RTD Project Title : Development of NMR Instrumentation and Software to Achieve Excitation and Detection of Large Bandwidths and Dipolar Couplings in High Resolution Spectra at High-Field
Contract N° : ERBFMGECT980107
Name of Contract Manager : Ivano Bertini
Contract Value (ECU) : 600,000
Contractual period : 01/05/98 to 01/05/2000
1 CONSORZIO INTERUNIVERSITARIO RISONANZE MAGNETICHE METALLOPROTEINE
2 BRUKER ANALYTIK GMBH
3 UNIVERSITY FRANKFURT
4 AGRICULTURAL UNIVERSITY WAGENINGEN
1. Prof. Claudio Luchinat
Consorzio Interuniversitario Risonanze Magnetiche Metalloproteine Paramagnetiche (CIRMMP)
2. Dr. Tony Keller, Bruker Analytik GmbH (DE)
3. Prof. Christian Griesinger, Johann Wolfgang Goethe Universitaet (DE)
4. Prof. Jacques Vervoort, Wageningen Agricultural University (NL)
The objective of this project is aimed at the development of novel probeheads,
digitizers and data acquisition routines to optimize signal detection and
heteronuclear information in NMR spectrometers at high-field. The goals
of these objectives are to develop two novel probes for 800 MHz instruments:
(A) large bandwidth detection probe; (B) probe for detection of quenched
dipolar couplings. This new hardware would be used under high-resolution
conditions and will drastically improve the performance of the currently
The short-pulse, flat-baseline probe for excitation and detection over large spectral widths will allow to analyze paramagnetic proteins with unparalleled effectiveness, thus addressing the increasing need to excite with an acceptable radiofrequency power and acquire larger and larger bandwidths, especially required for paramagnetic substances. The development of a large bandwidth detection probe requires optimization and further improvement of auxiliary electronic equipment and computer software. The fast sample-shuttling probe will allow polarization and detection at high fields as well as the measurement of heteronuclear dipole-dipole interaction at low fields. The detection of quenched dipolar couplings between different nuclear species will be a precious source of structural and dynamic information and will allow e.g. the direct detection of hydrogen bonds in proteins, which is a major obstacle in structure determination for all NMR structure biology groups in academia as well as in industry.
The successful realization of these two prototypes is expected to provide a tremendous increase in the quality, and a significant increase in the quantity of users access. Although no link exists between the present project and non-European facilities, both probes to be developed address technological challenges that when solved will be useful to many NMR users throughout the world. The successful completion of the present project will maintain European industry on the one hand and European researchers on the other at the forefront of NMR technology.
A. Large bandwidth detection probe
The development of a large bandwidth detection probe requires optimization
and further improvement of auxiliary electronic equipment and computer
The probe to be developed has to incorporate some features important for high resolution NMR (coil geometry, z gradient system), and some features which are typical for applications in solid NMR, especially the large spectral width to be excited by short 90° pulses as well as the capability of handling high rf power and high amplitude rf pulses including their suitable transient behavior.
The main requirements of such a probe are the following developments:
1. 1H observation channel, proton lock channel;
2. 90° pulse width of 2 us to ensure wide band excitation of the order of 500 kHz;
3. smaller dimension of the coil with respect to the standard 5mm probes;
4. z gradient capability;
5. improvement of noise filtering for detection of signals without baseline distortion;
6. development of analog to digital converter (16 bit over >500 kHz) for data acquisition;
7. rf circuitry which operates at high power.
The major problem for building an HR 800 MHz probe being capable to supply a 90° pulse of 2 microseconds consists in developing an appropriate resonance circuit as well as a suitable NMR coil for transmitting rf fields with an amplitude of 125 kHz (being equal to 1/(4·90° pulse length)). Such rf amplitudes are typical for solids NMR probes. The following issues are important to realize the construction of the desired probe:
1. The presence of a z gradient system and the ability to change samples as common with HR probes require a saddle-shaped coil as an NMR transmit/receive coil.
2. 90° pulses of the order of 2 us (equivalently, rf fields of 125 kHz) lead to certain boundary conditions with respect to:
(a) the geometric dimensions of the coil,
(b) the external Q factor of the probe, and
(c) the level of rf power supplied by the transmitter.
The electronic elements used in rf circuitry of standard HR probes are not suited to handle high power (e.g. power levels above 100 watts). However, Bruker's experience from solids NMR (including solids NMR probes at 800 MHz) indicates that it is very likely, if not mandatory, that 1H rf powers beyond 100 watts are necessary to achieve rf fields of 125 kHz.
