RECOMENDACIONES Y CONCLUSIONES

NMR spectra recorded at 500 MHz and 600 MHz proton frequency were recorded in the UCL/Ludwig Institute NMR laboratory on Varian UnityPlus spectrometers equipped with triple resonance Z-axis pulse field gradient (PFG) probes with, respectively, four and three radio frequency (RF) channels. All experiments performed on UCL spectrometers had been previously adapted from the cited pulse sequences by staff members and were set up by the author alone or under guidance of Dr R Harris. Spectra obtained at 800 MHz proton frequency were recorded at the MRC Biomedial NMR center. Mill Hill, London. The 800 MHz Varian spectrometer was equipped with four RF channels and single-axis pulsed field gradients and is equipped with a triple-resonance PFG probe. Experiments recorded at NIMR were performed by Dr. G. Kelly.

3.3.1 - Heteronuclear single quantum coherence experiments (HSQC)

[^H, ^^N]-HSQC pulse sequences were performed with gradient coherence selection, sensitivity enhancement and a water flip back pulse (Zhang et al. 1994).

3.3 .2 - Transverse relaxation optimised spectroscopy (TROSY)

[^H, ^^N]-TROSY experiments encompassing WATERGATE solvent suppression were downloaded with permission from Prof. Lewis Kay at the University of Toronto, Canada, (http://pound.med.utoronto.ca/pulse_reg.html) and modified in-house by Dr R. Harris.

3.3.3 -Measuringproton transverse relaxation rates

Proton transverse relaxation rates were measured using a ID 1-1 spin echo experiment similar to that described (Sklenaf and Bax 1987). The delay period T (see Figure 5.7(a)) was set to V4A, where A was the difference in Hz between the water signal and

the amide signals. Aa and Ab were set at 0.1 and 2.9 ms respectively. All experiments were recorded at 500 MHz and 25°C. The data were analysed as described in Section 3.4.3. A description of the principles of this experiment is given in Chapter 5, Section 5.4.1.

3.3.4 - Measuring translational diffusion coefficients using ID NMR

Translational diffusion coefficients were extracted from ID pulse field gradient experiments performed as described by Byrd and co-workers (Altieri et al. 1995) with the addition of a WATERGATE water suppression sequences prior to acquisition. Experiments were performed at gradient strengths between 6.5 and 51.7 G cm'^ and always at 500 MHz proton frequency and 25®C. The data were analysed as described in Section 3.4.4. A description of the principles of this experiment is given in Chapter 5, Section 5.4.2.

3 .3 .5 - Measuring Ti, T2 and heteronuclear-NOE

Ti, T2 and {^H}-^^N NOE experiments were based on published pulse sequences

(Kay et al. 1989) and incorporated gradient coherence selection and sensitivity enhancement (Zhang et al. 1994). All relaxation delay and pre-saturation periods are detailed in Tables 3.3 and 3.4. All relaxation data was analysed as described in

Table 3.3. Delay times for relaxation experiments conducted on ubiquitin samples (see Chapter 4). Isotope labelled? Temperature Experiment rc) % Glycerol (v/v) Relaxation Time (s) ^ [*'N] 25 Ti 0 0.010 0.060 0.151 0.301 0.452 0.753 1.004 1.406 0.151 25 '^NT2 0 0.015 0.045 0.075 0.105 0.135 0.165 0.196 0.226 0.271 0.316 0.361 0.075 25 { ‘H}-^^NN0E 0 0.000 2.997 2.997 0.000 25 ‘^NTi 0 0.010 0.060 0.151 0.301 0.452 0.753 1.004 1.406 0.151 25 *^NT2 0 0.017 0.050 0.083 0.116 0.149 0.182 0.215 0.248 0.298 0.347 0.397 25 {’H}-'®NN0E 0 0.000 2.997 15 Ti 50 0.126 0.251 0.502 1.506 2.008 3.012 4.016 0.502 15 ‘^NTz 50 0.004 0.008 0.012 0.016 0.023 0.031 0.039 0.047 0.070 0.016 20 *^NTi 50 0.126 0.251 0.502 0.753 1.004 1.506 2.008 3.012 4.016 0.502 1.004 3.012 20 *^NT2 50 0.004 0.008 0.016 0.023 0.031 0.039 0.047 0.068 0.093 0.016 25 *^NT, 50 0.126 0.251 0.502 0.753 1.004 1.506 2.008 3.012 4.016 0.502 25 *^NT2 50 0.004 0.008 0.016 0.023 0.031 0.039 0.047 0.070 0.097 0.136 0.016 25 {'H}-‘^NN0E 50 0.000 2.997 0.000 2.997 30 ‘^NTi 50 0.126 0.251 0.502 0.753 1.004 1.506 2.008 3.012 4.016 0.502 30 '^NT2 50 0.004 0.008 0.016 0.023 0.031 0.039 0.047 0.070 0.097 0.136 0.016 35 T, 50 0.126 0.251 0.502 0.753 1.004 1.506 2.008 2.510 3.012 0.502 35 '^NT2 50 0.004 0.008 0.016 0.023 0.031 0.047 0.051 0.093 0.117 0.016 35 {'H}-‘^NN0E 50 0.000 2.997 0.000 2.997 00 U)

