Supporting Information
|
|
- Rebecca Wilkinson
- 5 years ago
- Views:
Transcription
1 Supporting Information Arai et al /pnas SI Text Protein Expression and Purification. Myb3 (mouse, residues ) was expressed in Escherichia coli as a fusion with the B1 domain of protein G and purified using a Ni-NTA column. The fusion protein was cleaved on column using thrombin and Myb3 was separated using reverse-phase HPLC. The resulting protein (GYNDEDPEKEKRIKELELLLMSTENELKGQQAL) contains an additional Gly at the N terminus. Binding Simulations in the Absence of MLL. When the number of Myb3 binding sites on KIX is two, the binding can be described by an independent two-site binding model as shown below (4): CD Measurement. CD spectra of Myb3 and Myb5 (100 μm) were measured on an Aviv 6DS spectropolarimeter at 30 C in 0 mm Tris-acetate buffer (ph 7.0) containing 50 mm NaCl. ITC Measurement. Titrations of Myb3 (0.8 mm or 0.6 mm) into KIX (45 μm) in the presence or absence of MLL8 (90 μm) were performed at 30 C, using a MicroCal Omega VP-ITC instrument as previously described (1, 3). Samples were prepared in 0 mm Tris-acetate (ph 7.0) and 50 mm NaCl. Typically, two injections of 5 μl were followed by 8 injections of 10 μl until a molar ratio of was obtained. The dilution heats are typically small and were subtracted from the calorimetric data. Integration of the thermogram and subtraction of the blanks yielded a binding isotherm that was fitted to a one-site binding model using the MicroCal Origin software or to a two-site binding model using an in-house fitting program to determine the stoichiometric ratios, N 1 and N, the dissociation constants, K d1 and K d, and the changes in enthalpy of the interaction, ΔH 1 and ΔH, for the primary and secondary binding, respectively. To improve fits, correction for protein concentration was implemented in the fitting program (4). The change in entropy, ΔS, was then calculated according to Eq. S1, 1 ΔG = RT ln = ΔH TΔS, [S1] K d where R is the gas constant, T is the absolute temperature, and ΔG is the free energy change from the free to the bound form. All experiments were performed in duplicate. NMR Measurement. NMR spectra were recorded using Bruker 500-, 600-, and 800-MHz spectrometers and analyzed using NMRPipe (39) and NMRView (40). Backbone resonances of free Myb3 and the KIX domain in complex with Myb3 at a 1:1 ratio were assigned using standard 3D NMR experiments with 500 μm 13 C, 15 N-labeled protein in 0 mm Tris-d 11 -acetate-d 4 (ph 7.0), 50 mm NaCl, mm NaN 3, and 10% (vol/vol) D O at 30 C. Backbone resonances of Myb3 in complex with KIX at a 1:1 ratio in the presence of MLL8 were assigned using 1 H- 15 N HSQC, 15 N TOCSY-HSQC, and 15 N NOESY-HSQC spectra. 15 N R relaxation dispersion data were acquired on Bruker 500- and 800-MHz spectrometers, using relaxation-compensated constant-time Carr Purcell Meiboom Gill (CPMG) pulse sequences (10, 38) with a constant relaxation delay of 40 ms or 60 ms. Errors in R were estimated from duplicate measurements (10). The samples contained 0 mm Tris-d 11 -acetate-d 4 (ph 7.0), 50 mm NaCl, mm NaN 3, 10% (vol/vol) D O, 0.7 mm Myb3, mm KIX, and mm MLL8. The weighted q average ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 15 N/ 1 H chemical shift change was calculated as Δδ av = ðδδ H Þ + ðδδ N =5Þ (4). If K d1 and K d are known, the fractions of the F, B1, B, and B1 forms, f F, f B1, f B, and f B1, respectively, are calculated, using K d1 of 0.13 μm and K d of 46 μm, as f F = K d1 K d K d1 K d + ðk d1 + K d Þ½LŠ + ½LŠ K d ½LŠ f B1 = K d1 K d + ðk d1 + K d Þ½LŠ + ½LŠ K d1 ½LŠ f B = K d1 K d + ðk d1 + K d Þ½LŠ + ½LŠ f B1 = ½LŠ K d1 K d + ðk d1 + K d Þ½LŠ + ½LŠ, where [L] is the concentration of the free form of Myb3, which is a solution of the cubic equation ½LŠ 3 + a½lš + b½lš + c = 0 a = ½PŠ tot ½LŠ tot + K d1 + K d b = ½PŠ tot ½LŠ tot ðkd1 + K d Þ + K d1 K d c = K d1 K d ½LŠ tot, [S] where [P] tot and [L] tot are the total concentrations of KIX and Myb3, respectively (4). The closed-form solution of Eq. S has been reported (41), where a ½LŠ = 3 + qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ða 3 3bÞcos θ 3, θ = arccos a3 + 9ab 7c q ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi. ða 3bÞ 3 There are three Myb3 species; the free form (C0), Myb3 bound to the primary site (C1), and Myb3 bound to the secondary site Arai et al. 1of1
2 (C). The concentrations of these species, [C0], [C1], and [C], respectively, are obtained as ½C0Š = f F ½LŠ tot ½C1Š = ðf B1 + f B1 Þ½LŠ tot ½CŠ = ðf B + f B1 Þ½LŠ tot, which are shown in Fig. S4 A C. Binding Simulations in the Presence of MLL. When MLL8 was added to the Myb3:KIX complex, the binding model is described as shown below: The concentrations of three Myb3 species were calculated as ½C0Š = ½Myb3Š tot ½C1Š ½CŠ ½C1Š = ½S1Š + ½D1Š + ½DŠ ½CŠ = ½SŠ + ½D1Š + ½D3Š. Similarly, there are three MLL8 species: the free form (M0), MLL8 bound to the primary site (M1), and MLL8 bound to the secondary site (M). The concentrations of these species, [M0], [M1], and [M], respectively, are obtained as ½M0Š = ½MLLŠ tot ½M1Š ½MŠ ½M1Š = ½S3Š + ½D3Š + ½D4Š ½MŠ = ½S4Š + ½DŠ + ½D4Š, which are shown in Fig. S4 D G. To study the direct binding of Myb3 to the primary, high-affinity site on KIX, it is desirable to observe only the S1 and D forms, in which Myb3 binds only to the primary site. Here, the following equations hold, F + S1 + S + S3 + S4 + D1 + D + D3 + D4 ½KIXŠ tot = 0 C + S1 + S + D1 + D + D3 ½Myb3Š tot = 0 M + S3 + S4 + D + D3 + D4 ½MLL8Š tot = 0 F C K d1 S1 = 0 F C K d S = 0 F M K d3 S3 = 0 F M K d4 S4 = 0 S C K d1 D1 = 0 S4 C K d1 D = 0 S1 C K d D = 0 S3 C K d D3 = 0 S M K d3 D3 = 0 S4 M K d3 D4 = 0 S1 M K d4 D = 0 S3 M K d4 D4 = 0, where [KIX] tot, [Myb3] tot, and [MLL8] tot are the total concentrations of KIX, Myb3, and MLL8, respectively; C is the free Myb3 concentration; M is the free MLL8 concentration; and F, S1 S4, and D1 D4 denote the concentrations of the corresponding forms. By solving these simultaneous equations using the Newton Raphson method (4), the concentrations (fractions) of various KIX, Myb3, and MLL8 forms are obtained. Here, we used the following numbers: K d1 and K d are 0.13 μm and46μm, respectively, and K d3 and K d4 are.1 μmand 90 μm, respectively, as determined previously (3). Analysis of R Relaxation Dispersion Data. The R relaxation dispersion data were analyzed using the fortran version of the inhouse fitting program GLOVE (43), named GLOVEf. The fitting equation for the two-state model (including both models 1 and 3b) is where R eff = R ½KIXŠk ON + k OFF 1 cosh 1 ½D + coshðη τ + Þ CP D cosðη ÞŠ, " # D ± = 1 ±1 + Ψ + Δω FB pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Ψ + ξ sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 1 η ± = τ CP ±Ψ + Ψ + ξ Ψ = ð½kixšk ON + k OFF Þ Δω FB ξ = Δω FB ð½kixšk ON k OFF Þ, where R 0 is an intrinsic relaxation rate, [KIX] is the concentration of free KIX, k ON and k OFF are the association and dissociation rates, respectively, τ CP is the interval between 180 pulses in CPMG pulse sequences, and Δω FB is the chemical shift difference between the free and bound forms (44). The fitting equation for the induced-fit model (model ) and the conformational selection model (model 3a) is R eff = R 0 1 τ CP lnðλ 1 Þ, where λ 1 is the largest eigenvalue of the matrix 0 h Re exp h Im exp A τ CP A τ CP exp exp A p τ i CP A p τ i CP h Im exp h Re exp A τ CP A τ CP exp exp A p τ i CP 1 C A p τ i A, CP Arai et al. of1
