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Experimentation with N Enriched Ubiquitin
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David Rovnyak, Laura E. Thompson, and Katherine J. Selzler Department of Chemistry, Bucknell University, Moore Avenue, Lewisburg, PA 17837
Nuclear magnetic resonance (NMR) spectroscopy is of fundamental importance in the advancement of the life sciences. Students obtaining chemistry degrees are increasingly likely to enter the life sciences and to intersect with the application of NMR to the study of biological systems. Increased undergraduate laboratory instruction in biological NMR is needed to help bridge the gap to the sophisticated uses of NMR in the life sciences in graduate and industry settings. We present several biomolecular NMR experiments using human ubiquitin for upper level chemistry coursework. We review prior work and describe extensions, such as temperature variation, to illustrate protein dynamics in more detail. We exploit direct N detection of the NMR signal to achieve very high quality spectra on limited instrumentation and using a commercially available sample. 15
Introduction The National Institute of Health's "Roadmap for Medical Research" promotes the discovery of the detailed structural and functional relationships among all parts of cellular machinery in humans (1). NMR has a critical role in meeting the goals of the Roadmap since it is uniquely suited for studying membrane proteins, intermolecular interactions, dynamic processes, and macromolecules in native or near-native environments. © 2007 American Chemical Society
Rovnyak and Stockland; Modern NMR Spectroscopy in Education ACS Symposium Series; American Chemical Society: Washington, DC, 2007.
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220 Enhancing the NMR training of the next generation of scientists will help to meet a national priority and will reflect the wide scope of NMR utilization in the life sciences. There is also a dangerously widening chasm between the level of NMR instruction in undergraduate education and the complexity of NMR experimentation at the graduate level. To address these concerns, we have been developing and testing biomolecular NMR laboratories in upper level course work for majors in our 'Biochemistry and Cell Biology' program. We have set out to create robust, student-tested experiments that represent authentic training in biomolecular NMR and are accessible to the widest possible range of undergraduate educators. We decided upon directly observing the N NMR signal for N enriched proteins. Although the typical practice is to perform H observation, our approach provides compelling benefits to faculty and students. I5
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Goals for Protein NMR Laboratory Experiments Practical Considerations NMR instrumentation for performing structural biology research has additional components beyond those found in standard double-channel NMR instruments, and often requires additional levels of maintenance. Biomolecular NMR spectroscopy uses four independent RF channels, specialized probes, and proton ( H) signal detection. Unfortunately such instrumentation is not yet widely available to principally undergraduate institutions (PUIs). Moderate field double-channel instruments are commonly operated by PUIs, and are optimized for X channel signal detection, so-called 'direct detection' ( N through P). Operating at 200-400 MHzfrequenciesfor Ή , these instruments are not generally suitable for performing conventional protein NMR studies. We performed protein NMR via such "direct detection". Detecting a C or N NMR signal eliminates the need to suppress the water signal, which greatly simplifies experiments. Also, at the end of the pulse sequence, it is no longer necessary to return a coherencefroman X nucleus back to *H for observation. This so-called "back" step of "out and back" experiments involves several additional pulses and delay times. Eliminating this step further simplifies experiments and also saves time in the pulse sequence, which reduces some of the deleterious T! relaxation of the protein signals. Also, the transverse relaxation time constants (T ) of heteronuclei such as C or N proteins are larger for moderate fields (200-400 MHz for H) as shown in Figure 1. Larger values of T translate into narrower line shapes and higher signal to noise ratios. The effect is modest, but not insignificant. As thefrequencyof the signal decreases, so does the sensitivity of detecting that signal (3). In principle, detecting C nuclei is favored over detecting N !
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Rovnyak and Stockland; Modern NMR Spectroscopy in Education ACS Symposium Series; American Chemical Society: Washington, DC, 2007.
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Figure 1. Estimates of T time constants for prototypical proteins. Valu depend on numerous parameters and are approximate; rather the trend t larger T 's asfielddecreases should be noted. Left: C T 's for a deutera protein. Right: antiphase N{ H} T 's for a protonated amide nitrogen. 2
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nuclei since the C Larmorfrequencyis over twice as large as that for N. It is extremely expensive to purchase Cenriched proteins at this time, while Nenriched proteins are significantly more accessible for purchase (see notes in Experimental Details). The use of N enriched proteins leads also to a number of pedagogical advantages over C enriched samples 13
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Pedagogical Considerations The starting point of a protein NMR investigation is to acquire an experiment that correlates in a two-dimensional (2D) representation all directly bonded *H- N spin pairs of an N enriched sample. This experiment involves a heteronuclear single quantum coherence (HSQC) transfer among the nuclei (4). This 2D-HSQC experiment will confirm that the protein is folded, and is used to identify the first peak lists which help to complete the protein assigbnments. The HSQC is a critical building block for nearly all other protein NMR experiments and has value as a gateway experience into protein NMR, allowing students to experience how a NMR structural study begins. 15
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Rovnyak and Stockland; Modern NMR Spectroscopy in Education ACS Symposium Series; American Chemical Society: Washington, DC, 2007.
