Fourier transformed to generate a frequency domain data which provide characteristic property of various chemical entities

Bruker 750 WB MHz & its configuration :

• Make and Model : Bruker Biospin Switzerland, Avance III 750 MHz
• Magnetic Field : 17.61 Tesla, Wide bore (89 mm)
• Host Computer : HP make Linux PC equipped with Intel Xenon Dual core W3505, 2, 53 GHz, 4 GB RAM, 500 GB Hard Disk, DVD-RW, 20inch TFT monitor
• Software : Topspin 3.0 or higher for data processing and analysis
• Probes Available :

For solution-state NMR :

• 5mm Triple resonance H/C/N nuclei with 2H lock and 50G/cm gradient along Z axis

For Solid-State NMR :

• 4mm CP-MAS DVT Triple resonance H/X/Y probe with max spinning speed of 15KHz
• 3.2 mm CP-MAS Triple resonance (H/C/N) E-free probe (Bio-solid) with max. spinning speed of 24 KHz
• 1.9 mm H/C/N CP MAS Triple resonance (H/C/N) probe with max. spinning speed of 42 KHz

For wide-line static Probes : 

Low-temperature wide-line probe, solenoid coil 5 mm and 7.5 mm, can be tuned to various nuclei and can go upto 20K.

Decoupling :

• ¹H Homo decoupling
• Simultaneous ¹³C and ¹⁵N decoupling
• Deuterium decoupling by ²H transmitter

IIT Bombay HFNMR facility is one of the highest field in the country and suited for biological macro-molecular characterization. One can perform experiments in solid as well as in liquid phase. Following are special features:

• Experiments can be performed with wide temperature range.
• A series on nD experiments can be performed in the liquid phase.
• Suitable for multi-nuclei, multi-dimensional MAS experiments.
• Static solids can be investigated at various temperature (up to 20K)

2022

1. Dey A, Mitra D, Rachineni K, Paithankar H, Khatri LR, Vajpai N, Kumar A*. Mapping of methyl epitopes of a peptide-drug with its receptor by 2D STDD-Methyl TROSY NMR. Chembiochem. 2022 Oct 13. doi: 10.1002/cbic.202200489. Epub ahead of print. PMID: 36227643.

2. Shukla, Shivangi, and Ashutosh Kumar. Centromere Chromatin Dynamics at a Glance. Epigenomes 6, no. 4 (2022): 39.doi:10.3390/epigenomes6040039.

3. Shukla S, Agarwal P, Kumar A. Disordered regions tune order in chromatin organization and function. Biophys Chem. 2022 Feb;281:106716. doi: 10.1016/j.bpc.2021.106716. Epub 2021 Nov 17. PMID: 34844028.

4. Singh JS, Sajeev T K, Panigrahi R, Cherry P, Panchakshari NA, Shukla VK, Kumar A*, Mishra RK. Implications of critical node-dependent unidirectional cross-talk of Plasmodium SUMO pathway proteins. Biophys J. 2022 Apr 19;121(8):1367-1380. doi: 10.1016/j.bpj.2022.03.022. Epub 2022 Mar 21. PMID: 35331687; PMCID: PMC9072691.

5. Agadi N, Maity A, Jha AK, Chakrabarti R, Kumar A. Distinct mode of membrane interaction and disintegration by diverse class of antimicrobial peptides. Biochim Biophys Acta Biomembr 2022 Dec 1;1864(12):184047. doi: 10.1016/j.bbamem.2022.184047. Epub 2022 Sep 12. PMID: 36100074.

6. Bhattacharya A, Shukla VK, Kachariya N, Preeti, Sehrawat P, Kumar A. Disorder in the Human Skp1 Structure is the Key to its Adaptability to Bind Many Different Proteins in the SCF Complex Assembly. J Mol Biol. 2022 Sep 15;434(21):167830. doi: 10.1016/j.jmb.2022.167830. Epub ahead of print. PMID: 36116539.

7. Soumya Ranjan Pujahari, Pramod S. Mali, Rudra N. Purusottam, Ashutosh Kumar Combined liquid-state and solid-state NMR at natural abundance for comparative HOS assessment in the formulated-state of biphasic Biopharmaceutics. https://doi.org/10.1021/acs.analchem.2c05485

8. Sreya Das, Navin Khaneja, ``Composite pulse combinations for chirp excitation'' Journal of Magnetic Resonance , 347, 107359, (2022).