3 where Re[ ] and Im[ ] are functions to extract the real or imaginary elements, respectively, of the complex matrix (10). A is a 3 3 evolution matrix, and A * is its complex conjugate. For the induced-fit model, 0 A ½KIXŠk 1 ON k OFF 0 ½KIXŠk ON k OFF + k IB iδω FI k BI A, 0 k IB k BI iδω FB where k IB and k BI are the folding and unfolding rate constants between the intermediate and bound forms, respectively, and Δω FI is the chemical shift difference between the free and intermediate forms. For the conformational selection model, 0 A k 1 UH k HU 0 k UH k HU + ½KIXŠk ON iδω UH k OFF A, 0 ½KIXŠk ON k OFF iδω UB where k UH and k HU are the folding and unfolding rate constants between the unfolded and helical forms, respectively, and Δω UH and Δω UB are the chemical shift difference between the unfolded and helical forms and between the unfolded and bound forms, respectively. The free KIX concentration is obtained by ½KIXŠ = 1 K d + c K ½KIXŠ tot c M ½Myb3Š tot qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi + Kd c K ½KIXŠ tot + c M ½Myb3Š tot + 4cK ½KIXŠ tot K d, where c K and c M are correction factors for [KIX] tot and [Myb3] tot, respectively. For the two-state model, for the induced-fit model, K d = k OFF k ON ; K d = k OFF k ON k BI k BI + k IB ; and for the conformational selection model, K d = k OFF k ON k UH + k HU k UH. The fitting parameters are R 0, k ON, k OFF, Δω FB, c K, and c M for all three models and, in addition, k IB, k BI, and Δω FI for the inducedfit model and k UH, k HU, and Δω UH for the conformational selection model. Fitting Procedures. In the present study, 8 dispersion curves (four concentration ratios at two magnetic fields) were obtained for each residue, and each curve contained 17 data points. Because 1 residues show smooth dispersion curves, a total of 96 dispersion curves containing 1,63 data points were used for data analysis. Fits to the above fitting equations were done by the nonlinear least-squares method. There are three types of fitting as follows: Local fit: Dispersion curves for only one residue (8 curves) are globally fitted with common rate constants and common chemical shift differences. R 0 is a local parameter for each curve. Correction for protein concentrations was not done (c K and c M were fixed to 1). Full global fit: Dispersion curves for all residues (96 curves) are globally fitted with common rate constants and common correction factors for protein concentrations. Chemical shift differences are common for the curves of the same residue. R 0 is a local parameter for each curve. Subglobal fit: Dispersion curves for all residues (96 curves) are globally fitted with a common correction factor(s) for protein concentration(s). All rate constants and chemical shift differences are common for the curves of the same residue. R 0 is a local parameter for each curve. The nonlinear least-squares fitting requires good estimates of initial values. Therefore, many fitting runs were carried out from various initial parameter values (grid search). Then, the best fit was selected, that is, the fit with a small reduced χ, which indicates the goodness of fit, and with reasonable parameter sets. We initially performed local fits for each residue and searched for the best fit by a grid search. Then, the median of the rate constants obtained for all residues was calculated and was used as an initial value for the subsequent full global fit. In the full global fit, a grid search around the medians of local fits was performed. If the full global fit gives a reasonable fit, a subglobal fit was performed, in which only a correction factor(s) for protein concentration(s) is a global parameter(s). Here, the results of the full global fit were used as initial parameter values, and the random and Monte Carlo minimizations were performed as described previously (43). Fitting errors were obtained by a covariance matrix. Finally, we checked whether the fitted results fulfill the following: The K d value obtained by fitted rate constants is close to the K d obtained by ITC, the population of the bound form is larger than that of the free and intermediate forms, and the bound form is more structured than other forms. After the full global fits, we found that only the two-state model gives reasonable fits (main text and Fig. S7). Therefore, the subglobal fits were performed only for the two-state model. To obtain accurate estimates of correction factors for protein concentrations, c M and c K, in the subglobal fits to the two-state model, we performed a grid search by slightly changing the fixed c M and c K values. Here, c M and c K were fixed in each fit of the grid search, because fitting was unstable if both parameters were set as variables. The parameters obtained in the best fit among the grid searches are shown in the main text and in Fig. 5 (c M = 1.030; c K = 0.959). The apparent variation in k ON for different residues (Fig. 5A) is not statistically significant. On the other hand, residues L30, T305, N307, E308, and L309 in the C-terminal part of the c-myb helix have lower than average values of k OFF (Fig. 5B); the average k OFF is 1.7 ± 0.4 (SE) for residues and 16.6 ± 0.6 for residues Several variants of the two-state fits were performed in which clusters of residues were constrained to have the same values of k ON and k OFF. The results of these fits are given in Table S1. The best fits were obtained when k ON and k OFF were allowed to vary independently for each residue or when k ON was constrained globally (for residues ) or constrained separately for clusters of residues in the N- and C-terminal regions of c-myb. In all such fits, lower than average dissociation rates were observed consistently for the C-terminal residues. However, fits in which k OFF was constrained to a global value or constrained for separate clusters of residues in the N-terminal and C-terminal regions were noticeably poorer, as judged by χ (Table S1). Data Treatment for Fig. 5D. The correlation between the association rates of Giri et al. (7) and the predicted percentage of helix according to the program AGADIR () is valid only for some of the mutation sites. The association rates of the Lys/Ala, Lys/Gly, and Arg/Ala mutant peptides are systematically lower than wild type by an average 1.85 (± 0.31) μm 1 s 1, presumably because of the reduction in charge. In the complex, these residues, especially K93 and R94, are close to a negatively charged patch on the surface of KIX. The rates for the K93A, K93G, R94A, and K96A peptides were therefore corrected, k ON = k ON(obs) μm 1 1 s (Fig. 5D, green triangles). All other rates are as in table 1 of Giri et al. (7) (Fig. 5D, red circles). The line (slope = 0., R = 0.85) Arai et al. 3of1
