Aligning Ultrafast NMR Spectroscopy and Cold Probe Technology to Reveal the Mysteries of Protein Dynamics

The understanding of protein dynamics, the way these molecules fold to form a stable structure, interact with other molecules, or undergo changes during enzyme activity, etc., in their native environment is a key research area. The general aim is to understand the underlying fundamental principles that govern the workings of living organisms, most of which are carried out by proteins in interaction with other molecules. Nuclear magnetic resonance (NMR) spectroscopy, in particular, is capable of taking proteomics to the next level, where scientists study not only the structure, but also the dynamics and the interactions of proteins with other molecules.

With NMR, it is possible to gain insight into how proteins fulfill their functions in their native aqueous environment. However, these NMR studies are challenging and often require that researchers push the limits by using the combination of cutting-edge hardware, such as cold probes, and the latest innovative new methodology, such as very fast 2-D NMR.1–5

Figure 1 - Protein dynamics—time scale and NMR experiments.

Gathering insight into the dynamic properties of proteins is challenging. Very few analytical methods can provide local structural and dynamic information for the different protein segments comprising the protein, such as secondary structural elements, individual residues, or even individual nuclei. Further, besides the need for site-resolved studies, the time scale of the dynamic process is also an important factor that needs to be addressed. The time scale of the experimental method has to be comparable to the time scale of the dynamic process studied (Figure 1). NMR spectroscopy is uniquely suited to the study of dynamics of proteins, since it fulfills both of the above requirements.

Figure 2 - Comparison of 1-D and 2-D NMR (heteronuclear single quantum coherence [HSQC]) of 3 mM NuiA in 90% H2O 10% D2O.

Figure 1 shows the many types of motions in proteins that need to be studied and the matching NMR methods that make these studies possible. It is clear that NMR offers a wide range of options to better understand the dynamic world of proteins. One significant issue faced by NMR spectroscopists is that the 1-D NMR spectrum that can be collected in a few seconds does not provide the needed atomic resolution information on large molecules such as proteins. Researchers have to turn to 2-D NMR to achieve site-specific resolution, but the experiment is too time consuming to meet the time-scale requirement (Figure 2). The acquisition time of typical 2-D NMR spectra ranges from several minutes to tens of minutes, depending on sample concentration and the resolution required in the indirect dimension.

In the past 3–5 years, several exciting fast NMR methods have emerged.1–5 Most of these methods are intended to accelerate higher-dimensional (3-D and 4-D) NMR data collection for protein structure determination. Unfortunately in most cases the increase in speed comes at a price. Namely, shorter experiment times generally mean that less signal is acquired. As a consequence, the signal-to-noise ratio (SNR) in fast experiments in general is dramatically reduced. Hence, the technology that increases the SNR is very beneficial. A very fast 2-D NMR technique, the SOFAST HMQC (band-selective optimized-flip-angle short transient heteronuclear multiple quantum coherence) was introduced in 2005.4,5 The method is distinctive because it offers a considerable advantage in speed and potentially increases the sensitivity per unit time of many conventional experiments. This allows the recording of 2-D NMR spectra of proteins in less than 10 sec, and thus breaks down the barriers to real-time investigation of dynamic events in proteins. Several research groups have recently demonstrated that the time scale of hydrogen/deuterium (H/D) exchange measurements in proteins can be dramatically increased by combining the sensitivity advantages of cold probes with fast NMR methodologies.4–7

SOFAST method

Figure 3 - 2-D 1H-15N correlation spectra of Rb. capsulatus cytochrome C′ (128 residues) recorded on a Varian 600-MHz NMR spectrometer. a) SOFAST-HMQC with a recycle time of 1 msec and a total experimental time of 6 sec. b) Standard HSQC using a recycle time of 1 sec and a total experimental time of 180 sec.

In order to reduce the recycling delay that accounts for most of the experiment time in conventional multidimensional NMR spectroscopy, the SOFAST technique employs both longitudinal relaxation optimization8 and optimized flip-angle9 band-selective excitation. For instance,the SOFAST-HMQC4,5 experiment selects the NH (amide) region using a shaped polychromatic excitation pulse10 that provides a variable flip-angle capability for the first excitation pulse. This pulse preserves the +Z magnetization of the other protein protons (aliphatic and aromatic sites), which serves as a reservoir of magnetization to rapidly relax the NH protons back to thermal equilibrium during the acquisition time. This setup makes it possible to set the relaxation delay to as short as 1 msec versus the traditionally used relaxation delays of over 1 sec. The reduction in the relaxation delay from ca. 1 sec to 1 msec decreases experiment duration by about a factor of 20 (Figure 3), without diminishing the sensitivity per unit time with respect to conventional methods.4,5

Unfortunately, most proteins that are involved in relevant biological processes are usually available only in very limited quantities. This problem is further exacerbated by the fact that protein samples enriched with at least one magnetically active isotope (N15 and/or C13) are required for the basic 2-D correlation experiments (linking H1 and neighboring C13 or N15 atoms). The goal of researchers is to collect data on dilute protein samples that most closely resemble the native biological environment, but in turn they are faced with the detection limit of their hardware.