9. Sreya Das, Justin Jacob, Navin Khaneja, ``Mechanism of chirp excitation'' Journal of Magnetic Resonance Open , Volumes 10-11, 100026, (2022).

10. Manoj Gautam, Govind Kumar Mishra,Aakash Ahuja,Supriya Sau,Mohammad Furquan,Sagar Mitra‘Direct-Contact Prelithiation of Si–C Anode Study as a Function of Time, Pressure, Temperature, and the Cell Ideal Time”

11. S Yadav, AK Sam, C Venkataraman, A Kumar, HC Phuleria, “1H NMR structural signatures of source and atmospheric organic aerosols in India” Chemosphere 301, 134681

12. A Anand, S Yadav, HC Phuleria, “Chemical characteristics and oxidative potential of indoor and outdoor PM2. 5 in densely populated urban slums” Environmental Research 212, 113562

2021-22

1. Joshi S, Khatri L K, Kumar A*, Rathore S A. Monitoring size and oligomeric-state distribution of therapeutic mAbs by NMR and DLS: Trastuzumab as a case study J Pharm Biomed Anal 2021, 195, 113841. doi: 10.1016/j.jpba.2020.113841

2. Joshi S, Khatri L K, Kumar A*, Rathore S A. NMR based quality evaluation of mAb therapeutics: A proof of concept higher order structure biosimilarity assessment of trastuzumab biosimilars J Pharm Biomed Anal 2022, 214, 114710 doi:/10.1016/J.JPBA.2022.114710

3. Khosravi F, Upadhyay M, Kumar A, Shahsavani MB, Akbarian M, Najafi H, Tamaddon AM, Yousefi R. A novel method for the chaperone aided and efficient production of human proinsulin in the prokaryotic system J Biotechnol. 2022 Feb 20;346:35-46. doi:10.1016/j.jbiotec.2022.01.002. Epub 2022 Jan 21.PMID: 35066065

4. Sarkar S, Purusottom N R, Kumar A, Khaneja N. Chirp pulse sequences for broadband π rotation J Magn Reson. 2021, 38, 107002. doi: 10.1016/j.jmr.2021.107002

5. Pravin N, Kumar R, Tripathi S, Kumar P, Mohite GM, Navalkar A, Panigrahi R, Singh N, Gadhe LG, Manchanda S, Shimozawa M, Nilsson P, Johansson J, Kumar A, Maji SK, Shanmugam M. Benzimidazole‐based Fluorophores for the Detection ofAmyloid Fibrils with Higher Sensitivity than Thioflavin‐T J. Neurochem. 2021, 156(6), 1003-
1019. doi: 10.1111/jnc.15138

6. Marathe S, Dhamija B, Kumar S, Jain N, Ghosh S, Dharikar JP, Srinivasan S, Das S, Sawant A, Desai S, Khan F, Syiemlieh A, Munde M, Nayak C, Gandhi M, Kumar A, Srivastava S, Venkatesh KV, Barthel SR, Purwar R. (2021) Multiomics Analysis and Systems Biology Integration Identifies the Roles of IL-9 in Keratinocyte Metabolic
Reprogramming. J Invest Dermatol. doi: 10.1016/j.jid.2021.02.013.

7. AS Sawner, S Ray, P Yadav, S Mukherjee, R Panigrahi, M Poudyal, K Patel, D Ghosh, E Kummerant, A Kumar, R Riek and SK Maji (2021), Modulating α-Synuclein Liquid–Liquid Phase Separation. Biochemistry, 60(48): 3676-3696

8. Singh JS, Sajeev TK, Panigrahi R, Shukla VK, Kumar A*, Mishra RK Implications of critical nodes-dependent unidirectional cross-talk between Plasmodium and Human SUMO pathway proteins in Plasmodium infection Biophy. J.(2022) https://doi.org/10.1016/j.bpj.2022.03.022

  2017-18

1. Dubey R, Minj P, Malik N, Sardesai DM, Kulkarni SH, Acharya JD, Bhavesh NS, Sharma S, Kumar A (2017) Recombinant human islet amyloid polypeptide forms shorter fibrils and mediates β-cell apoptosis via generation of oxidative stress. Biochem J. 16:3915-3934

2. Jha NN, Kumar R, Panigrahi R, Navalkar A, Ghosh D, Sahay S, Mondal M, Kumar A Maji SK (2017) Comparison of α-Synuclein Fibril Inhibition by Four Different Amyloid Inhibitors. ACS Chem Neurosci DOI:10.1021/acschemneuro.7b00261