4 is a linear least-squares fit of all data except L300A and L301A. These latter mutants, which bind faster than wild type and have negative Φ values, are clear outliers (Fig. 5D, black circles) and were excluded from the least-squares fit. It should be noted that the slope is unchanged and the correlation coefficient is improved (R = 0.94) if the Lys and Arg mutants are also excluded. The linear correlation between association rate and predicted helicity is robust and is also observed when the population of helix is predicted for mutant Myb5 peptides with the N terminus acetylated (R = 0.8) or preceded by Gly residues (R = 0.86) to mimic the fusion protein used by Giri et al. (7) for stopped-flow kinetics. Arai et al. 4of1
5 Fig. S1. (A) 1 H- 15 N HSQC spectra of 15 N-labeled Myb3 showing chemical shift changes upon titration with KIX. Assignments are shown for residues showing fast-exchange shifts. The cross-peak color changes gradually from blue (free) to red (bound) according to the concentration ratios shown. (B) 1 H- 15 N HSQC spectra of 15 N-labeled KIX showing chemical shift changes upon titration with Myb3. The cross-peak color changes gradually from black (free) to magenta (bound) according to the concentration ratios shown. (C and D) Histograms of the averaged chemical shift differences Δδ av for the primary (C) and secondary (D) binding of Myb3 to KIX. The black horizontal line shows the mean of all Δδ av (0.144 ppm and ppm for primary and secondary binding, respectively). The residues are categorized into the following groups: red, greater than or equal to mean + SD; orange, mean + 1 SD to mean + SD; yellow, mean to Legend continued on following page Arai et al. 5of1
6 mean + 1 SD; and gray, less than mean. (C) 1 H and 15 N chemical shifts of KIX for the c-myb bound form at a 1:0.8 KIX:Myb3 ratio were subtracted from those of free KIX to get Δδ H and Δδ N. The data at a 1:0.8 ratio (rather than at 1:1) were used for primary binding to eliminate the effect of secondary c-myb binding to KIX. (D) Δδ H and Δδ N obtained from the titration analysis (Fig. S3A) were used to calculate Δδ av. The data at a 1:0.8 ratio were used as a reference. (E) 1 H- 15 N HSQC spectra of 15 N-labeled Myb3 showing chemical shift changes upon titration with the KIX-L68A mutant. The cross-peak color changes gradually from blue (free) to red (bound) according to the concentration ratios shown. Fast-exchange shifts are observed for some peaks in the titrations of small amounts of KIX, suggesting the presence of secondary c-myb binding on KIX. Fig. S. (A and B) ITC titration profiles (Upper) and binding isotherms (Lower) for the Myb3:KIX interactions in the absence (A) and the presence (B) of MLL8 [0 mm Tris-acetate (ph 7.0), 50 mm NaCl, 30 C]. (A) 0.8 mm Myb3 was titrated into 45 μm KIX. Two titration curves from duplicate measurements were globally fitted assuming a two-site binding model. Thermodynamic parameters are K d1 = 0.3 ± 0.06 μm, N 1 = 1.1 ± 0., ΔG 1 = 9. ± 0. kcal/mol, ΔH 1 = 5 ± 1 kcal/mol, TΔS 1 = 4 ± 1 kcal/mol, K d = 43 ± 14 μm, N = 0.8 ± 0., ΔG = 6.1 ± 0. kcal/mol, ΔH = 7 ± 3 kcal/mol, TΔS = 1 ± 3 kcal/mol. A correction factor for the Myb3 concentration was 1.1 ± 0.. (B) 0.6 mm Myb3 was titrated into 45 μm KIXinthepresenceof90μM MLL8. The titration curve was fitted to a one-site binding model. Thermodynamic parameters are K d = 0.13 ± μm, N = 1.04 ± 0.01, ΔG = 9.3 ± 0. kcal/mol, ΔH = 9.5 ± 0.1 kcal/mol, and TΔS = 0.3 ± 0.1 kcal/mol. Arai et al. 6of1
7 Fig. S3. (A) Global fitting of the titration curves for the 15 N-KIX titration with unlabeled Myb3, referenced to the shifts at a 1:0.8 KIX:Myb3 ratio (4). 1 H(A, Left) and 15 N(A, Right) chemical shift changes of HSQC cross-peaks of 15 N-KIX are plotted as a function of the Myb3/KIX concentration ratio. Only the peaks showing clear fast-exchange shifts were used for fitting (154 curves for 77 assigned peaks). Color codes are shown (A, Right) along with the residue number. In the fitting, we assumed that K d1 is 0.13 μm, which was obtained by the ITC experiment for the Myb3 binding to the primary site on KIX in the presence of excess MLL8 and that Δδ H and Δδ N in the primary binding site are zero (3). The global fitting gave a K d of 46 ± 1 μm. A correction factor for the Myb3 concentration was ± 0.00 (4). (B) 15 N R relaxation dispersion curves for 15 N-Myb3 in the free form (B, Left) and for 15 N-Myb3 in the presence of twofold excess of MLL8 (B, Right) measured with an 800-MHz spectrometer at 0.7 mm Myb3. Color codes are shown in each panel along with the residue number. Arai et al. 7of1
8 Fig. S4. (A) Concentrations of various Myb3 species dependent on a KIX:Myb3 concentration ratio from 0 to 1.5 at 0.7 mm Myb3. Red, Myb3 bound to the primary c-myb binding site on KIX; blue, Myb3 bound to the secondary c-myb binding site on KIX; and green, free Myb3. (B) Fractions of various Myb3 species under the conditions for R relaxation dispersion experiments (from 1:0.95 to 1:1.10 Myb3:KIX concentration ratios at 0.7 mm Myb3), plotted in a logarithmic scale. (C) The results show that 3 9% of Myb3 binds to the secondary, low-affinity site. See SI Text for details of the calculations. (D F) Fractions of various KIX (D), MLL8 (E), and Myb3 (F) species under the conditions for R relaxation dispersion experiments plotted on a logarithmic scale (the KIX:Myb3: MLL8 concentration ratio = 1:0.95:1.90, 1:1.00:.00, 1:1.05:.10, and 1:1.10:.0 at 0.7 mm Myb3). (D) The D form, in which Myb3 and MLL bind at the primary (c-myb/pkid) and secondary (MLL) sites, respectively, is the most dominant form under these conditions. See SI Text for the definitions of various KIX forms. (E) Almost half of MLL8 binds to the MLL site, and the rest is mostly in the free form. Less than % of MLL8 binds to the primary site. Such a small amount of secondary MLL8 binding to KIX will not affect the Myb3 binding to the primary site on KIX. (F and G) More than 94% of Myb3 is bound to the primary site, % is in the free form, and only less than 0.5% is bound to the secondary site. Such a small fraction of secondary Myb3 binding to KIX will not be detected in the R relaxation dispersion experiments. Arai et al. 8of1
