The CypD-CL1 complex system is a very interesting system for structural investigations of the CypD and its catalytic activity. CL1 enhances or induces large scale motions in CypD that are believed to represent the catalytic cycle. These motions and conformational changes could now be characterized using relaxation dispersion experiments or room temperature crystallography in
discussion
order to allow the population of both conformations. One of the drawbacks of the system is the motion in the intermediate exchange regime in the NMR experiments, leading to line broadening and bad signal to noise. Here mutations that influence the dynamics would be very helpful in order to move the dynamics to another timescale to improve the NMR conditions. Fraser et al describe that the S99T mutant in CypA slows down the protein dynamics (Fraser 2009). Due to the high ho-mology between CypA and CypD this mutation could serve the same purpose in CypD. In addition the experiments could be expanded to a more detailed ligand observed analysis during catalysis, identifying the structural features of the ligand that are involved in the process. This information could be very valuable for a further ligand design, because it could be exploited to trap certain states of the protein or to induce a certain conformation.
appendix
Table 7: Assignment table for CypD apo All assignments were done manually. ResidueNHCAHA*CBHB*CGHG*CDHD*CEHE*CZHZ* SER1117.98.5158.24.52643.9 GLY2111.18.5544.83.97 ASN3122.18.3851.237.9 PRO463.24.7332.30/1.7427.12.15/2.1450.94.15 LEU5121.48.5453.25.5544.81.7226.81.91 VAL6115.29.1558.35.4235.42.671.14/0.93 TYR7116.68.72556.1942.82.80/2.566.586.43 LEU8116.39.0153.84.7645.126.2 ASP91269.1954.55.6641.92.59 VAL10118.98.1460.25.5635.31.75 ASP11127.39.5452.95.2245.32.31 ALA12118.68.6450.25.0821.60.78 ASN13125.99.3453.74.4637.43.39/2.62 GLY141048.8345.74.12/3.71 LYS15122.27.7352.533.2 PRO1664.14.632.42.36/1.9027.62.1251.14.01/3.71 LEU17122.79.854.84.5743.61.7125.91.410.96/0.77 GLY18104.97.6944.14.65/3.73 ARG19121.48.3254.75.5634.31.627.443 VAL20126.69.55614.6134.42.07 VAL21126.88.9261.74.9933.51.921.00/0.88 LEU22128.99.6453.54.9543.10.961.59 GLU23122.68.6454.84.9731.71.736.92.08/1.76 LEU24124.68.5152.14.8842.92.02/1.3126.91.25 LYS25125.28.953.74.5327.71.66/1.5823.41.36/1.251.941.62.92/2.83 ALA26128.68.1954.13.7318.81.48 ASP27114.19.0455.14.2438.42.84/2.74
6 . Appendix
appendix
ResidueNHCAHA*CBHB*CGHG*CDHD*CEHE*CZHZ* VAL28119.97.6263.94.235.41.931.12/0.99 VAL29113.78.2757.631.7 PRO3066.14.3931.42.65/1.9927.52.150.93.61/3.07 LYS31122.610.7960.24.0631.61.71/1.43261.39/1.2129.21.49/1.3941.92.67/2.60 THR32124.710.3567.53.9868.74.160.85 ALA33125.69.27564.1218.61.46 GLU34117.58.04584.528.22.27/1.7333.72.49/2.02 ASN351157.1156.64.0239.62.87/2.33 PHE36117.37.0661.94.0540.33.21/3.147.176.99128.95.9 ARG37119.28.960.63.6230.31.8424.541.3 ALA38118.18.4454.118.11.22 