C7995 Advanced Methods of Biomolecular Chemistry Name: Zuzana Trosanova, 356857 Task 1 - Preparation for Measurement TASK 1A: Shimming Chloroform in acetone (1%) was used as a sample with parameter set lineshape. Using command lock we took choice acetone. Signal was tunned automatically with command atma as well as manually with command atmm manwbsw. Automatic command topshim was used for shimming in z - direction. Manual shimming (in x, y, xy, xz, yz, ... ) was performed until lock signal was stabilized. Command loopadj was used for optimalization of lock phase and lock gain. Resulting peak was evaluated using command humpcal. Results are shown in Table 1. Before shimming After shimming 0.11 % 19.8 Hz 13.3 Hz 0.55 % 16.3 Hz 6.4 Hz 50 % half width 3.16 Hz 0.64 Hz Tab. 2: Results from manual shimming procedure. TASK IB: Pulse Calibration For this task, a dopped water was used as a sample and zg as a parameter set. Using command lock we used choice #### Spectrum was measured immediately after shimming and wobbling procedures. Using command paropt, intensity of acquired peaks was modulated by sinus function. Phase 360° was observed in time 34,4 |is, length of 90° pulse is calculated as follow: 34,4 |is/4 = 8,6 |is. (Fig. 1.) Using command pulsecal, which calculates the 90° pulse automatically, we obtained value 8,3 |is. i n i Fig. 1: Set of peaks modulated by sinus function obtained from command paropt. C7995 Advanced Methods of Biomolecular Chemistry Name: Zuzana Trosanova, 356857 TASK 1C: Temperature Calibration For last task, a 4% methanol was used as a sample, zg was used as a parameter set. First, we lock the signal (form the lock table we choose solvent ###) and we also provided wobbling and shimming procedures using commands atmm manwbsw and topshim, respectively. Methanol gives 2 signals - one from methyl group with chemical shift 5i and one from hydroxyl group with chemical shift 52. Exact peak position is given by command pp. The difference in position of peaks is a temperature function. This function allows temperature calibration. First, we used NMR-TempCal.xls, an equation in Excel's table. With help of this spreadsheet, we were able to translate chemical shift difference into temperature. Another way is to use command calctemp. Results are shown in Table 2. Tha aim of this task was to calibrate the temperature at 25 °C (298,15 K) and 10 °C (283,15 K). However, we couldn't set temperature to 10 °C due to the technical reasons. Therefore we used temperature 20 °C (293,15 K). T [°C] / [K] Si [ppm] 52 [ppm] TNMR-Temp.calc.xls [K] Tcalctemo [K] 20/ 293,15 3,3676 4,9217 293,39 294,87 25/ 298,15 3,3686 4,8729 299,11 301,24 Tab. 2: Results from temperature calibration using command calctemp and spreadsheet NMR-TempCalc.xsl. SUMMARY: Optimization and calibration methods were performed. We met several technical problems resulting in not perfect optimization of shimming and temperature calibration. C7995 Advanced Methods of Biomolecular NMR Name: Zuzana Trosanova, 356857 TASK 2 - ID spectroscopy in water TASK 2A: Solvent suppression test The very high concentration of water compared to the very low concentration of biomolecules necessitates the use of solvent suppression methods. Solvent suppression techniques are very efficient techniques used to suppress strong water signals from proton. 