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Furthermore, the relationships between relaxation properties of N nuclei and the global and local dynamics of proteins are very well known. It is common in an NMR structural study to characterize the global reorientation and backbone dynamics by N relaxation. Similar to the usage of the HSQC, investigators often begin a structural study with the determination of the protein rotational correlation time by the use of N relaxation studies. Broadly, it is our impression that most undergraduate curricula do not include experimentation with hydrodynamics or protein dynamics. It is highly desirable tofindways of allowing students to experimentally measure protein hydrodynamic properties and to directly observe that proteins are not at all rigid, but instead have substantial motionalfreedomwith functional significance. l5
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Experimental Details 15
A sample of approx. 11 mg uniformly N-enriched human ubiquitin was purchasedfromVLI Research (Malvern, PA, http://www.vli-research.com) at 1 mM concentration in 50 mM potassium phosphate buffer at pH 5.8. Additional evaporative concentration was performed and the sample transferred to a Shigemi (Allison Park, PA) sample tube. Additional vendors also providing isotopically enriched ubiquitin are Spectra Stable Isotopes (Columbia, MD, 21046, http://www.spectrastableisotopes.com) and Cambridge Isotope Laboratories (Andover, MA, http://www.isotope.com). All pulse sequences were implemented on a Bruker ARX spectrometer operating at 300 MHz for Ή (Figure 2), and may be downloaded at (http://facstaff.bucknell.edu/drovnyak/). The Ti and especially T measurements should be acquired with sample spinning off. 2
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N Detected 2D-HSQC
This first laboratory has been used for three years in an upper level chemistry course entitled 'Biological Physical Chemistry' involving mainly senior students in a Biochemistry and Cell Biology major who are intending to proceed in the life sciences in medicine or graduate research. The NMR instrument is initially demonstrated to groups of 3-5 students who then carry out a series of short exercises that facilitate a guided inquiry approach to some fundamentals of protein NMR. The instructor should be present to help facilitate discussion, but all experiments can be independently acquired and processed by the students. Students first acquire a Ή NMR spectrum with no water suppression and are asked to discuss why they do not observe signalfromthe protein (e.g. role of receiver gain and dynamic range). Students enable a flag that implements water suppression via presaturation and acquire another ID proton spectrum, then
Rovnyak and Stockland; Modern NMR Spectroscopy in Education ACS Symposium Series; American Chemical Society: Washington, DC, 2007.
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(a) Directly detected HSQC
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Figure 2. Ν detected pulse sequences for proteins. Thin lines are 90° pu and thick bars are 180°pulses. Pulses labeled with φ are applied with χ pha Sequences are (a) a 2D-HSQC experiment, and (b-c) experiments fo measuring T\ and T time constants. The coherence transfer uses τ= 2 1/4J , ( tH-^N J-92 Hz). In (b) we use values of δ between 0.45 - 0.60 In (c) we use value of /between 5-7 ms. Adapted with permission from χ
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Rovnyak and Stockland; Modern NMR Spectroscopy in Education ACS Symposium Series; American Chemical Society: Washington, DC, 2007.
224 discuss the changes in the spectrum. Presaturation avoids the use of gradients or shaped pulses, and gives more than sufficient water suppression to appreciate the proton NMR spectrum of a protein. Students should then be given the typical signal regimes for proteins (5), which we summarize in Table 1. The signal regions for aqueous proteins are generally consistent with the qualitative rules that students have previously learned for organic compounds in chloroform. These initial H NMR exercises help to build confidence in handling protein NMR spectra. !
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Table 1. Typical Signal Regions for Protein NMR Spectra Protein Moiety
Chemical Shift Range (ppm)
backbone amide 10.0^7.0 aromatic 8.0-6.5 backbone alpha 6.0-3.5 aliphatic side chain 3.5-1.0 methyl