LEU39122.27.9557.23.8140.926.60.910.56/0.91 CYS40120.17.9163.64.44273.31/2.15 THR41107.18.0363.44.1769.24.471.32 GLY42108.27.845.73.84/3.55 GLU43118.58.0158.54.1430.12.22/2.0435.32.10/1.80 LYS44118.79.1554.34.3230.31.66/0.9324.91.08/1.0327.51.4642.22.85/2.68 GLY45105.67.9744.64.34/3.58 PHE46113.86.4154.24.6539.53.15/2.706.647 GLY47104.77.7245.84.28/2.76 TYR48113.26.8657.74.3738.73.11/3.06 LYS49124.58.46613.7531.62.0425.41.36301.98/1.8342.23.31/3.11 GLY50117.99.7445.54.47/3.65 SER51116.48.5858.564.8 THR52107.19.6161.45.8973.54.341.24 PHE53120.88.4657.44.938.96.96.97128.77.43 HIS54119.87.4356.94.7531.83.27/2.786.82 ARG551246.854.84.9632.51.0528.243.43.17/2.99
appendix
ResidueNHCAHA*CBHB*CGHG*CDHD*CEHE*CZHZ* VAL56128.89.3861.94.535.21.821.10/0.76 ILE57127.78.6257.440.8 PRO5862.832.327.152.3 SER591159.69593.8761.64.15/4.03 PHE60114.77.5957.25.0538.33.27/2.997.227.21 MET61112.18.1554.85.1935.228.41.03/0.78 CYS62115.48.4756.84.8430.23.15/2.73 GLN63127.89.4654.930.833.62.57/2.05 ALA64126.28.0150.222.4 GLY65103.98.447.14.04/2.83 ASP66124.510.0951.538.8 PHE67115.76.6356.139 THR68109.87.35624.5568.74.040.74 ASN69120.58.6652.839.4 HIS7057.428.56.77 ASN71112.67.6252.638.9 GLY72110.89.7345.24.66/3.39 THR73112.58.0262.64.4670.94.271.09 GLY74113.38.6245.54.50/3.57 GLY75109.48.143.2 LYS76115.77.03564.6334.61.89/1.5623.40.8729.41.1841.22.15 SER77114.67.8156.85.2469.64.27/4.26 ILE78111.78.6563.74.2337.31.710.720.47 TYR79120.78.1156.24.7438.93.44/2.366.446.13 GLY80106.77.1744 SER8162.8 ARG82115.77.9253.75.5834.31.5526.91.54/1.4743.53.18/2.90 PHE831189.175539.57.327.58
appendix
ResidueNHCAHA*CBHB*CGHG*CDHD*CEHE*CZHZ* PRO8462.6432.92.25/1.6927.41.98/1.9252 ASP85119.88.854.840.9 GLU86132.19.3760.33.831.52.23/1.80352.40/2.32 ASN871077.0552.94.1239.63.38/2.74 PHE88113.28.34566.0138.43.57/2.56 THR89118.28.5766.83.7468.91.18 LEU901198.1654.54.4640.81.5127.31.810.83/0.91 LYS91118.68.3254.24.6934.32.22/1.6225.51.49/1.4228.21.64/1.5742.73.05 HIS92123.510.9156.925.93.10/2.866.6 VAL93113.97.0563.44.0132.82.360.99/0.84 GLY94105.77.3245.7 PRO9562.53227.549.8 GLY96109.99.0444.94.49/3.26 VAL97119.76.6664.53.6333.72.041.39/0.97 LEU98129.97.3853.14.8344.225.7-0.04-1.5625 SER99121.18.3155.45.2365 MET100123.48.5753.731.231.9 ALA101126.58.2551.919.91.39 ASN102113.88.4754.34.640.4 ALA103123.38.8750.34.8119.21.32 GLY104109.38.243.5 PRO105644.3832.12.33/1.8227.82.14/1.9849.33.64/3.52 ASN106118.88.9754.54373.11/2.66 THR107110.810.460.34.4168.94.420.9 ASN108120.27.1355.74.2539.71.54/0.98 GLY1091119.21464.61/3.65 SER110117.38.9257.74.7766.2 GLN111124.98.5257.85.332.135.71.87
appendix