A rough estimate of the rf power requirements is as follows:
A standard 5mm 800MHz 1H probe with Q = 350 has a 90° pulse of about 10 us at a probe rf input power of ca. 30 watts. A 3mm coil (with the same principal geometry) would give about 1.5 times higher rf fields (all other quantities kept constant) thus arriving at 7us. Since a lower Q factor when using elements withstanding higher rf voltages of the probe is expected, e.g. Q = 250, this would correspond to a 8.5 us 90° pulse.
Hence, for a 2 us pulse, an rf power at the probe input of the order of 500 watts may be necessary. In any case, low-power transmitters are not sufficient.
For short pulses, the pulse shape (deviation from the ideal rectangular shape due to transient effects) becomes important. During the transient response (i.e. during pulse rise and decay) the rf phase is not well defined. Thus depending on the rf phase sensitivity of the experiment (pulse sequence) to be performed, this issue has to be taken into account. Pulse rise and decay times depend on the Q factor of the probe. For Q=350 and f0=800MHz, the rise time (to 90% pulse amplitude) amounts to 0.35 microseconds, which is already a significant portion of the pulse length of 2 microseconds. For Q=200 the rise time is equal to 0.18 us. Therefore, although a high Q is preferable for high rf fields (and also high signal sensitivity), a lower Q might be advisable for the sake of short pulse transients (relative to the overall pulse length).
Finally, with respect to 1H background signals, possible materials are either fluorinated plastics materials or, when there is not too much disadvantageous influence of rf features, the use of certain ceramics materials.
During the development of the probe, the main tools for evaluating the progress will be electronic tools. In the final stages, the assessment of the performance will be based on paramagnetic NMR samples containing fast-relaxing signal shifted over a broad spectral region.
Excitation profile for a typical radio-frequency pulse.
B. Probe for detection of quenched dipolar couplings.
Dipolar couplings can be used to study the structural and dynamic
features of nuclei within a molecule as well as of nuclei on different
molecules. The structure is inferred from the so called NOE between nuclei
which can be used only between nuclei of the same sort at high field (homonuclear
NOE) since the NOE between different nuclei (heteronuclear NOE) is quenched
due to the large field and slow motion of biomacromolecules. The goal in
the project is to recover this NOE. This allows the following applications:
- Direct measurement of hydrogen bridges in biomacromolecules (so far impossible)
- Slow motion in proteins (so far impossible)
- Interactions of atoms of the solute with the solvent at low field with atomic resolution.
The quenched NOE is recovered by locating the sample for a limited time in a much reduced field without compromising the high sensitivity and resolution of the high field spectrometer. The initial magnetization (Boltzmann magnetization) is determined by the high field. After excitation the sample is transferred from the high field part to the low field part where quenched dipolar couplings are now active due to the low field. Then the sample is transferred back to the high field part and the detection takes place. The sensitivity during detection is determined by the high field (B03/2) and the resolution is linearly dependent on B0.
The probe to be developed must achieve a transfer of the sample between the high field and low field part within approx. 100 us. When transferring from low field to high field, the homogeneity of 10-9 must be regained after not more than 100 us.
These requirements pose considerable technological challenges. The following two designs will be tried after each other:
1) Liquid state NMR is very successful in the use of LC-NMR in which a solution is dislocated without moving the container. The technological challenge is posed here by the problem to transfer identical volumes between scans and make the capillaries such that there is no degradation of sensitive proteins after many thousands of scans. The two magnetic fields are created by two independent magnets that are positioned close to each other.
2) Solid state NMR has developed so called zero field NMR spectrometers in which a solid sample could be shuttled between two fields within 100 us. However, the homogeneity obtained after shuttling the sample in the high field part had to be only 10-7. The technological challenge is the development of robust sample tubes for liquids and the dumping of mechanical vibrations after shuttling the sample back to obtain field homogeneity of 10-9.
The first phase will be devoted to building the transfer line and assessing the speed of transfer, the precision of locating samples and the stability of proteins under the condition of the transfer.
The second phase will apply NMR experiments to establish the amount of magnetization that survives the shuttling of the sample with abundant proteins (BPTI, lysozyme) without labeling.
The third phase will apply the experiments established in phase 2 to labeled biomacromolecules, e.g. deuterated calmodulin.