3. Ranjan P, Kumar A (2017) Perturbation in long-range contacts modulates kinetics of amyloid formation in α-Synuclein familial mutants. ACS Chem. Neurosci. 18: 2235-2246

4. Khaneja N, Kumar A (2017) Two pulse recoupling. J Magn Res 281: 162-171.

5. Khaneja N, Kumar A (2017) Broadband excitation by method of double sweep. Applied  MagnRes 281: 162-171

6. Shukla VK, Singh JS, Vispute N, Ahmad B, Kumar A, Hosur RV (2017) A functionally  relevantintermediate in the unfolding pathway of Cyclophilin. Biophysical J 112: 605-619

7. Ranjan P, Ghosh D, Yarramala DS, Maji SK, Kumar A (2017) Differential copper binding to alphasynucleinand its disease-associated mutants affect the aggregation and amyloid formation. Biochim Biophys Acta 1861: 365-374

8. N. Khaneja, Chirp excitation Journal of Magnetic Resonance 282, 32-36 (2017).

9. N. Khaneja, Electron dynamics in solid state via time varying wavevectors Physica B 539, 29-34 (2018).

10. N. Khaneja, Chirp Mixing Chemical Physics Letters 704, 62-67 (2018) 11. N. Khaneja, Conservation of Energy, Density of States and Spin Lattice relaxation Concepts in

Magnetic Resonance: Part A (2018) https://doi.org/10.1002/cmr.a.21457

2016-17

1.Khaneja N and Kumar A, "Two Pulse Recoupling" Journal of Magnetic Resonance 281, 162-171 (2017).

2. Khaneja N and Kumar A, "Broadband excitation by method of double sweep", Applied Magnetic Resonance 48(8), 771-782 (2017).

3. Khaneja N "Rf-inhomogeneity compensation using method of Fourier synthesis" Journal of Magnetic Resonance, 277, 113-116 (2017).

4. Ranjan P, Ghosh D, Yarramala DS, Maji SK, Kumar A (2017) Differential copper binding to alpha-synuclein and its disease-associated mutants affect the aggregation and amyloid formation Biochim Biophys Acta 1861: 365-374

5. Khaneja N, Kumar A (2016) Four pulse recoupling. J Magn Res 272: 158-165.

6. Singh JS, Shukla VK, Gujrati M, Mishra RK, Kumar A (2016) Backbone and side chain resonance assignments of Plasmodium falciparum SUMO. Biomol NMR Assign 11(1):17-20. doi: 10.1007/s12104-016-9712-9.

7. Sagar N, Singh AK, Temgire MK, Vijayalaxmi S, Dhawan A, Kumar A, Chattopadhyay N, Bellare JR (2016) 3D scaffold induces efficient bone repair: in vivo studies of ultra-structural architecture at the interface. RSC Advance

8. Jacob RS, Das S, Ghosh S, Anoop A, Jha NN, Khan T, Singru P, Kumar A, Maji SK (2016) Amyloid formation of growth hormone in presence of zinc: Relevance to its storage in secretory granules. Sci Rep 23; 6:23370

9. Khaneja N, Kumar A (2016) Recoupling Pulse Sequences with Constant Phase Increments. J Magn Res 271:75-82

10. Malik N, Kumar A (2016) Resonance assignment of disordered protein with repetitive and overlapping sequence using combinatorial approach reveals initial structural propensities and local restrictions in the denatured state. J Biomol NMR 66:21-35

11. Kachariya NN, Dantu SC, Kumar A (2016). Backbone and side-chain assignments of human cell-cycle regulatory protein Skp1. Biomol NMR Assignment

12. Ranjan P, Kumar A (2016). The Involvement of His50 during Protein Disulfide Isomerase binding is essential for inhibiting α-Syn fibril formation. Biochemistry 55(19): 2677-80.

13. Shukla VK, Singh JS, Trivedi D, Hosur RV, Kumar A (2016). NMR assignments of mitochondrial cyclophilin Cpr3 from Saccharomyces cerevisiae. Biomol NMR Assign 10(1): 203-6.

14. Singh AK, Gajiwala AL, Rai RK, Khan P, Singh C, Barbhuyan T, Vijayalakshmi S, Chattopadhyay N, Sinha N, Kumar A, Bellare JB (2016). Cross-correlative 3D micro-structural investigation of human bone processed into bone allografts. Mater Sci Eng C Mater Biol Appl 62: 574-84.