2 mM sucrose in water (90 % H2O , 10 % D2O) was used for measurement. Shimming was provided carefully using automatic command topshim 3D and manual approach. Water signal was suppressed using presaturation and WATERGATE with parameter set zgpr and p3919gp, respectively (fig. 1 and 2). The dublet of anomeric proton is used for shimming quality evaluation. The dublet is more separated for better shimming, the shimming quality is defined as ratio of the least intensity in dublet to maximal intensity in dublet. Using macro suppcal we obtained result with value 0.27 (27 %). Signal to noise ratio (SINO) was 293.0. r General DATE> 2015/10/22 TIME = 13:43 INSTRUM = spent PULPROC = zgpr Fl [1H) 51 = SF = SW_p 32768 500.22 = S012.S21 0 0 0 5.44 5.42 5.40 5.38 5.36 5.34 5.32 5.30 [ppm] Figure 1: Detail for anomeric proton from ID spectrum of 2 mM sucrose using presaturation, parameter set: zgpr, 25 °C. C7995 Advanced Methods of Biomolecular NMR Name: Zuzana Trosanova, 356857 wa er suppresion2 Topsh im3d General DATE = 2015/10/22 TIME = 14:11 IN5TRUM = spect PULPROC - zgpr Fl a 51 = SF = SW_p H) 32768 500.22 = S012.S21 5.44 5.42 5.40 5.3B 5.36 5.34 5.32 5.30 Ippmj Figure 2: Detail for anomeric proton from ID spectrum of 2 mM sucrose using WATERGATE, parameter set: p3919gp, 25 °C. TASK 2B: Proton ID spectra in water Different approaches for water suppression were measured also with dsDNA (sequence: TCTTGTGTTCT * AGAACACAAGA). In case of presaturation (pulse sequence zgpr), water frequence is irradiated by a long low power pulse. In addition to removing of water signal, exchangeable protons are also eliminated. WATERGATE is based on the gradient spin echo technique. We used two pulse sequences for WATERGATE zgpwg and p3913gp. Resulted spectra are shown in Fig. 3, detail for imino region is shown in Fig. 4. Both WATERGATEs, zgpwg and p3913gp, achieve similar results and higher intensities in imino region compared to presaturation. C7995 Advanced Methods of Biomolecular NMR Name: Zuzana Trošanová, 356857 Zuzka 20 1 /dl/data/flala TOvhiB Scale : 8.1577 .... ruika.. 22. .1.. /dl/data/l i iv,=:-.l Zuzka 21 1 /dl/data/f1ala/700ml [ppml Figure 3: Water supression using presaturation zgpr (green), WATERGATE p3919gp (blue) and flip - backzggpwg (red) . Sample: dsDNA (sequence: TCTTGTGTTCT * AGAACACAAGA), 10 °C. Zuzka 20 1 /dl/jdata/fi al a/700m|Z| Iscale : 8.1577 Zuzka 22 1 /dl/data/fial a/700 : Scale : 1.163 /dl/data/fiala/700m|Zf [ppml Figure 4; Detail for imino region. Water supression using presaturation zgpr (green), WATERGATE p3919gp (blue) and standart W zggpwg (red). Sample: dsDNA (sequence: TCTTGTGTTCT * AGAACACAAGA), 10 °C. C7995 Advanced Methods of Biomolecular NMR Name: Zuzana Trosanova, 356857 TASK 3 - 2D Homonuclear Spectroscopy TASK 3A: Through - bond correlation experiments For this experiment a dsDNA (sequence: TCTTGTGTTCT * AGAACACAAGA) in D20 was used as a sample. Spactra were measured at 25 °C. For ID spectrum was used zgpr pulse sequence with spectral width of 9.9925 ppm (Fig. 1). zgpr Dna inD20 700 8 6 4 2 [ppm] Figure 1: ID spectrum for dsDNA (sequence: TCTTGTGTTCT * AGAACACAAGA). 