The sensitivity of NMR experiments has always been a challenge. Over the past 3–5 years, several techniques have evolved that are helping to push the barriers of NMR sensitivity and throughput to new levels. One of the most exciting and recent developments in this area is the use of cryogenically cooled probes.11 These devices have the electronics within the NMR probe cooled to approximately –250 °C (comparable to the temperature on the surface of Pluto), which results in an increase in sensitivity by a factor of 2–4 as compared to a conventional NMR probe. At cryogenic temperatures, the thermal noise of the radiofrequency (RF) electronics is significantly reduced while the probe’s quality factor is improved. This results in reduced noise and greater signal, thereby significantly increasing the overall SNR of the probe. The improved sensitivity of the cryogenic probe translates to 4–16 times shorter experiments compared to data collected on a conventional probe. This technology was introduced to the marketplace by Varian (Palo Alto, CA).12 More importantly, the company’s cold probe technology has been developed to achieve the sensitivity increase without any compromise in NMR performance.11 As new NMR methods emerge, however, the robustness and applicability of the cold probe becomes challenged.

Combining cold probe technology and the ultrafast NMR technique of SOFAST is not only very important but is also very difficult. For instance, reducing the recycling delay in the SOFAST experiments means that the duty cycle of the NMR pulse sequence increases dramatically, from the typical 8% to over 80% (RF pulses and N15 decoupling). Imagine pushing this increased RF load into a tiny antenna (RF coil) that needs to be maintained at cryogenic temperatures with a tight precision (higher then 0.1K stability) using a closed-cycle cryogenic refrigerator system.11 This challenge seemed so significant that researchers at the Institute of Structural Biology (IBS) in Grenoble, France, have developed a modified version of the SOFAST experiment with no N15 decoupling, and thus a reduced duty cycle, to ensure that the sequence could be run on a cryogenic probe.5

Figure 4 - SOFAST HMQC collected on N15, C13 labeled 1 mM ubiquitin (90% H2O, 10% D2O) using a 5-mm Varian triple resonance Z-PFG cold probe. Experimental time was 8 sec. Data courtesy of Dr. Ashish Arora (Central Drug Research Institute, Lucknow, Uttar Pradesh, India).

The rewards of being able to completely align the cold probe technology and the very fast SOFAST method are very significant, and the technology was carefully evaluated by the Varian applications and R&D team. They have shown that on the very robust Varian cold probes it is possible to run the SOFAST-HMQC experiments with N15 decoupling without any special setup or hardware requirements. As an example, data recently collected on a newly installed 600-MHz Varian cold probe at the Molecular & Structural Biology Division, Central Drug Research Institute (CDRI) in Lucknow, India, are shown in Figure 4.

One can envision maintaining the NMR “antenna” at 25K while almost continuously bombarding it with high-power RF pulses and decoupling, and doing so not for a few seconds, but for hours. This would be of particular significance for high-throughput laboratories that require performing series of back-to-back 2-D experiments. Studies at the Varian NMR laboratories have shown that the duration of such power-intensive experiments can be extended considerably. Thus, impressive power handling capabilities of the cold probe open new avenues for detailed studies of protein dynamics and noticeably increase the throughput of similar experiments.

Overall, the combined use of the SOFAST NMR method and the robust Varian cold probe is revolutionizing the way researchers can study fast protein dynamics and gain better insight into the world of proteomics.

References

  1. Freeman, R.; Kupče, Ē. J.  Biomol. NMR2005, 27, 101–13.
  2. Malmodin, D.; Billeter, M. Prog. Nucl. Magn. Reson. Spectrosc.2005, 46, 109–29.
  3. Kupče, Ē .; Nishida, T.; Freeman, M. Prog. Nucl. Magn. Reson. Spectrosc.2003, 42, 95–122.
  4. Schanda, P.; Brutscher, B. J.Am. Chem. Soc. 2005, 127, 8014.
  5. Schanda, P.; Kupče, Ē.; Brutscher, B. J. Biomol. NMR 2005, 33, 199–211.
  6. Gal, M.; Mishkovsky, M.; Frydman, L. J. Am. Chem. Soc., in press (2006).
  7. Bougault, C.; Feng, L.; Glushka, J.; Kupče, Ē .; Prestegard, J.H. J. Biomol. NMR2005, 28, 385–90.
  8. Pervushin, K.; Vögeli, B.; Eletsky, A. J. Am. Chem. Soc. 2002, 124, 12, 898–902.
  9. Ernst, R.R.; Bodenhausen, G.; Wokaun, A. Principles of Nuclear Magnetic Resonance in One and Two Dimensions. Oxford Science Publications: Oxford, U.K., 1987.
  10. Kupče, Ē; Freeman, R. J. Magn. Reson. 1993, 102A, 122–6.
  11. Losonczi, J.; Green, I. Am. Lab. 2004, 36, 26–9.
  12. Flynn, P.F.; Mattiello, D.L.; Hill, H.D.W.; Wand, A.J. JACS 2000, 122A, 4823–4

Dr. Losonczi is Marketing Manager, NMR Probes; Dr. Kupče is a Principal Applications Scientist and Varian Fellow; and Dr. Gray and Dr. Sandor aredor are Senior Applications Scientists, Varian Inc., 3120 Hansen Way, Palo Alto, CA 94303, U.S.A.; tel.: 650-424-3826; fax: 650-494-7186; e-mail: [email protected]. Dr. Brutscher is a Research Scientist, Institut de Biologie Structurale Jean-Pierre Ebel CNRS-CEAUJF, Grenoble, France.

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