9 Fig. S5. 15 N R ex of Myb3 in the presence of KIX and MLL8 measured by the R relaxation dispersion experiments with 500-MHz (Left) and 800-MHz (Right) spectrometers. 15 N-Myb3:KIX:MLL8 concentration ratios are shown in each panel. R ex was estimated from the difference in R eff at the lowest and highest 1/τ cp values. Arai et al. 9of1
10 Fig. S6. 15 N R relaxation dispersion curves for 1 residues of 15 N-Myb3 in complex with KIX and MLL8 used for global analysis. In each panel, the Myb3:KIX concentration ratios and the color codes are shown. The MLL8 concentration was twofold excess of the KIX concentration. Solid lines are the fits to the twostate binding model. Arai et al of 1
11 Fig. S7. Correlation of 15 N chemical shift differences of 15 N-Myb3 determined from the R relaxation dispersion experiments (Δω N ) with equilibrium chemical shift differences of 15 N-Myb3 between the free and KIX-bound forms in the presence of MLL8 determined from HSQC spectra (Δδ N ). (A) The results obtained by the fits to the induced-fit mechanism (model ). Fitted parameters are k ON = (1.8 ± 0.1) 10 7 M 1 s 1, k OFF = 15.5 ± 0.3 s 1, K d = 0.81 ± 0.06 μm, k IB = 0.74 ± 0.09 s 1, k BI = 15.4 ± 0.4 s 1, and a correction factor for the Myb3 concentration = 1.07 ± k IB < k BI, indicating that the population of c-myb in the intermediate state (88%) is much higher than that in the fully bound state (4%), which is unreasonable. The Δω N values between the free and bound forms obtained by R dispersion experiments (red) were significantly larger than Δδ N obtained from equilibrium HSQC. However, the Δω N values between the free and intermediate forms (blue) were well correlated with Δδ N between the free and bound forms, which is inconsistent. Therefore, the fits to the induced-fit model are unreasonable. (B) The results obtained by the fits to the slow conformational selection mechanism (model 3a). Fitted parameters are k ON = (.1 ± 0.) 10 8 M 1 s 1, k OFF = 1.0 ± 0.6 s 1, K d = 0.7 ± 0.1 μm, k HU = 1,000 ± 1,000 s 1, k UH = 1,900 ± 100 s 1, and a correction factor for the Myb3 concentration = ± The Δω N values between the unfolded and helical forms obtained by fitting the R dispersion experiments (blue) were significantly larger than Δδ N between the free and bound forms obtained from equilibrium HSQC spectra, suggesting that the conformational ensemble of free c-myb contains a state that is more highly structured than KIX-bound c-myb. Therefore, the fits to the conformational selection model 3a are physically unrealistic. Fig. S8. (A and B) Helicities of the TADs of c-myb (A) and pkid (B) predicted by AGADIR () at 303 K and 88 K, respectively. (C and D) Deviations of 13 C α (red) and 13 C (blue) chemical shifts from sequence-corrected (19) random coil shifts for intrinsically disordered proteins (0), applied to Myb3 (C, chemical shifts at 303 K) and pkid (D, chemical shifts at 88 K) (9). The predicted population of helix between residues 90 and 301 of Myb3 (66%) is in close agreement with experiment (70%, estimated from 13 C α and 13 C shifts). For pkid, the population of helix between residues 117 and 19 (spanning helix αa of pkid bound to KIX) (6) estimated from the secondary chemical shifts is 46%. Residues (helix αb of bound pkid) have no measurable propensity to adopt regular helical structure in the free pkid. Arai et al of 1
12 Table S1. Variants of the subglobal fits to the two-state model in which clusters of residues are constrained to have the same values of k ON and k OFF Fitted parameter values Correlation between Δω FB and Δδ FB Fit type Global parameters, in addition to c M and c K Local parameters, in addition to 0 Δω FB and R No. Average k ON parameters Reduced χ ( 10 7 M 1 s 1 ) N-terminal average k OFF,s 1 * C-terminal average k OFF,s 1 c M c K Slope r 1 k ON, k OFF ± ± ± ± a k ON1 (90 303), k ON ( ) k OFF ± ± ± ± b k ON1 (90 301), k ON (30 309) k OFF ± ± ± ± k ON (90 309) k OFF ± ± ± ± a k OFF1 (90 303), k OFF ( ) k ON ± ± ± ± b k OFF1 (90 301), k OFF (30 309) k ON ± ± ± ± a k ON (90 309), k OFF1 (90 303), k OFF ( ) ± ± ± ± b k ON (90 309), k OFF1 (90 301), k OFF (30 309) ± ± ± ± a k ON1 (90 303), k ON ( ), k OFF1 (90 303), k OFF ( ) ± ± ± ± b kon1(90 301), kon(30 309), koff1(90 301), koff(30 309) ± ± ± ± k OFF (90 309) k ON ± ± ± k ON (90 309), k OFF (90 309) ± ± ± *k OFF1 or the average of k OFF for residues k OFF or the average of koff for residues Correlation coefficient. The results of this type of fit are shown in Fig.. Arai et al. 1 of 1
Supporting Information
Supporting Information Boehr et al. 10.1073/pnas.0914163107 SI Text Materials and Methods. R 2 relaxation dispersion experiments. 15 NR 2 relaxation dispersion data measured at 1 H Larmor frequencies of
More informationSupporting Information
Supporting Information Li et al. 10.1073/pnas.1314303110 SI Text Preparation of NMR Samples. Mutagenesis, protein expression, and purification were performed as previously described (1), except that the
More informationSupplementary Information. Overlap between folding and functional energy landscapes for. adenylate kinase conformational change
Supplementary Information Overlap between folding and functional energy landscapes for adenylate kinase conformational change by Ulrika Olsson & Magnus Wolf-Watz Contents: 1. Supplementary Note 2. Supplementary
More informationSupporting Information. Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2009
Supporting Information Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2009 Helical Hairpin Structure of a potent Antimicrobial Peptide MSI-594 in Lipopolysaccharide Micelles by NMR Anirban
More informationSupplementary Figures
1 Supplementary Figures Supplementary Figure 1 Type I FGFR1 inhibitors (a) Chemical structures of a pyrazolylaminopyrimidine inhibitor (henceforth referred to as PAPI; PDB-code of the FGFR1-PAPI complex:
More informationProtein dynamics from NMR Relaxation data
Protein dynamics from NMR Relaxation data Clubb 3/15/17 (S f2 ) ( e ) Nitrogen-15 relaxation ZZ-exchange R 1 = 1/T 1 Longitudinal relaxation (decay back to z-axis) R 2 = 1/T 2 Spin-spin relaxation (dephasing
More informationSubstrate-dependent switching of the allosteric binding mechanism of a dimeric enzyme
Supplementary Information: Substrate-dependent switching of the allosteric binding mechanism of a dimeric enzyme Lee Freiburger, 1 Teresa Miletti, 1 Siqi Zhu, 1 Oliver Baettig, Albert Berghuis, Karine
