Volume 8, Issue 12, Pages 1279-1287 (December 2000) Structural Changes Induced in p21Ras upon GAP-334 Complexation as Probed by ESEEM Spectroscopy and Molecular-Dynamics Simulation  Christian T. Farrar, Jianpeng Ma, David J. Singel, Christopher J. Halkides  Structure  Volume 8, Issue 12, Pages 1279-1287 (December 2000) DOI: 10.1016/S0969-2126(00)00532-3

Figure 1 Two-Pulse Modulus Fourier Transform ESEEM Spectrum of the p21·GAP-334 Complex with [15N]Gly, [13Cγ]-Asx, and [2Hβ]-Thr Isotope-Labeled p21·Mn2+GMPPNP The peaks that are centered at 1.37, 2.74, 3.71, and 15.43 MHz are, respectively, the 15N, 2H, 13C, and 1H Zeeman frequencies. The peaks evident at 4.49 MHz and 8.07 MHz are the low- and high-frequency 31P fundamental lines. The antiphase peak centered at 12.44 MHz is the 31P sum combination line. The spectrum was acquired with an excitation frequency of 10.443 GHz and a magnetic field strength of 3624 Gauss. The experimental dead time was 200 ns, and the temperature was 4.4 K Structure 2000 8, 1279-1287DOI: (10.1016/S0969-2126(00)00532-3)

Figure 2 Two-Pulse Modulus Fourier Transform ESEEM Spectrum of [15N]Gly, [13Cγ]Asx, and [2Hβ]Thr Isotope-Labeled p21·Mn2+GMPPNP in the Presence of TIM The peaks that are centered at 2.28, 3.58, and 14.65 MHz are, respectively, the 2H, 13C, and 1H Zeeman frequencies. The peaks evident at 4.30 MHz and 7.62 MHz are the low- and high-frequency 31P fundamental lines. The antiphase peak that is centered at 11.79 MHz is the 31P sum combination line. No 15N signal, which would give rise to a peak centered at 1.48 MHz, is observed. The spectrum was acquired with an excitation frequency of 9.890 GHz and a magnetic field strength of 3441 Gauss. The experimental dead time was 200 ns, and the temperature was 4.4 K Structure 2000 8, 1279-1287DOI: (10.1016/S0969-2126(00)00532-3)

Figure 3 Detailed Stereoview of the p21 Active Site Structure Including Residues Gly-60, Thr-35, Ser-17, Gly-13, the Attacking Water Molecule, the Nucleotide, and the Metal Ion for the ESEEM-Constrained MD Simulation of p21·GAP-334 For clarity Gln-61 of p21 and Arg-789 of GAP-334 are omitted Structure 2000 8, 1279-1287DOI: (10.1016/S0969-2126(00)00532-3)

Figure 4 Overlay of the Molecular-Dynamics Active Site Structures of p21·GAP Before and After the Incorporation of the ESEEM Distance Constraint Red indicates the structure before the incorporation of the ESEEM distance contraint, while green indicates the structure after this incorporation. The active site residues Gly-60, Thr-35, Ser-17, Gly-13, the phosphates of the GTP, and the metal ion and its coordinating water molecules are shown Structure 2000 8, 1279-1287DOI: (10.1016/S0969-2126(00)00532-3)

Figure 5 Two-Dimensional Plot of the Active Site Structure from the ESEEM-Constrained MD Simulation Shows the Hydrogen Bonding Interactions and Distances for Gln-61, Gly-60, Thr-35, Ser-17, Gly-13, and the Attacking Water Molecule The hydrogen bond distances indicated are those between the proton and the heavy-atom acceptor Structure 2000 8, 1279-1287DOI: (10.1016/S0969-2126(00)00532-3)

Figure 6 Two-pulse Modulus and Cosine Fourier Transform ESEEM Spectra of the p21·GAP-334 Complex with [15Nϵ]Lys and [13Cβ]Thr Isotope-labeled p21·Mn2+GMPPNP (A) Two pulse modulus Fourier transform ESEEM spectrum. (B) Cosine Fourier transform ESEEM spectrum. The peak centered at 14.04 MHz is the 1H Zeeman fundamental frequency. The peaks evident at 4.04 MHz and 7.94 MHz are the low- and high-frequency 31P fundamental lines. The peak at 12.44 MHz is the 31P sum combination line. The shoulder observed in (A) on the edge of the low-frequency 31P line at approximately 3.71 MHz is the 13C fundamental peak. The antiphase peak that is centered at 7.29 MHz is the 13C sum combination peak. The spectrum was acquired with an excitation frequency of 9.956 GHz and a magnetic field strength of 3440 Gauss. The experimental dead time was 200 ns, and the temperature was 4.4 K Structure 2000 8, 1279-1287DOI: (10.1016/S0969-2126(00)00532-3)