Shuttle probe-magnet design.
RF DESIGN FOR 800MHz - 2 CHANNEL PROBE
(one channel 1H, the second for lock)
MECHANICAL FRAME CONSTRUCTION
UPPER END FLANGE CARRYING RF CIRCUITRY ELEMENTS
The first probe (objectives position A) will contain high power circuitry
(voltage stable elements) in order
to achieve short pulses for 300kHz excitation band width. The coil will be a 3mm saddle coil.
The second probe (objectives position B) will be similar in appearance,
however not necessarily with circuitry for
high-power requirements. In order to realize the fast sample transfer, a system similar to the MAS transfer system (for 2.5mm o.d. samples already existing) is planned. It will be decided which control unit for pneumatic insertion and ejection will be used.
It is planned that a milestone will be reached at the end of four six-monthly
periods of the RTD project. Contract deliverables will be produced in the
form of reports upon completion of each of the outlined tasks represented
in Table 1. Technical deliverables will be in the form of instrumentation
hardware design, software, novel pulse sequences and test samples.
Milestone M1 will be reached upon the design and availability of the 16 bit ADC and the establishment of a shuttling device with a transfer time of less than 100 us and a reproducibility of translocation ensuring that high resolution detection is possible.
Milestone M2 will be met with the synthesis and characterization of paramagnetic, diamagnetic and isotopically labeled test samples.
Milestone M3 will be reached when Bruker provide the two novel probes and when pulse sequences have been written to minimize phase and baseline distortion and to allow the recovery of quenched dipolar interactions. It is planned that this milestone will be passed 6 months before completion of the RTD project.
The final milestone M4 will be reached upon completion of the project and will indicate the availability of all sythesised test samples and pulse sequences.
The deliverables in the form of instrumentation hardware, software and test samples are outlined in greater detail below.
(1) 16 bit ADC covering a frequency range > 500 kHz. With an increased bandwidth, an ADC (analog to digital converter) of much higher capability is required to handle the increased data sampling rate required by the experiment. Filtering software has to be written for this large bandwidth ADC.
(2) Short 90 pulse probe. This is the most important part of the project to achieve large bandwidth NMR.
(3) Establishment of shuttling device with a transfer time of less than 100 us and a reproducibility of translocation ensuring that high resolution detection is possible. This is the most important part of the project to achieve recovering of quenched dipolar couplings, that requires shuttling the sample between high and low field.
(4) Preparation/synthesis of test samples for the large bandwidth probe. The samples prepared, being both paramagnetic and diamagnetic in nature, will be designed in order to test a broad range of frequencies.
(5) Deuterated calmodulin in quantities of 20 mg for the detection of quenched dipolar couplings. The samples prepared, being 15N and 15N, 13C, 2H labelled will be designed in order to measure heteronuclear NOE's at low field.
(6) Pulse sequences to minimize phase distortion and baseline distortion with the new probe. Again, this will optimize the usage of the new probe design.
(7) Pulse sequences to suppress strong solvent signal with the new probe. This has great importance in the investigation of molecules of biological interest which are generally sampled in water. This gives rise to a strong signal due to its much greater relative concentration and must be minimized. The novel probe will most probably require design of tailored sequences for water signal suppression.
(8) Pulse sequences that allow the recovery of quenched dipolar interactions. Direct detection of hydrogen bridges in proteins; direct detection of slow motion in proteins; interactions of atoms of the solute with the solvent at low field with atomic resolution.
It is intended that upon completion of the RTD project the novel probes will remain at the large scale facilities where they will be installed. These probes will be available to external users of the European life science community already during the testing phase to optimize feedback to the manufacturer. As these developments will be of great significance to the NMR community at large, the results will be published promptly.
ROLE OF PARTICIPANTS
The participants include three large scale facilities of the EU in the
area of life sciences. The fourth participant represents the manufacturer
of currently installed instrumentation.
Eight tasks have been outlined and are represented in tables 1 and 2, below. The involvement of large scale facilities will be mainly through postdoctoral research projects.
(1) The role of the coordinator will be to manage and coordinate all eight tasks. The Florence group will be responsible for the preparation and synthesis of paramagnetic (and diamagnetic) samples for use in testing the prototype large bandwidth probe. They will also be responsible for the development of novel pulse sequences specifically designed for the suppression of strong solvent signal with the new probe as well as pulse sequences which minimize phase distortion and baseline distortion. Upon construction of the short pulse probe by Bruker it will be initially installed at the Florence facility.