More informationChapter 6. The interaction of Src SH2 with the focal adhesion kinase catalytic domain studied by NMR
The interaction of Src SH2 with the focal adhesion kinase catalytic domain studied by NMR 103 Abstract The interaction of the Src SH2 domain with the catalytic domain of FAK, including the Y397 SH2 domain
More informationMapping Protein Folding Landscapes by NMR Relaxation
1 Mapping Protein Folding Landscapes by NMR Relaxation P.E. Wright, D.J. Felitsky, K. Sugase, and H.J. Dyson Abstract. The process of protein folding provides an excellent example of the interactions of
More informationProblem solving steps
Problem solving steps Determine the reaction Write the (balanced) equation ΔG K v Write the equilibrium constant v Find the equilibrium constant using v If necessary, solve for components K K = [ p ] ν
More informationSensitive NMR Approach for Determining the Binding Mode of Tightly Binding Ligand Molecules to Protein Targets
Supporting information Sensitive NMR Approach for Determining the Binding Mode of Tightly Binding Ligand Molecules to Protein Targets Wan-Na Chen, Christoph Nitsche, Kala Bharath Pilla, Bim Graham, Thomas
More informationSupporting Information
Supporting Information Micelle-Triggered b-hairpin to a-helix Transition in a 14-Residue Peptide from a Choline-Binding Repeat of the Pneumococcal Autolysin LytA HØctor Zamora-Carreras, [a] Beatriz Maestro,
More informationSUPPLEMENTARY INFORMATION
Figure S1. Secondary structure of CAP (in the camp 2 -bound state) 10. α-helices are shown as cylinders and β- strands as arrows. Labeling of secondary structure is indicated. CDB, DBD and the hinge are
More informationK ex. Conformational equilibrium. equilibrium K B
Effects of Chemical Exchange on NMR Spectra Chemical exchange refers to any yprocess in which a nucleus exchanges between two or more environments in which its NMR parameters (e.g. chemical shift, scalar
More informationNMR in Medicine and Biology
NMR in Medicine and Biology http://en.wikipedia.org/wiki/nmr_spectroscopy MRI- Magnetic Resonance Imaging (water) In-vivo spectroscopy (metabolites) Solid-state t NMR (large structures) t Solution NMR
More informationSupplementary Figure 1. Stability constants of metal monohydroxides. The log K values are summarized according to the atomic number of each element
Supplementary Figure 1. Stability constants of metal monohydroxides. The log K values are summarized according to the atomic number of each element as determined in a previous study 1. The log K value
More informationSUPPLEMENTARY INFORMATION
doi:10.1038/nature10458 Active Site Remodeling in the Bifunctional Fructose-1,6- bisphosphate aldolase/phosphatase Juan Du, Rafael F. Say, Wei Lü, Georg Fuchs & Oliver Einsle SUPPLEMENTARY FIGURES Figure
More informationTable 1. Kinetic data obtained from SPR analysis of domain 11 mutants interacting with IGF-II. Kinetic parameters K D 1.
Kinetics and Thermodynamics of the Insulin-like Growth Factor II (IGF-II) Interaction with IGF-II/Mannose 6-phosphate Receptor and the function of CD and AB Loop Solvent-exposed Residues. Research Team:
More informationThe Fic protein Doc uses an inverted substrate to phosphorylate and. inactivate EF-Tu
The Fic protein Doc uses an inverted substrate to phosphorylate and inactivate EF-Tu Daniel Castro-Roa 1, Abel Garcia-Pino 2,3 *, Steven De Gieter 2,3, Nico A.J. van Nuland 2,3, Remy Loris 2,3, Nikolay
More informationBiology Chemistry & Physics of Biomolecules. Examination #1. Proteins Module. September 29, Answer Key
Biology 5357 Chemistry & Physics of Biomolecules Examination #1 Proteins Module September 29, 2017 Answer Key Question 1 (A) (5 points) Structure (b) is more common, as it contains the shorter connection
More informationTable S1. Primers used for the constructions of recombinant GAL1 and λ5 mutants. GAL1-E74A ccgagcagcgggcggctgtctttcc ggaaagacagccgcccgctgctcgg
SUPPLEMENTAL DATA Table S1. Primers used for the constructions of recombinant GAL1 and λ5 mutants Sense primer (5 to 3 ) Anti-sense primer (5 to 3 ) GAL1 mutants GAL1-E74A ccgagcagcgggcggctgtctttcc ggaaagacagccgcccgctgctcgg
More informationSupporting Information
Supporting Information Smith et al. 10.1073/pnas.1519609113 SI Methods Sample Preparation. Perdeuterated, 15 N-labeled WT and mutant ubiquitin was expressed in Escherichia coli adapted to 100% D 2 O Toronto
More informationSupplementary Materials for
advances.sciencemag.org/cgi/content/full/4/1/eaau413/dc1 Supplementary Materials for Structure and dynamics conspire in the evolution of affinity between intrinsically disordered proteins Per Jemth*, Elin
More informationSupplementary Figure 1. SDS-PAGE analysis of GFP oligomer variants with different linkers. Oligomer mixtures were applied to a PAGE gel containing
Supplementary Figure 1. SDS-PAGE analysis of GFP oligomer variants with different linkers. Oligomer mixtures were applied to a PAGE gel containing 0.1% SDS without boiling. The gel was analyzed by a fluorescent
More informationEffects of Chemical Exchange on NMR Spectra
Effects of Chemical Exchange on NMR Spectra Chemical exchange refers to any process in which a nucleus exchanges between two or more environments in which its NMR parameters (e.g. chemical shift, scalar
More informationTHE TANGO ALGORITHM: SECONDARY STRUCTURE PROPENSITIES, STATISTICAL MECHANICS APPROXIMATION
THE TANGO ALGORITHM: SECONDARY STRUCTURE PROPENSITIES, STATISTICAL MECHANICS APPROXIMATION AND CALIBRATION Calculation of turn and beta intrinsic propensities. A statistical analysis of a protein structure
More informationSupplementary Materials for
advances.sciencemag.org/cgi/content/full/3/4/e1600663/dc1 Supplementary Materials for A dynamic hydrophobic core orchestrates allostery in protein kinases Jonggul Kim, Lalima G. Ahuja, Fa-An Chao, Youlin
More informationMillisecond Time-scale Protein Dynamics by Relaxation Dispersion NMR. Dmitry M. Korzhnev
Millisecond Time-scale Protein Dynamics by Relaxation Dispersion NMR Dmitry M. Korzhnev Department of Molecular, Microbial and Structural Biology University of Connecticut Health Center 263 Farmington
More informationSupplementary Information
Supplementary Information Adenosyltransferase Tailors and Delivers Coenzyme B 12 Dominique Padovani 1,2, Tetyana Labunska 2, Bruce A. Palfey 1, David P. Ballou 1 and Ruma Banerjee 1,2 * 1 Biological Chemistry
More informationSUPPLEMENTARY FIGURES
SUPPLEMENTARY FIGURES Supplementary Figure 1 Protein sequence alignment of Vibrionaceae with either a 40-residue insertion or a 44-residue insertion. Identical residues are indicated by red background.