ResidueNHCAHA*CBHB*CGHG*CDHD*CEHE*CZHZ* PHE1121198.8155.35.9143.13.36/3.266.837.81128.86.81 PHE113115.29.7454.95.7343.92.746.756.84 ILE114117.58.7959.25.0540.81.60.760.97 CYS115125.69.1460.429.23.71/2.98 THR116113.78.8961.14.4267.50.82 ILE117118.37.6459.84.3844.11.810.961.13 LYS1181218.3357.433.1224.61.6629.91.77423.04/3.01 THR119121.47.8657.13.55681.06 ASP120121.78.4356.44.239.72.90/2.70 TRP121118.57.459.94.6926.93.40/3.357.057.87.41/6.80 LEU122120.17.1554.34.338.91.3138.90.270.44/0.85 ASP123122.97.5955.95.2439.32.75 GLY124111.69.54453.92/2.87 LYS125115.77.956.44.1535.31.8425.21.27/1.2329.31.6942.43.02 HIS126119.37.5654.93.9731.83.246.91 VAL127124.78.663.84.2133.321.00/1.14 VAL128133.69.762.94.130.91.680.80/-0.00 PHE129117.78.2755.25.13423.10/2.516.627.52131.66.08 GLY130107.47.3146.94.11/4.04 HIS131119.18.5155.55.1234.73.12/2.826.95 VAL132122.88.7264.23.9432.41.960.95/0.74 LYS133132.29.6556.64.5135.11.71/1.67251.58/1.52291.78/1.67423.03 GLU134115.77.9555.14.7733.62.13/1.78362.29/2.18 GLY1351098.946.34.94/4.17 MET136122.69.0656.74.5329.82.0533.12.77/2.64 ASP137116.89.1956.84.2338.92.75/2.59 VAL138124.27.4866.13.3930.92.41.03/0.56 VAL139120.97.5866.93.330.92.561.05/0.75
appendix
ResidueNHCAHA*CBHB*CGHG*CDHD*CEHE*CZHZ* LYS140117.38.19584.1530.21.6624.31.67/1.5726.91.69/1.6041.32.96/2.88 LYS1411217.5659.53.9632.72.14/1.9025.51.75/1.4930.11.6742.32.92 ILE142121.18.16673.33382.050.470.06 GLU143117.78.2758.73.5130.61.7636.72.14 SER144113.57.3560.84.1562.93.71 PHE145119.87.7357.5539.23.57/2.997.27 GLY146105.47.8544.34.48/3.66 SER147110.38.295866.4 LYS148121.59.0860.2 SER149109.88.1559.24.4164.54.14/3.99 GLY150113.48.1244.44.25/3.88 ARG151119.47.3856.74.4530.61.7527.31.75/1.6743.33.26 THR152116.58.8459.85.4871.44.61.31 SER153116.79.52594.4263.54.19/4.04 LYS154118.27.3954.24.44381.26260.829.61.0342.52.92/2.84 LYS155122.78.4756.84.2332.21.88/1.77251.44/1.2129.41.6541.92.90/2.84 ILE156133.89.3959.55.1337.52.360.820.77 VAL157128.38.6961.44.5135.62.140.82/0.76 ILE1581269.0660.74.7137.12.070.910.71 THR159125.68.8564.33.9568.94.011.23 ASP160115.38.1253.44.9644.72.96/2.92 CYS161115.58.2355.14.5230.73.05 GLY162101.66.5745.53.84/3.37 GLN163120.99.1654.85.0630.81.95/1.64332.60/2.46 LEU164127.39.1755.64.5543.51.6727.41.660.89/0.77 SER165120.48.156064.4
appendix
Table 8: Assignment table for CypD in complex with CL1 All NH assignments were obtained manually. The carbon assignments were produced by an automated signal assignment using FLYA. ResidueNHCAHA*CBHB*CGHG*CDHD*CEHE*CZHZ* SER1118,28,458,54,53643.90/3.904,84 GLY2111,18,4744,83.98/3.98 ASN3122,18,3151,15,12382.66/3.548.01/8.39 PRO463,24,732,91.74/2.3227,12.19/2.19514.03/4.17 LEU5121,28,5153,25,5544,91.91/1.91271,720.89/0.90 VAL6115,29,1358,35,435,42,690.93/1.13 TYR7116,58,71556,1942,82.57/2.806.57/6.576.43/6.43 LEU8116,28,9753,64,95451.39/1.9226,40,920.89/0.92 ASP9125,99,1554,45,6641,92.60/2.62 VAL10118,88,160,25,5235,31,740.87/0.90 ASP11127,19,4552,95,2345,32.29/2.29 ALA12119,18,63505,0821,60,76 ASN13125,79,3153,84,4637,42.64/3.387.97/8.11 GLY14104,28,8145,73.69/4.13 LYS15122,27,7252,54,933,11.85/1.9424,71.38/1.5029,11.73/1.7342,23.04/3.047,74 PRO1663,94,5732,51.89/2.3627,62.06/2.1451,23.73/4.03 LEU17122,69,7154,74,5743,51.67/1.68261,410.75/0.94 GLY18104,87,6644,13.73/4.64 ARG19121,38,2354,75,5634,41.60/1.6127,21.60/1.60433.13/3.348,32 VAL20126,59,5261,14,5834,42,070.94/0.95 VAL21126,88,8961,84,9933,41,940.89/1.00 LEU221299,6353,44,9443,20.94/0.9426,81,530.68/0.74 GLU23122,68,6654,84,9631,71.71/1.7136,91.76/2.10 LEU24124,68,4552,34,88431.33/2.01271,270.55/0.55