(2) Bruker, as manufacturing representative will be responsible for the technical and engineering aspects of the NMR instrumentation design. The tasks under their responsibility are the design and construction of a 16 bit broad band Analog to Digital Converter (ADC), the design of the probes (a) a short 900 pulse probe for use in large bandwidth NMR, (b) a fast sample shuttling probe for the recovery of high field quenched dipolar couplings. The construction of the shuttling devices for probe (B) and the establishment of the speed of transfer of the sample volume as well as the precision of locating of the sample after translocation is the responsibility of Bruker.
(3) Partner 3, UNIFRA, will be responsible for the preparation and synthesis of isotopically labeled samples for use in the detection of quenched dipolar coupling. They will also be responsible for setting up the experiments in phase B2 on the established equipment as well as in phase B3 and for writing pulse sequences used in the measurement of heteronuclear NOE's. Both of these tasks are ultimately connected with a novel fast sample shuttling probe. This probe will therefore be initially installed at the Frankfurt facility when Bruker makes it available.
(4) Partner 4, WAU, will continue the task involving the preparation and synthesis of test samples for both probes in the second half of the RTD project (heme and non-heme iron-proteins for the large bandwidth probe and of a labeled NIFL domain for the dipolar probe). This will allow the other facilities to concentrate on the testing and optimal usage of the new probes.
ANNUAL REPORT May 31, 1999
Participants: (1) CIRMMP, Florence, Italy
(2) BRUKER, Rheinstetten, Germany
(3) UNIFRA, Frankfurt, Germany
(4) WAU, Wageningen, The Netherlands
The objective of this project is aimed at the development of novel probeheads,
digitizers and data acquisition routines to optimize signal detection and
heteronuclear information in NMR spectrometers at high-field. The goals
of these objectives are to develop two novel probes for 800 MHz instruments:
A. large bandwidth detection probe;
B. probe for detection of quenched dipolar couplings.
In this first year of the 2 year project, as planned in the project programme, the following deliverables have been successfully produced:
1) the 16 bit ADC (analog to digital converter) covering a frequency range > 500 kHz was built by Bruker and installed at the Florence LSF. Such increased bandwidth has required an ADC of much higher capability to handle the increased data sampling rate required by the experiment;
2) paramagnetic test samples for the large bandwidth probe have been synthesized/prepared, in order to test a broad range of frequencies;
3) deuterated calmodulin in quantities of 50 mg for the detection of quenched dipolar couplings was prepared, being 15N and 15N, 13C, 2H labelled in order to measure heteronuclear NOE's at low field.
During this period, a large part of the work has already been done in order to achieve the following deliverables in the next six months, according to the project program:
1) development of a short 90o pulse probe, the most important part of the project to achieve large bandwidth NMR;
2) establishment of shuttling device with a transfer time of less than 100 us and a reproducibility of translocation ensuring that high resolution detection is possible. This is the most important part of the project to achieve recovering of quenched dipolar couplings, that requires shuttling the sample between high and low field;
3) development of pulse sequences to minimize phase distortion and baseline distortion with the new probe, to optimize the usage of the new probe design.
Progresses made till now (see detailed reports from the partners) make us confident that such deliverables will be successfully achieved. The first prototype of the wideband probe (A) will be probably available in June, i.e. about three months before the scheduled time, while the first prototype of the shuttle probe (B) will be probably delayed by about two months with respect to the scheduled time.
Therefore, milestone M1 (the design and availability of the 16 bit ADC and the establishment of a shuttling device with a transfer time of less than 100 us and a reproducibility of translocation ensuring that high resolution detection is possible) is completed as far as the first part is concerned and it is at a good stage as far as the second part is concerned. Milestone M2 (synthesis and characterization of paramagnetic, diamagnetic and isotopically labeled test samples) is achieved as far as Partner 1 is concerned, as planned in the project program.
Report of contractor (1): CIRMMP, Florence, Italy
CIRMMP, the coordinator, has been responsible of coordinating the works
of all partners involved in the development and testing of the two probes.
Furthermore, the Florence group has been responsible for the preparation
and synthesis of paramagnetic samples for use in testing the prototype
large bandwidth probe. Three kinds of samples have been prepared:
1. Nickel complexes, as 5Br-NiSAL-Me-DPT samples, which have a spectrum with signals from –50 up to 500 ppm. In particular, sharp signals between 400 and 500 ppm have been measured at the 200 MHz, and at 800 MHz by irradiating different regions of the spectrum. The new probe should allow us to acquire the whole spectrum of this sample at 800 MHz in the same spectral window.
