High-power relaxation dispersion NMR data set at different ligand concentrations: a litmus test for classification of recognition mechanism

High-power relaxation dispersion of ubiquitin (with SH3c titrated in) The high-power relaxation dispersion experiments were measured using the 15N based constant time E-CPMG experiment for quantifying kinetics of exchange processes in ubiquitin happening on a micro-to-millisecond time-scale. The measurements were conducted in Bruker Avance 600 MHz and 950 MHz spectrometers fitted with cryoprobe-TCI (Neo console running Topspin 4.x (Bruker Biospin corporation)) at 277 K. The constant time (CT) CPMG delay is divided into two equal halves, sandwiching the U-element that ensures the equal contribution of anti-phase and in-phase relaxation to R2,eff. In the E-CPMG experiment performed here, refocusing pulses are applied with (hard pulse) γB1/2π (7143 Hz and 7407 Hz for 15N in the 950 MHz and 600 MHz spectrometers respectively) fields (corresponding to 15N hard pulses) for all refocusing frequencies, thus reducing any off-resonance effects that can affect the measurement of R2,eff. The R2,eff values were measured at CPMG frequencies (νCPMG) of 66.7 Hz, 133 Hz, 267 Hz, 400 Hz, 533 Hz, 667 Hz, 1333 Hz, 2000 Hz, 2667Hz, 3333 Hz, 4000 Hz, 4667 Hz, 5333 Hz and 6000 Hz. The constant volume of 200 μL of NMR samples was put inside 3 mm tubes (Hilgenberg 299 GmbH) in 20 mM sodium phosphate buffer, pH 6.5, containing 100 mM NaCl, 10mM TCEP, 0.05% (w/v) sodium azide, and 10% D2O. In all experiments the ubiquitin (15N labeled) concentration was 1 mM. The SH3c (unlabeled) concentration was varied from 0, 0.02 mM, 0.05mM, 0.1 mM, 0.25 mM, 0.5 mM up to 1 mM. The probe temperature was calibrated using a digital thermometer and standard methanol sample. The reference spectra were collected without the CPMG delay period (τ). The R2,eff was calculated as R2,eff(νCPMG) = −1/T log(I(νCPMG)/I0) where νCPMG is the effective frequency of the CPMG field, (νCPMG = 1/(4τ), where the time between the centers of consecutive 180◦ pulses is 2τ), T is the constant delay during which CPMG pulses were applied (60 ms), I0 is the intensity of the peak in reference experiment (no constant-time delay) and I(ν) is the intensity of the peak at that particular CPMG frequency. The CPMG delay (60 ms) was chosen such that the residual intensity was approximately 50% of maximum intensity. The experiment was performed with 3 s recycle delay between increments using 12 different refocusing field strengths between 0 to 6000 Hz collected in scrambled and interleaved manner with 1024 (1H) and 130 (15N) complex points, respectively. For each increment, 16 transients were measured following the Echo-AntiEcho scheme for indirect frequency sign discrimination. There is a heat compensation block in the middle of the recycle delay that contains additional 180 degree pulses so that the total number of CPMG 180◦ refocusing pulses at fixed B1 field strength is constant irrespective of inter-pulse delay. The E-CPMG experiments took 3 days to complete, and standard 1H, 15N TROSY-HSQC spectra were collected before and after each experiment to monitor sample stability (there were no changes in the spectra before and after the experiments). The error-bars on individual data points reflect error-propagation of signal to noise ratio from duplicate measurement at one CPMG frequency. High-power relaxation dispersion of SH3c (with ubiquitin titrated in) The high-power relaxation dispersion on the 15N labeled SH3c were measured at 277 K in Bruker Avance-III 800 MHz spectrometer equipped with cryoprobe-TCI. The refocusing pulses were applied with γB1/2π ~5 kHz for 15N in an interleaved manner with 3 s recovery delay. The spectra were recorded with 1024 and 156 complex points in the direct and indirect dimensions, respectively. The NMR experiments were performed with the 15N-labeled CIN85-SH3 and unlabeled ubiquitin complex in 20 mM sodium phosphate buffer, pH 6.5, containing 100 mM NaCl, 10mM TCEP, 0.05% (w/v) sodium azide, and 10% D2O. In this experiment the SH3c (15N labeled) concentration was kept fixed at 1 mM, and the ubiquitin (unlabeled) concentration was varied from 0, 0.02 mM, 0.05 mM, 0.075 mM, 0.1 mM, 0.15 mM, 0.25 mM, 0.5 mM, up to 1mM. The R2,eff values were measured at the same frequencies as the previous experiment. In all other aspects, the experiment was performed under identical experimental setup as described above. Chromatograms and mass specs of 15N-Glu24-labelled G53(D)Thr ubiquitin are included.