appendix
ResidueNHCAHA*CBHB*CGHG*CDHD*CEHE*CZHZ* LYS251258,953,84,5227,41.59/1.6723,51.26/1.3627,51.91/1.9141,62.84/2.916,96 ALA26128,68,1854,13,7418,81,49 ASP27114,19,0655,24,2538,62.74/2.84 VAL28119,67,5963,84,2235,31,941.00/1.12 VAL29114,18,2357,74,5931,62,581.00/1.21 PRO3066,14,431,42.00/2.6427,42.00/2.1050,83.09/3.62 LYS31122,510,7660,14,0831,61.45/1.72261.22/1.4229,21.42/1.50422.62/2.6810,79 THR32124,610,3767,54,0268,74,170.88/2.58 ALA33125,79,27564,1518,71,48 GLU34117,68,0358,34,5228,21.75/2.2933,62.04/2.49 ASN35114,87,1756,74,0339,72.33/2.887.16/7.17 PHE36117,67,14624,0840,43.15/3.297.13/7.146.99/7.00128,95,81 ARG37118,98,8160,63,6530,11.85/1.9524,40.94/1.6641,32.85/2.954,84 ALA38118,38,454,24,118,31,24 LEU39121,98,0257,13,8641,31.57/1.5726,60,920.59/0.92 CYS401207,9263,64,48272.17/3.320,94 THR41107,18,0263,54,1569,14,471.33/3.84 GLY42108,27,7745,63.52/3.84 GLU43118,68,0158,54,14302.06/2.2235,31.81/2.10 LYS44118,79,0954,24,3230,30.93/1.6524,91.05/1.0727,51.46/1.4642,12.69/2.854,83 GLY45105,67,9544,73.56/4.32 PHE46113,76,37544,6239,52.67/3.126.64/6.646.97/6.97130,57,46 GLY47104,57,5445,82.72/4.31 TYR48112,76,7458,64,339,43.00/3.126.64/7.106.79/6.86 LYS49123,78,2760,83,7431,71.98/1.9925,11.37/1.3930,11.82/1.9542,23.12/3.284,94 GLY50117,79,7445,53.64/4.45
appendix
ResidueNHCAHA*CBHB*CGHG*CDHD*CEHE*CZHZ* SER51116,48,6558,64,9665,44.29/4.294,95 THR52107,59,24615,73734,31.27/4.30 PHE53120,68,3957,64,9839,31.88/3.006.90/6.916.97/7.05128,77,45 HIS541207,3556,84,97312.78/3.126.86/6.866,94 ARG551226,7254,94,9632,60.98/1.1328,21.66/1.6643,42.98/3.149,14 VAL56129,49,23624,5135,11,820.75/1.09 ILE57126,88,5557,45,17411,86
1.14/1.53/ 1.63
1,14 PRO5862,54,3632,81.71/2.2627,41.91/2.0151,83.75/3.84 SER59114,89,6593,8561,73.97/4.079,61 PHE60113,87,4456,94,9637,33.08/3.397.11/7.117.15/7.15129,96,54 MET61112,38,254,55,13351.61/2.6928,20.95/0.9520,31,14 CYS62114,98,4956,74,9630,72.80/3.090,75 GLN63128,49,41554,5230,72.32/2.5533,42.04/2.487.17/7.17 ALA64125,88,150,54,8523,20,87 GLY65102,67,747,22.74/4.02 ASP66123,69,9451,64,22392.59/2.99 PHE67116,26,3555,74,56392.60/3.967.09/7.107.06/7.45130,45,88 THR68110,27,3662,24,5768,74,040.75/7.36 ASN691218,7952,84,1439,32.77/3.428.08/8.56 HIS70112,16,657,34,5128,33.12/3.287.53/6.725,81 ASN71112,47,5152,84,1439,42.77/3.557.30/8.56 GLY721089,4745,63.37/4.56 THR73112,17,9562,34,5671,34,321.10/1.10 GLY74111,68,1545,63.43/4.57 GLY751108,1143,33.57/4.66
appendix