More informationBiological Thermodynamics
Biological Thermodynamics Classical thermodynamics is the only physical theory of universal content concerning which I am convinced that, within the framework of applicability of its basic contents, will
More informationSUPPLEMENTARY INFORMATION
5 N 4 8 20 22 24 2 28 4 8 20 22 24 2 28 a b 0 9 8 7 H c (kda) 95 0 57 4 28 2 5.5 Precipitate before NMR expt. Supernatant before NMR expt. Precipitate after hrs NMR expt. Supernatant after hrs NMR expt.
More informationSupporting Information
Supporting Information German Edition: DOI: Sampling of Glycan-Bound Conformers by the Anti-HIV Lectin Oscillatoria agardhii agglutinin in the Absence of Sugar** Marta G. Carneiro, Leonardus M. I. Koharudin,
More informationSupplementary Materials: Probing the Ion Binding Site in a DNA Holliday Junction Using Förster Resonance Energy Transfer (FRET)
S1 of S5 Supplementary Materials: Probing the Ion Binding Site in a DNA Holliday Junction Using Förster Resonance Energy Transfer (FRET) Jacob L. Litke, Yan Li, Laura M. Nocka and Ishita Mukerji (a) (b)
More informationEffects of Chemical Exchange on NMR Spectra
Effects of Chemical Exchange on NMR Spectra Chemical exchange refers to any process in which a nucleus exchanges between two or more environments in which its NMR parameters (e.g. chemical shift, scalar
More informationNMR study of complexes between low molecular mass inhibitors and the West Nile virus NS2B-NS3 protease
University of Wollongong Research Online Faculty of Science - Papers (Archive) Faculty of Science, Medicine and Health 2009 NMR study of complexes between low molecular mass inhibitors and the West Nile
More informationNature Structural and Molecular Biology: doi: /nsmb Supplementary Figure 1. Definition and assessment of ciap1 constructs.
Supplementary Figure 1 Definition and assessment of ciap1 constructs. (a) ciap1 constructs used in this study are shown as primary structure schematics with domains colored as in the main text. Mutations
More informationLABORATORY OF ELEMENTARY BIOPHYSICS. Isothermal Titration Calorimetry as a tool for determining thermodynamic parameters of chemical reactions
LABORATORY OF ELEMENTARY BIOPHYSICS Experimental exercises for III year of the First cycle studies Field: Applications of physics in biology and medicine Specialization: Molecular Biophysics Isothermal
More informationTimescales of Protein Dynamics
Timescales of Protein Dynamics From Henzler-Wildman and Kern, Nature 2007 Dynamics from NMR Show spies Amide Nitrogen Spies Report On Conformational Dynamics Amide Hydrogen Transverse Relaxation Ensemble
More informationThe Diffusion Coefficient for PGK Folding in Eukaryotic Cells
Biophysical Journal, Volume 99 Supporting Material The Diffusion Coefficient for PGK Folding in Eukaryotic Cells Apratim Dhar, Simon Ebbinghaus, Zhen Shen, Tripta Mishra, and Martin Gruebele 1 Supplementary
More informationBMB/Bi/Ch 173 Winter 2018
BMB/Bi/Ch 173 Winter 2018 Homework Set 8.1 (100 Points) Assigned 2-27-18, due 3-6-18 by 10:30 a.m. TA: Rachael Kuintzle. Office hours: SFL 220, Friday 3/2 4:00-5:00pm and SFL 229, Monday 3/5 4:00-5:30pm.
More informationTimescales of Protein Dynamics
Timescales of Protein Dynamics From Henzler-Wildman and Kern, Nature 2007 Summary of 1D Experiment time domain data Fourier Transform (FT) frequency domain data or Transverse Relaxation Ensemble of Nuclear
More informationBiophysical Journal, Volume 96. Supporting Material
Biophysical Journal, Volume 96 Supporting Material NMR dynamics of PSE-4 β-lactamase: an interplay of ps-ns order and μs-ms motions in the active site Sébastien Morin and Stéphane M. Gagné NMR dynamics
More informationTITAN: Two-dimensional lineshape analysis
TITAN: Two-dimensional lineshape analysis Chris Waudby Christodoulou Group c.waudby@ucl.ac.uk Andres Ramos Lisa Cabrita John Christodoulou Inhibition of fatty acid synthesis for treatment of tularemia
More informationSupplementary material
Supplementary material Phosphorylation of the mitochondrial autophagy receptor Nix enhances its interaction with LC3 proteins Vladimir V. Rogov 1,*, Hironori Suzuki 2,3,*, Mija Marinković 4, Verena Lang
More informationSUPPLEMENTARY MATERIAL FOR
SUPPLEMENTARY MATERIAL FOR THE LIPID-BINDING DOMAIN OF WILD TYPE AND MUTANT ALPHA- SYNUCLEIN: COMPACTNESS AND INTERCONVERSION BETWEEN THE BROKEN- AND EXTENDED-HELIX FORMS. Elka R. Georgieva 1, Trudy F.
More informationSupplemental data for
Supplemental data for A Real-Time Guanine Nucleotide Exchange Assay using NMR: Activation of RhoA by PDZ- RhoGEF. Geneviève M.C. Gasmi-Seabrook 1,3, Christopher B. Marshall 1,3, Melissa Cheung 1,3, Bryan
More informationTable S1. Overview of used PDZK1 constructs and their binding affinities to peptides. Related to figure 1.
Table S1. Overview of used PDZK1 constructs and their binding affinities to peptides. Related to figure 1. PDZK1 constru cts Amino acids MW [kda] KD [μm] PEPT2-CT- FITC KD [μm] NHE3-CT- FITC KD [μm] PDZK1-CT-
More informationSupporting Protocol This protocol describes the construction and the force-field parameters of the non-standard residue for the Ag + -site using CNS
Supporting Protocol This protocol describes the construction and the force-field parameters of the non-standard residue for the Ag + -site using CNS CNS input file generatemetal.inp: remarks file generate/generatemetal.inp
More informationMicrocalorimetry for the Life Sciences
Microcalorimetry for the Life Sciences Why Microcalorimetry? Microcalorimetry is universal detector Heat is generated or absorbed in every chemical process In-solution No molecular weight limitations Label-free
More informationStructural basis for catalytically restrictive dynamics of a high-energy enzyme state
Supplementary Material Structural basis for catalytically restrictive dynamics of a high-energy enzyme state Michael Kovermann, Jörgen Ådén, Christin Grundström, A. Elisabeth Sauer-Eriksson, Uwe H. Sauer
More informationSUPPLEMENTARY INFORMATION
Parallel Allostery by camp and PDE Coordinates Activation and Termination Phases in camp Signaling Srinath Krishnamurthy, 1 Nikhil Kumar Tulsian, 1 Arun Chandramohan, 1 and Ganesh S. Anand 1, * 1 Department
More informationSUPPLEMENTARY ONLINE DATA
SUPPLEMENTARY ONLINE DATA Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interaction with Different Types of Acetylcholine Receptors E.N. Lyukmanova 1,2,*, M.A. Shulepko
More informationMCB100A/Chem130 MidTerm Exam 2 April 4, 2013
MCBA/Chem Miderm Exam 2 April 4, 2 Name Student ID rue/false (2 points each).. he Boltzmann constant, k b sets the energy scale for observing energy microstates 2. Atoms with favorable electronic configurations
More informationStructural characterization of NiV N 0 P in solution and in crystal.