高功率弛豫散布实验针对泛素（含SH3c等摩尔量）进行，该实验采用基于15N的恒定时间E-CPMG实验以量化泛素在微秒至毫秒时间尺度上发生的交换过程动力学。实验在Bruker Avance 600 MHz和950 MHz核磁共振光谱仪（配备低温探头-TCI，Neo控制台运行Topspin 4.x，Bruker Biospin公司）的277 K温度下进行。恒定时间CPMG延迟被分为两等分，夹持U元素以确保反相和同相弛豫对R2,eff的贡献相等。在本实验中进行的E-CPMG实验中，对所有重聚焦频率应用了（硬脉冲）γB1/2π（950 MHz和600 MHz光谱仪中15N的频率分别为7143 Hz和7407 Hz）场（对应于15N硬脉冲）的复聚焦脉冲，从而减少了可能影响R2,eff测量的任何失谐效应。R2,eff值在CPMG频率（νCPMG）为66.7 Hz、133 Hz、267 Hz、400 Hz、533 Hz、667 Hz、1333 Hz、2000 Hz、2667 Hz、3333 Hz、4000 Hz、4667 Hz、5333 Hz和6000 Hz处进行测量。200 μL NMR样品的恒定体积置于3 mm管（Hilgenberg 299 GmbH）中，在20 mM磷酸钠缓冲液，pH 6.5，含有100 mM NaCl、10 mM TCEP、0.05%（w/v）亚硝酸钠和10% D2O中。所有实验中，泛素（15N标记）的浓度为1 mM。SH3c（未标记）的浓度从0、0.02 mM、0.05 mM、0.1 mM、0.25 mM、0.5 mM直至1 mM不等。探头温度使用数字温度计和标准甲醇样品进行校准。参考光谱在没有CPMG延迟期（τ）的情况下收集。R2,eff计算公式为R2,eff(νCPMG) = −1/T log(I(νCPMG)/I0)，其中νCPMG是CPMG场的有效频率，（νCPMG = 1/(4τ)，其中连续180°脉冲中心的间隔为2τ），T是应用CPMG脉冲的恒定延迟时间（60 ms），I0是参考实验（无恒定时间延迟）中峰的强度，I(ν)是该特定CPMG频率处的峰强度。CPMG延迟（60 ms）被选择，使得残留强度约为最大强度的50%。实验在每次增量之间使用3 s回波延迟，采用12种不同的复聚焦场强（0至6000 Hz）以交错方式收集，分别以1024（1H）和130（15N）复数点进行。对于每个增量，按照回波-反回波方案测量16个瞬态，以进行间接频率符号区分。在回波延迟中包含一个热补偿块，其中包含额外的180°脉冲，以确保在固定的B1场强下，CPMG 180°复聚焦脉冲的总数不随脉冲延迟而变化。E-CPMG实验耗时3天完成，在每个实验前后均收集了标准的1H、15N TROSY-HSQC光谱，以监测样品稳定性（实验前后光谱无变化）。个别数据点的误差条反映了从重复测量中信号与噪声比误差传播。SH3c的高功率弛豫散布实验（含泛素等摩尔量）在Bruker Avance-III 800 MHz核磁共振光谱仪上测量，该光谱仪配备低温探头-TCI。复聚焦脉冲以γB1/2π ~5 kHz的频率对15N进行交错应用。光谱以1024和156复数点分别在直接和间接维度上进行记录。NMR实验在20 mM磷酸钠缓冲液，pH 6.5，含有100 mM NaCl、10 mM TCEP、0.05%（w/v）亚硝酸钠和10% D2O中，使用15N标记的CIN85-SH3和未标记的泛素复合物进行。在本实验中，SH3c（15N标记）的浓度保持为1 mM，而泛素（未标记）的浓度从0、0.02 mM、0.05 mM、0.075 mM、0.1 mM、0.15 mM、0.25 mM、0.5 mM直至1 mM不等。R2,eff值在与前一个实验相同的频率处进行测量。在其他所有方面，实验均按照上述描述的相同实验设置进行。包含15N-Glu24标记的G53(D)Thr泛素的色谱图和质量谱。