25 °C Pulse sequence cosyphr was used for COSY measurement. Acqu- and Proc-parameters are shown in Tab. 1. Obtained spectrum is shown in Fig. 2. C7995 Advanced Methods of Biomolecular NMR Name: Zuzana Trosanova, 356857 Acqus Parameters Nucleus 1H 1H Dimension direct indirect Number of real points 2048 1600 Spectral width [ppm] 9.0084 9.0084 Observed frequency [MHz] 700.80329 700.80329 Carrier shift [ppm] 4.701 4.701 Proc Parameters Nucleus 1H H Dimension direct indirect Size of real spectrum 4096 4096 Spectrometer frequency [MHz] 700.8 700.8 Window function SINE SINE Table 1: Selected acquisition and processing parameters for COSY spectrum. 8 6 4 2 F2 Ippm] Figure 2: COSY spectrum for dsDNA (sequence: TCTTGTGTTCT * AGAACACAAGA). TOCSY spectrum was measured using pulse sequence dipsi2phr. Acquisition and processing parameters are shown in Tab. 2. Obtained spectrum is shown in Fig. 3. C7995 Advanced Methods of Biomolecular NMR Name: Zuzana Trosanova, 356857 Acqus Parameters Nucleus 1H 1H Dimension direct indirect Number of real points 2048 800 Spectral width [ppm] 9.0084 9.0084 Observed frequency [MHz] 700.80329 700.80329 Carrier shift [ppm] 4.701 4.701 Proc Parameters Nucleus 1H H Dimension direct indirect Size of real spectrum 1024 1024 Spectrometer frequency [MHz] 700.8 700.8 Window function QSINE QSINE Table 2: Selected acquisition and processing parameters for COSY spectrum. C7995 Advanced Methods of Biomolecular NMR Name: Zuzana Trosanova, 356857 -----. . JL 1 11, ... j —-□ c ■ ' 1-1 i i + -- i *x I 'til.}-■ ^ f > * ■ ^ ■ i f 1 ■ 1 -f Figure 4: Comparison of COSY (shown in red) and TOCSY (shown in blue) spectra for dsDNA (sequence: TCTTGTGTTCT * AGAACACAAGA). Highlighted regions: A) base-to-base - CH3-H6 from Thymine (violet), H5-H6 from Cytosine (red), B) sugar-to-sugar H1'-H2',H2" and H2',H2"-H3' from sugar (blue), H3'-H4' from sugar (yellow). 25 °C. C7995 Advanced Methods of Biomolecular NMR Name: Zuzana Trosanova, 356857 TASK 3B: 2D NOE Spectra Pulse sequence noesyhsqcetgpsi3d was used for measurement of through-space correlation. Acqus Parameters Nucleus 1H 13C 1H Dimension direct indirect indirect Number of real points 2048 64 1 Spectral width [ppm] 13.9994 75 13.9994 Obserwd frequency [MHz] 500.22235 125.785324 500.22235 Carrier shift [ppm] 4.706 39 4.706 ProcParameters Nucleus 1H 13C Dimension direct indirect Size of real spectrum 2048 128 Spectrometer frequency [MHz] 500.22 125.780419 Window function QSINE QSINE Table 3: Selected acquisition and processing parameters for NOESY spectrum. 14 12 10 8 6 4 2 F2 [ppm] Figure 5: NOESY spectrum ofdsDNA (sequence: TCTTGTGTTCT * AGAACACAAGA). 10 °C. C7995 Advanced Methods of Biomolecular NMR Name: Zuzana Trosanova, 356857 □ F2 Ippm] Figure 6: Detail for imino - imino region in NOESY spectrum of measured dsDNA. 10 °C. □ NOESYFPCPPHWC noesy long General DATE = 2015/11/19 TINE = 17:23 INSTRUM - spect PULPRDG = rioesyfpgpphng F2 £1H) 51 = 2G4S ' SF = 503.22 SWLp = 10000 SI = 204S --5F-=---500 = 22---5W_p - 1000C 12.0 F2 [ppmj Figure 7: Detail for imino - amino region in NOESY spectrum of measured dsDNA. 10 °C. C7995 Advanced Methods of Biomolecular NMR Name: Zuzana Trosanova, 356857 z □ NOESYFPCPPHWC noesy long General DATE = 2015/11/19 TIME = 17:23 INSTP.UH = spect PULPROC ■ noesyfpgpphwg F2 C1H) SI - 2048 SF = 500.22 SW_p - 10000 Fl C1H) SI - 2048 SF - 500.22 SW_p = 10000 > F2 |ppm] Figure 8: Detail for imino - methyl group region in NOESY spectrum of measured dsDNA. 