Supplementary Figure 1 Structural characterization of NiV N 0 P in solution and in crystal. (a) SAXS analysis of the N 32-383 0 -P 50 complex. The Guinier plot for complex concentrations of 0.55, 1.1,
More informationBiochemistry 530 NMR Theory and Practice. Gabriele Varani Department of Biochemistry and Department of Chemistry University of Washington
Biochemistry 530 NMR Theory and Practice Gabriele Varani Department of Biochemistry and Department of Chemistry University of Washington 1D spectra contain structural information.. but is hard to extract:
More informationSimulative and experimental characterization of a ph-dependent
Simulative and experimental characterization of a ph-dependent clamp-like DNA triple-helix nanoswitch Federico Iacovelli, # Andrea Idili, # Alessandro Benincasa, Davide Mariottini, Alessio Ottaviani, Mattia
More informationLS1a Fall 2014 Problem Set #2 Due Monday 10/6 at 6 pm in the drop boxes on the Science Center 2 nd Floor
LS1a Fall 2014 Problem Set #2 Due Monday 10/6 at 6 pm in the drop boxes on the Science Center 2 nd Floor Note: Adequate space is given for each answer. Questions that require a brief explanation should
More informationSUPPLEMENTARY INFORMATION
SUPPLEMENTARY INFORMATION doi:10.1038/nature11524 Supplementary discussion Functional analysis of the sugar porter family (SP) signature motifs. As seen in Fig. 5c, single point mutation of the conserved
More informationChemical Exchange and Ligand Binding
Chemical Exchange and Ligand Binding NMR time scale Fast exchange for binding constants Slow exchange for tight binding Single vs. multiple binding mode Calcium binding process of calcium binding proteins
More informationPresentation Microcalorimetry for Life Science Research
Presentation Microcalorimetry for Life Science Research MicroCalorimetry The Universal Detector Heat is either generated or absorbed in every chemical process Capable of thermal measurements over a wide
More informationChapter 3. Dynamics in a high-affinity peptide-sh2 domain complex
Dynamics in a high-affinity peptide-sh2 domain complex 47 Abstract The interaction between the tyrosine kinases Src and focal adhesion kinase (FAK) is a key step in signalling processes from focal adhesions.
More informationSlow symmetric exchange
Slow symmetric exchange ϕ A k k B t A B There are three things you should notice compared with the Figure on the previous slide: 1) The lines are broader, 2) the intensities are reduced and 3) the peaks
More informationUsing NMR to study Macromolecular Interactions. John Gross, BP204A UCSF. Nov 27, 2017
Using NMR to study Macromolecular Interactions John Gross, BP204A UCSF Nov 27, 2017 Outline Review of basic NMR experiment Multidimensional NMR Monitoring ligand binding Structure Determination Review:
More informationSupplemental Information for. Quaternary dynamics of B crystallin as a direct consequence of localised tertiary fluctuations in the C terminus
Supplemental Information for Quaternary dynamics of B crystallin as a direct consequence of localised tertiary fluctuations in the C terminus Andrew J. Baldwin 1, Gillian R. Hilton 2, Hadi Lioe 2, Claire
More informationProteins are not rigid structures: Protein dynamics, conformational variability, and thermodynamic stability
Proteins are not rigid structures: Protein dynamics, conformational variability, and thermodynamic stability Dr. Andrew Lee UNC School of Pharmacy (Div. Chemical Biology and Medicinal Chemistry) UNC Med
More informationA Single Outer Sphere Mutation Stabilizes apo- Mn Superoxide Dismutase by 35 C and. Disfavors Mn Binding.
Supporting information for A Single Outer Sphere Mutation Stabilizes apo- Mn Superoxide Dismutase by 35 C and Disfavors Mn Binding. Anne-Frances Miller* and Ting Wang Department of Chemistry, University
More informationStudying Invisible Excited Protein States in Slow Exchange with a Major State Conformation
pubs.acs.org/jacs Studying Invisible Excited Protein States in Slow Exchange with a Major State Conformation Pramodh Vallurupalli,*,, Guillaume Bouvignies,, and Lewis E. Kay*,, Departments of Molecular
More informationIntroduction solution NMR
2 NMR journey Introduction solution NMR Alexandre Bonvin Bijvoet Center for Biomolecular Research with thanks to Dr. Klaartje Houben EMBO Global Exchange course, IHEP, Beijing April 28 - May 5, 20 3 Topics
More informationI690/B680 Structural Bioinformatics Spring Protein Structure Determination by NMR Spectroscopy
I690/B680 Structural Bioinformatics Spring 2006 Protein Structure Determination by NMR Spectroscopy Suggested Reading (1) Van Holde, Johnson, Ho. Principles of Physical Biochemistry, 2 nd Ed., Prentice
More informationActa Crystallographica Section D
Supporting information Acta Crystallographica Section D Volume 70 (2014) Supporting information for article: Structural basis of the heterodimerization of the MST and RASSF SARAH domains in the Hippo signalling
More informationMCB100A/Chem130 MidTerm Exam 2 April 4, 2013
MCB1A/Chem13 MidTerm Exam 2 April 4, 213 Name Student ID True/False (2 points each). 1. The Boltzmann constant, k b T sets the energy scale for observing energy microstates 2. Atoms with favorable electronic
More informationPROTEIN NMR SPECTROSCOPY
List of Figures List of Tables xvii xxvi 1. NMR SPECTROSCOPY 1 1.1 Introduction to NMR Spectroscopy 2 1.2 One Dimensional NMR Spectroscopy 3 1.2.1 Classical Description of NMR Spectroscopy 3 1.2.2 Nuclear
More informationSupplementary Figure 1.