1) What is NMR?

Nuclear Magnetic Resonance (NMR) is a spectroscopic technique that can be used to obtain structural and dynamic properties of molecules by utilizing the behaviour of certain atoms when placed in very powerful superconducting magnets. When NMR active nuclei are placed in a strong magnetic field they can align with the field and begin to precess at a frequency dependent on the isotopes gyromagnetic ratio and the strength of the applied magnetic field. The component of the frequency that is dependent on the chemical and physical environment of the nuclei is called the chemical shift. NMR experiments perturb the alignment by applying short pulses of radio frequency (RF) energy to determine the chemical shift of each of the NMR active atoms in the molecule being studied. By using combinations of pulses and delays (what is known as a pulse sequence) additional information may be ascertained such as which atoms are bonded to each other and which atoms are spatially close to each other. By utilizing many different experiments, it is possible to determine the three-dimensional structure of molecules, including large bio-molecules such as proteins, nuclei acids etc.

2) What types of biological questions can I answer with NMR?

There are too many uses for NMR of biological samples to list, but here are some of the more common biological questions one can routinely address: 1) Determining the three-dimensional structure of proteins and protein complexes in solution 2) Using NMR relaxation to probe the molecular motions at various timescales 3) To determine the site of binding for small molecules, metals, other proteins, nucleic acids, etc. 4) To monitor the protein stability for site directed mutants 5) For studying conformational exchange in proteins and probing low-populated intermediate states

3) Why do I need to label my protein by 15N or 13C?

The experiments that are used to study proteins depend on observing the 15N and 13C chemical shifts in addition to protons. The natural abundance of 15N and 13C are 0.3% and 1.0% respectively. At such low natural abundance the sensitivity of the experiments is too low to detect. To resolve this issue protein can be expressed on enriched media allowing full incorporation of these stable isotopes.

4) How long does it take to run an NMR experiment?

The answer to this question depends on the type of NMR experiment that is being run, the concentration of the sample, and the overall behaviour of the system being studied. For a simple 1D spectrum of a small molecule data can typically be collected in 15 minutes. For very insensitive 3D and 4D NMR experiments a single experiment may take several days to a week. To collect an entire set of experiments necessary to calculate a three-dimension structure data collection time can vary from a week to a few months.

5) How much sample do need?

This question is difficult to answer, as it is dependent on the questions being asked and the behaviour of the sample. Typically, 300 l (with specialized 3mm NMR tubes) to 600 l of sample is needed. Protein concentrations for well-behaved systems should be above 500 M for structural studies, but lower concentrations may be used for other non-structural studies. While 500 M is a rough estimate for the lowest concentration to use for structural studies it is advisable to make protein concentrations as high as possible and should be limited by solubility and protein behaviour, not the amount of protein prepared. The amount of time running longer experiments to compensate for low concentrations and the increased time to interpret NMR spectra will almost certainly be longer then the time it takes to prepare additional sample.

5) Can I do multidimensional experiments with unlabelled sample?

Yes. You can do multidimensional homonuclear experiments like COSY, NOESY, TOCSY and heteronuclear experiment like 13C 1H-/15N-1H HSQC but we can’t do three dimensional experiments at natural abundance. On the other hand with istopically enriched sample one can do up to 6Dexperiments, if required.

Instructions for sample preparation/submission

Instructions for Users

• First time users should contact the operator to understand the requirements.
• Sample should be completely soluble in 600 μl of the given solvent.
• For low molecular weight (MW < 250) compounds a minimum of 1-3 mg and for high molecular weight 2-5 mg of samples should be provided for proton spectra.
• For protein samples, concentration should be minimum 500 M for 2D and 3D experiments.
• For solid state NMR sample requirement is ~ 100 mgs.
• For proton decoupled Carbon-13 spectra, 50-100 mg of sample will be required.

Solvent Requirement :
For ensuring magnetic field stability and adjusting field homogeneity, a deuterium lock channel is provided. Therefore, samples are to be dissolved in a deuterated solvent. Deuterated solvents like D2O, CDCl3, are provided by the Facility free of cost. However Users have to pay for expensive solvents such as CD3COCD3, DMSOd6, C6D6, etc.. The User should check the solubility of the sample in any of these solvents (of course, in undeuterated solvent) and suggest best solvent.

Instructions for Registration