10 °C. C7995 Advanced Methods of Biomolecular NMR Name: Zuzana Trosanova, 356857 TASK 4 - 2D heteronuclear spectroscopy of isotopically labeled protein sample TASK 4A: *H - 15N correlation Heterouclear Single Quantum Coherence, where 1H and 15N atoms are correlated, was measuerd using pulsesequence hsqctfpf3gp. Spectrum was measured two times: 1) wider spectral witdh, which includes also signal from Arginines (spectral width in 15N dimension: 31.999 ppm). However, arginines are not visible in obtained spectrum (Fig. 1) and 2) spectral width, which is sufficient for amide signals only (spectral width in 15N dimension: 70.0009 ppm, Fig. 2). Selected acquision and processing parameters are listed in Tab. 1. Acq us Param ete rs Nucleus 1H 15N Dimension direct indirect Number of real points 2048 256 Spectral width [ppm] 16,0185 31,9999 Observed frequency [MHz] 500,222351 50,692833 Carrier shift [ppm] 4,7 118 ProcParameters Nucleus 1H 15N Dimension direct indirect Size of real spectrum 2048 2048 Spectrometer frequency [MHz] 500,22 50,6868524 Window function QSINE QSINE Table 1: Selected acquisition and processing parameters for hsqctfpf3gp experiment. C7995 Advanced Methods of Biomolecular NMR Name: Zuzana Trosanova, 356857 Figure 1: 1H-15N HSQC for 15N13C labeled ubiquitin with wider spectral width. Signal for arginine's sidechains are not visible. 25 °C. □ N HSQC General DATE = 2015/11/19 TIME - 13:12 IN5TRUM = spect PULPROC = hsacetf3gosi si: = 2043 SF' = 500.22 SlnLp = S012.S21 Fl: (15N) Si: = 256 SF = 50.6S7 5W_p = 3041.363 ■ *fc .... 4* F2 Ippm] Figure 2: ^-^N HSQC of15N13C labeled ubiquitin for amide region. 25 °C. C7995 Advanced Methods of Biomolecular NMR Name: Zuzana Trosanova, 356857 TASK 4B: *H - 13C correlation hsqceptg was used as a pulse sequence for Heterouclear Single Quantum Coherence with 1H - 13C correlation. Selected acquision and processing perameters are listed in Tab. 2, obtained spectrum is shown in Fig. 2. Acq us Param ete rs Nucleus 1H 13C Dimension direct indirect Number of real points 1024 256 Spectral width [ppm] 13,015 79,9995 Observed frequency [MHz] 500,222351 125,7854502 Carrier shift [ppm] 4,7 40 ProcParameters Nucleus 1H 13C Dimension direct indirect Size of real spectrum 1024 1024 Spectrometer frequency [MHz] 500,22 125,780419 Window function QSINE QSINE Table 2: Selected acquisition and processing parameters for 1H - 13C HSQC spectrum. I □ C7995 Advanced Methods of Biomolecular NMR Name: Zuzana Trosanova, 356857 □ □ 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 F2 [ppm] Figure 4: Detail for slitting of signal in 1H - 13C HSQC spectrum for double labeled ubiquitin. 25 °C We can observe splitting of signals because of strong scalar coupling interaction (J interaction, Fig. 3 and 4). To remove splitting of signals we measured the 2D Constant-Time HSQC (CT-HSQC). CT-HSQC experiment is a version of the conventional 2D HSQC experiment in which the typical variable 13C evolution period is replaced by a constant-time evolution period in which homonuclear 13C-13C coupling constants are refocused. Evolution period was held constant at value 28 ms. Negative peaks correspond to carbons with none or 2 bounded Hydrogens (Fig. 4). Aromatic carbons are shown in Figure 5. C7995 Advanced Methods of Biomolecular NMR Name: Zuzana Trosanova, 356857 6 4 2 0 F2 Ippm] Figure 4: 1H - 13C CT - HSQC spectrum for double labeled ubiquitin. 25 °C 11 10 9 8 7 6 F2 Ippm] Figure 5: 1H - 13C CT - HSQC spectrum for aromatic carbons in double labeled ubiquitin. 25 °C