2. Horseradish peroxidase samples, a hemoprotein of 44,000 Da, containing high spin Fe(III) and thus presenting a spectrum with signals in the range –20 – 100 ppm. At low fields (200 MHz), spectra have been easily acquired, whereas at 800 MHz the non-ideal pulse shape determines phase and baseline distortions over the 100,000 Hz of spectral width.
3. Three blue copper protein samples (azurin, stellacyanin and plastocyanin) in the oxidized state. The signals of the cysteine ligand are shifted up to 1,000 ppm and broad beyond detection even at low field, and are thus not detectable with the present instrumentation at 800 MHz. The signals, too broad to be detected, were revealed , by assuming that they were present in a certain region of the spectrum and observing saturation transfer effects in the spectrum of their copper(I) form in a sample containing both the Cu(I) and Cu(II) forms. Figure 1 shows the obtained 1H NMR spectrum of (A) P. aeruginosa azurin, (B) spinach plastocyanin and (C) cucumber stellacyanin. The position and linewidth of the signals where obtained using taylored saturation transfer experiments by plotting the intensity of the respective exchange connectivities with the reduced species as a function of the decoupler irradiation frequency, as non observable in the normal spectrum. These samples could then represent the forefront for assessing the performances of the new large bandwidth probe, that should be optimized by the development of pulse sequences to minimize phase and baseline distortion.
Therefore the deliverable “Preparation/synthesis of test samples for the large bandwidth probe“ has been successfully achieved.
The Florence group is also responsible for the development of pulse sequences to minimize phase distortion and baseline distortion with the new large bandwidth probe. A review of the pulse sequences already published has been performed and some of them have been implemented on the Avance 800 MHz spectrometer. In particular, the saturation transfer pulse sequence have been optimized to reduce the effect of bad subtraction artifacts due to the high sensitivity of the very high field and its stability requirements. New pulse sequences have been projected and will be developed as soon as the new probe will be available.
Report of contractor (2): BRUKER, Rheinstetten, Germany
Bruker has been involved in the achievement of the following three deliverables.
1. 16 bit Analog to Digital Converter
A 16 bit broad band Analog to Digital Converter (ADC) was developed to be used in conjunction with the large bandwidth detection probe. The ADC covers a spectral range up to 500 kHz using digital filtering and up to 1000 kHz using analog filtering.
2. Wideband excitation probe for 800 MHz
The wide-band excitation probe (A) has been built and tested at the 800 MHz NMR spectrometer in Karlsruhe. In order to achieve the goals stated in the project program, i.e., to unite unique features typical for high-resolution NMR and those known from wideline solids NMR, the following procedures have been followed and results have been obtained:
1. The probe has been constructed and built to contain high power circuitry
(elements withstanding high rf voltages) in order to achieve short pulses
for 300 kHz excitation bandwidth.
2. The NMR excitation and detection coil is realized by a 3mm saddle coil in order to enable the advantage of the vertical cryomagnet geometry for pneumatically inserting and ejecting the sample.
3. Although the rf circuitry has been chosen equivalently to solids-NMR probe construction principles, special care was taken in selecting low-susceptibility and/or susceptibility compensated materials, in particular for the NMR coil and in the closest neighborhood of the coil. Thus the shim properties of the probe are like those of a 800 MHz high-resolution probe.
4. In order to obtain highly efficient proton rf irradiation, the 1H circuit of the probe contains a l/4 transmission-line resonator. Thus Q values on the order of 200 could be realized being significantly higher than values obtained in traditional probe circuitry. Beside the 1H channel, the probe contains a 2H lock channel.
5. The following NMR results were obtained upon the first NMR tests in April/May 99:
· Nonspinning lineshape (1%CHCl3/acetone-d6): better than
0.4/10/20 Hz (see Figure 2)
· 90° pulse at ca. 150 W rf power: 3µs
· Shorter pulses are feasible at higher rf transmitter powers
· Residual 1H background signal (without z gradient system):negligibly small (see Figure 3)
· Sensitivity (2mM sucrose, anomeric proton, in 90% H2O/10%D2O, with water suppression): 107:1 (see Figure 4)
The first prototype is thus almost finished and will be probably available in July for the tests at the Florence Large Scale Facility.