“ In Ti and T2 experiments, the time periods stated refer to the length of time permitted for relaxation in the pulse sequence. In the case of { NOE

Table 3.4. Delay times for relaxation experiments conducted on PaDDAH samples (see Chapters 5 and 7)

00

Sample Temperature (®C) Experiment Relaxation Delay Period (s)a

WT 25 Ti 0.000 0.126 0.251 0.502 0.753 1.004 1.255 1.506 1.757 2.008 2.510 25 ^^NT2 0.000 0.008 0.012 0.016 0.023 0.031 0.051 0.070 0.098 0.125 25 {*H}-*^NNOE 0.000 3.011 30 *^NT2 0.000 0.008 0.012 0.016 0.023 0.031 0.051 0.070 0.098 0.125 35 ^^NT2 0.000 0.008 0.012 0.016 0.023 0.031 0.051 0.070 0.098 0.125 N36W 25 *^NTi 0.126 0.502 1.004 1.506 2.008 2.510 3.012 4.016 0.502 25 ^^NT2 0.004 0.008 0.016 0.023 0.031 0.039 0.047 0.062 0.078 0.097 0.031 25 NOE 0.000 2.997 R40E 25 *^NTi 0.126 0.251 0.502 1.004 2.008 3.012 0.251 25 ‘^NT2 0.004 0.008 0.016 0.031 0.047 0.070 0.078 0.124 0.016 25 NOE 0.000 2.997

* In Ti and T2 experiments, the time periods stated refer to the length of time permitted for relaxation in the pulse sequence. In the case of NOE

Section 3.4.2. A brief description of the theory of NMR relaxation and the principles of these experiments are given in Chapter 4, Section 4.2.1.

3.3.6 - NMR experiments recorded to facilitate sequence-specific resonance assignment

All 3D triple resonance pulse sequences used (see Chapter 4, Table 4.2 and Chapter 5, Table 5.3) were based on the sequences published by Kay and co-workers and employed TROSY selection in and dimensions (Yang and Kay 1999a; Yang and Kay 1999b). In the case of WT PaDDAH, active suppression of the undesired cross peak components was performed (Yang and Kay 1999b) due to the presence of intense signals from the highly flexible polyhistidine purification tag. The length of the recycle delay (ideally ca. 5 seconds for deuterated proteins) was optimised for experiment time against the numbers of scans acquired. All experiments were recorded at 600 MHz and the temperatures stated in the text.

3 .3 .7 -3 D [’H, ’^N]NOESYHSQC

A 3D ['^]-edited NOESY HSQC experiment (Zhang et al. 1994) was recorded with gradient coherence selection and sensitivity enhancement and a mixing time of 100 ms.

3.3.8- Processing NMR data

All raw NMR data were processed using the nmrPipe program of Delaglio and co­ workers (Delaglio et al. 1995). Standard manipulations of data sets included zero- filling to the nearest 2" points, application of window functions, base line corrections, linear prediction of indirect dimensions where necessary, and first and second order phase corrections. The precise processing steps varied between data sets. Spectra were initially visualised in nmrDraw (Delaglio et al. 1995).

Following processing, spectra were exported into AZARA (Boucher 2002) format using the PIPE2AZARA command. Multiple spectra were visualised and cross- referenced in Plot2 (Boucher 2002). Sequential assignment was performed using the ANSIG analysis package (Kraulis 1991) with contour and cross peak files generated by AZARA.

s. 5 .9 - Estimation o f amide proton solvent exchange rates

A [^H, ^^N]-TROSY spectrum (see Section 3.3.2; see Figure 5.6 for experiment details) was recorded from a 600 pi, 1 mM sample of -labelled WT PaDDAH in 20 mM sodium phosphate buffer, pH 7.0, lOOmM NaCl, 10 % D2O. Following

acquisition of the spectrum, the sample was diluted 1:10 with 20 mM sodium phosphate buffer, pH 7.0, 100 mM NaCl prepared in D2O and concentrated to 600 pi.

An identical [^H, ^^N]-TROSY spectrum with identical parameters was recorded. Again, following acquisition of the spectrum, the sample was diluted 1:10 with 20 mM sodium phosphate buffer, pH 7.0, 100 mM NaCl prepared in D2O an

concentrated to 600 pi and a final identical [^H, ^^]-TROSY spectrum recorded.