a b c d e f g 1 Supplementary Figure 1. Identification of unfolded regions in the Chz1-H2A.Z-H2B complex and structure and dynamics of Chz.core-sH2B_H2A.Z. (a) 1 H- 15 N HSQC spectrum of Chz1. All backbone
More informationSupplementary figure 1 Application of tmfret in LeuT. (a) To assess the feasibility of using tmfret for distance-dependent measurements in LeuT, a
Supplementary figure 1 Application of tmfret in LeuT. (a) To assess the feasibility of using tmfret for distance-dependent measurements in LeuT, a series of tmfret-pairs comprised of single cysteine mutants
More informationMore NMR Relaxation. Longitudinal Relaxation. Transverse Relaxation
More NMR Relaxation Longitudinal Relaxation Transverse Relaxation Copyright Peter F. Flynn 2017 Experimental Determination of T1 Gated Inversion Recovery Experiment The gated inversion recovery pulse sequence
More informationSUPPLEMENTARY INFORMATION
doi:10.1038/nature20798 Supplementary Tables Table S1: Kinetic parameters for porphyrin torsional rotation in c-p6 6+ and c-p6 12+ from 1 H EXSY. Table S2: Calculated (NICS, ppm) and experimental ( δ,
More informationNB-DNJ/GCase-pH 7.4 NB-DNJ+/GCase-pH 7.4 NB-DNJ+/GCase-pH 4.5
SUPPLEMENTARY TABLES Suppl. Table 1. Protonation states at ph 7.4 and 4.5. Protonation states of titratable residues in GCase at ph 7.4 and 4.5. Histidine: HID, H at δ-nitrogen; HIE, H at ε-nitrogen; HIP,
More informationA rule of seven in Watson-Crick base-pairing of mismatched sequences
A rule of seven in Watson-Crick base-pairing of mismatched sequences Ibrahim I. Cisse 1,3, Hajin Kim 1,2, Taekjip Ha 1,2 1 Department of Physics and Center for the Physics of Living Cells, University of
More informationElectronic Supplementary Information for
Electronic Supplementary Material (ESI) for Metallomics. This journal is The Royal Society of Chemistry 2015 Electronic Supplementary Information for Metal ion mediated transition from random coil to β-sheet
More informationSupplementary Materials for
advances.sciencemag.org/cgi/content/full/1/9/e1500511/dc1 Supplementary Materials for Contractility parameters of human -cardiac myosin with the hypertrophic cardiomyopathy mutation R403Q show loss of
More informationPresenter: She Zhang
Presenter: She Zhang Introduction Dr. David Baker Introduction Why design proteins de novo? It is not clear how non-covalent interactions favor one specific native structure over many other non-native
More informationLigand Controlled Assembly of Hexamers, Dihexamers, and Linear Multihexamer Structures by the Engineered, Acylated, Insulin Degludec
Supplementary material Ligand Controlled Assembly of Hexamers, Dihexamers, and Linear Multihexamer Structures by the Engineered, Acylated, Insulin Degludec D. B. Steensgaard*, G. Schluckebier, Holger M.
More information1. Use the Data for RNAse to estimate:
Chem 78 - - Spr 1 03/14/01 Assignment 4 - Answers Thermodynamic Analysis of RNAseA Denaturation by UV- Vis Difference Absorption Spectroscopy (and Differential Scanning Calorimetry). The accompanying excel
More informationCholera Toxin Invasion
Protein-carbohydrate interactions: Isothermal Titration Calorimetry Dr Bruce Turnbull School of Chemistry and Astbury Centre for Structural Molecular Biology University of Leeds Cholera Toxin Invasion
More informationMolecular Modeling lecture 2
Molecular Modeling 2018 -- lecture 2 Topics 1. Secondary structure 3. Sequence similarity and homology 2. Secondary structure prediction 4. Where do protein structures come from? X-ray crystallography
More informationLongitudinal-relaxation enhanced fast-pulsing techniques: New tools for biomolecular NMR spectroscopy
Longitudinal-relaxation enhanced fast-pulsing techniques: New tools for biomolecular NMR spectroscopy Bernhard Brutscher Laboratoire de Résonance Magnétique Nucléaire Institut de Biologie Structurale -
More information- Basic understandings: - Mapping interactions:
NMR-lecture April 6th, 2009, FMP Berlin Outline: Christian Freund - Basic understandings: Relaxation Chemical exchange - Mapping interactions: -Chemical shift mapping (fast exchange) Linewidth analysis
More informationS2004 Methods for characterization of biomolecular interactions - classical versus modern
S2004 Methods for characterization of biomolecular interactions - classical versus modern Isothermal Titration Calorimetry (ITC) Eva Dubská email: eva.dubska@ceitec.cz Outline Calorimetry - history + a
More informationSedimentation Velocity Analysis of Interacting Systems using c(s) Peter Schuck
Sedimentation Velocity Analysis of Interacting Systems using c(s) Peter Schuck simulated sedimentation of rapidly self-association systems monomer/dimer monomer/trimer 5 concentration (Kd) 4 2 concentration
More informationISoTherMal TITraTIon Calorimetry
ISoTherMal TITraTIon Calorimetry With the Nano ITC, heat effects as small as 1 nanojoules are detectable using one nanomole or less of biopolymer. The Nano ITC uses a solid-state thermoelectric heating
More information1. 3-hour Open book exam. No discussion among yourselves.
Lecture 13 Review 1. 3-hour Open book exam. No discussion among yourselves. 2. Simple calculations. 3. Terminologies. 4. Decriptive questions. 5. Analyze a pulse program using density matrix approach (omonuclear
More informationInterpreting and evaluating biological NMR in the literature. Worksheet 1
Interpreting and evaluating biological NMR in the literature Worksheet 1 1D NMR spectra Application of RF pulses of specified lengths and frequencies can make certain nuclei detectable We can selectively
More informationBiophysical Chemistry: NMR Spectroscopy
Relaxation & Multidimensional Spectrocopy Vrije Universiteit Brussel 9th December 2011 Outline 1 Relaxation 2 Principles 3 Outline 1 Relaxation 2 Principles 3 Establishment of Thermal Equilibrium As previously
More informationElectronic Supporting Information
This journal is The Royal Society of Chemistry 211 Electronic Supporting Information to: Unspecific ligand binding yielding stable colloidal ITO-nanoparticle dispersions by C. Grote, K. J. Chiad, D. Vollmer,
More informationAleksandr V. Mikhonin, Sanford A. Asher,* Sergei V. Bykov, and Adrian Murza
3280 J. Phys. Chem. B 2007, 111, 3280-3292 UV Raman Spatially Resolved Melting Dynamics of Isotopically Labeled Polyalanyl Peptide: Slow r-helix Melting Follows 3 10 -Helices and π-bulges Premelting Aleksandr
More information1 What is energy?
http://www.intothecool.com/ 1 What is energy? the capacity to do work? (Greek: en-, in; + ergon, work) the capacity to cause change to produce an effect? a certain quantity that does not change in the
More informationExp.3 Determination of the Thermodynamic functions for the Borax Solution
Exp.3 Determination of the Thermodynamic functions for the Borax Solution Theory: The relationship between Gibb s energy (ΔG), Enthalpy (ΔH), Entropy (ΔS) and the equilibrium constant (K) for a chemical
More informationBiochemistry 530 NMR Theory and Practice
Biochemistry 530 NMR Theory and Practice Gabriele Varani Department of Biochemistry and Department of Chemistry University of Washington 1D spectra contain structural information.. but is hard to extract:
More informationHSQC spectra for three proteins
HSQC spectra for three proteins SH3 domain from Abp1p Kinase domain from EphB2 apo Calmodulin What do the spectra tell you about the three proteins? HSQC spectra for three proteins Small protein Big protein
More information