3 . NMR probe enabling fast sample transfer for 600 MHz ("shuttle probe")
Detection of quenched dipolar couplings (B) is based on the development of an NMR High-resolution probe setup that allows a fast sample transfer between two well-defined positions within the NMR magnet. During the past three months the principal design and construction has been decided. The probe will consist of two parts inside the cryomagnet. The lower probe, a 600 MHz Selective-Inverse probe, located in its usual position, will be connected by a transfer tube with the upper part (inserted into the cryomagnet from the top). After transferring the sample pneumatically to the upper probe, the sample is located in a field corresponding to an 1H resonance frequency of 60 MHz (or less). The construction of a first prototype is planned within the end of 1999.
Report of contractor (3): UNIFRA, Frankfurt, Germany
The task of the partner in Frankfurt is to provide samples for the measurement of quenched relaxation in biomolecules, provide pulse sequences for the new probe to be developed by Bruker and to apply those pulse sequences to the samples. Since the probe is not yet available, protein samples have been prepared that contain the appropriate labeling and full characterization is under way.
1) UNIFRA has concentrated on the preparation of HgiCI, a protein that
binds DNA and is involved in the restriction/methylation of DNA of Herpetosiphon
giganteus. Protein has been prepared in quantities of approximately 50
mg. The assignment of the protein has been performed in the DNA free form.
It is being performed at the moment also for the DNA bound form. The protein
exhibits parts with different mobility that has been characterized by conventional
methods for the detection of motion of various time scales. The protein
changes its dynamics upon binding of DNA (Fig. 2). It also exhibits interesting
properties with respect to polymerisation upon DNA binding (Fig. 3).
2) Ubiquitin was characterized with 2H,13C,15N and 13C,15N labeling with various experiments especially for the study of dynamics of the peptide backbone. Hydrogen bond mediated J-couplings between the donor 15N and the acceptor 13C for the deuterated sample were measured in order to characterize these hydrogen bridges. Similar measurements have also been performed on the sample of HgiCI.
3) Pulse sequences that allow the recovery of quenched dipolar interactions have not been developed yet, since they require the probe to do it on.
Report of contractor (4): WAU, Wageningen, The Netherlands
The task of the partner in Wageningen is to provide samples for the detection of quenched dipolar couplings and for the large bandwith detection probe.
1. We have cloned and overproduced the single lipoyl domain of the E3 component from N. meningitis. The protein is a dimer of more than 100 kDa with a flexible linker region of 42 amino acids. The linker region and especially the dynamical behavior of the linker region is very important for functional catalysis of this enzyme. In HSQC spectra of 15N labeled protein about 40 clear resonances are visible which on the basis of 3D experiments can be linked to the flexible linker. In 3D TOCSY-HSQC and NOESY-HSQC we can identify many spinsystems which are likely to result from the linker region. We succeeded in labeling the protein with 13C and 15N and hope to measure the structural and dynamical characteristics of the protein using the new probe designed for the detection of quenched dipolar couplings.
2. We have isolated an intermediate of the degradation pathway of microperoxidase 8, an iron containing heme-protein. This intermediate is formed upon catalytic turn over conditions. The intermediate has on the basis of MS data the extra mass of one oxygen as compared to normal MP8. We succeeded in stabilizing the intermediate and it can be obtained in large amounts (3-5 mg's). Initial measurements have been performed at the CIRMMP facility in Florence and it is intended to extend and improve these measurements using the new large bandwith detection probe. We hope that we will be able to assign all resonances and thereby detect exactly where in the protein molecule a modification has occurred.
It is intended that when the new probes will become available both measurements at UNIFRA and at CIRMMP will performed. These measurements are scheduled late 1999.
The large bandwidth detection probe has been installed
at the NMR lab in Florence together with an amplifier able to deliver 500
W, and it has been tested on the selected samples:
1. Nickel complex, 5Br-NiSAL-Me-DPT, which have a spectrum with signals from –50 up to 500 ppm;
2. Horseradish peroxidase samples, a hemoprotein of 44,000 Da, containing high spin Fe(III) and thus presenting a spectrum with signals in the range –20 – 100 ppm;
3. Three blue copper protein samples (azurin, stellacyanin and plastocyanin) in the oxidized state.
On very large spectral width some baseline distortion of the spectrum occurs, and the Florence Group is now working on designing taylored pulse sequences to reduce this effect.