ResidueNHCAHA*CBHB*CGHG*CDHD*CEHE*CZHZ* LYS76115,16,8655,94,65351.55/1.8423,40.85/0.9629,51.15/1.1541,12.12/2.127,18 SER77114,37,7156,65,3270,14.25/4.257,13 ILE781128,6563,94,2137,31,74
0.73/- 0.39/1.23
0,49 TYR79120,58,0856,14,76392.38/3.466.43/6.436.15/6.15 GLY80106,27,1243,73.90/5.12 SER81114,37,762,94,162,93.98/4.103,99 ARG82114,27,7453,45,6634,41.59/1.6826,31.48/1.4943,42.98/3.144,37 PHE83117,69,0355,15,1839,43.00/3.127.32/7.327.61/7.61132,86,43 PRO8462,6432,81.71/2.2627,41.92/2.0051,93.75/3.84 ASP85119,98,7754,84,1640,81.93/2.40 GLU86132,39,3660,43,831,61.82/2.2534,92.32/2.40 ASN87106,97,0852,94,1439,52.77/3.427.08/8.36 PHE88113,18,3156,16,0238,42.60/3.577.29/7.296.51/7.29130,45,88 THR89118,28,5666,83,7669,13,761.20/8.59 LEU90118,98,1654,54,4840,91.50/1.5027,31,660.82/0.93 LYS91118,78,2854,34,734,41.61/2.2325,71.42/1.4928,31.59/1.6542,73.07/3.076,07 HIS92123,710,8756,74,4225,92.88/3.090.93/6.516,9 VAL931147,0863,63,9732,82,350.84/1.00 GLY94105,77,2945,63.63/4.40 PRO9562,63,7132,21.87/1.9727,51.86/2.1649,73.44/3.62 GLY96109,98,9644,93.25/4.46 VAL97119,76,6564,73,6133,42,020.93/1.38 LEU98130,17,3753,24,7844,40.33/0.5325,6-0,09-1.63 SER99121,38,21555,29642.74/3.801,38 MET100123,68,65545,58322.29/2.7932,12.42/2.4216,92,24
appendix
ResidueNHCAHA*CBHB*CGHG*CDHD*CEHE*CZHZ* ALA101127,48,2252,45,2119,61,39 ASN102114,39,5554,5241,11.47/2.038.22/8.03 ALA103124,78,74,6518,31,24 GLY104105,97,7843,43.57/3.60 PRO10563,94,3732,31.88/2.3627,61.97/2.1649,73.45/3.61 ASN1061198,954,54,0237,22.64/3.098.80/8.90 THR107110,410,2160,44,3868,74,30.82/3.14 ASN108118,46,9555,64,3639,91.04/1.666.95/6.95 GLY109110,89,5945,63.48/4.63 SER110118,29,3956,84,6466,13.67/3.822,74 GLN111125,48,6258,35,432,91.71/2.2635,31.73/1.938.51/8.64 PHE112118,78,8255,66,09433.43/3.446.88/6.887.84/7.84128,76,79 PHE113115,79,9655,35,7743,62.69/2.706.75/6.886.78/6.97130,45,87 ILE114117,48,8659,15,0441,21,57
0.74/0.85/ 1.85
0,95 CYS1151259,0560,84,4129,42.93/3.724,69 THR116113,48,95614,4267,64,40.80/1.96 ILE117118,77,6259,84,33441,77
0.93/1.28/ 1.40
1,08 LYS118121,48,1957,53,7533,11.93/2.1324,51.66/1.72301.76/1.7642,13.00/3.007,94 THR119120,57,6957,23,5468,12,871.01/3.80 ASP120122,18,4156,74,2339,52.72/2.88 TRP121119,67,59594,58273.33/3.467,057.75/9.287.37/6.55 LEU122117,97,354,24,4438,31.27/1.2738,80,330.33/0.75 ASP1231247,7755,95,2539,52.72/2.87 GLY124111,69,6144,82.94/3.96
appendix
ResidueNHCAHA*CBHB*CGHG*CDHD*CEHE*CZHZ* LYS125115,77,9356,94,2135,21.82/1.93261.22/1.3329,51.61/1.6242,23.05/3.114,84 HIS126119,77,72553,9131,33.34/3.337.92/6.937,28 VAL127124,48,4763,94,2333,52,041.01/1.16 VAL128133,29,6634,01311,67-0.02 PHE129117,68,2655,55,1242,12.55/3.126.63/6.637.46/7.60131,76,16 GLY130107,47,2946,93.99/4.12 HIS1311198,5155,55,1134,62.81/3.108.72/6.946,91 VAL132122,98,7264,13,9132,41,960.74/0.94 LYS1331329,6256,54,5135,11.68/1.6924,91.51/1.5429,11.68/1.7942,13.01/3.049,66 GLU134115,87,9355,24,7733,51.79/2.12362.19/2.29 GLY135109,18,8546,34.16/4.93 MET136122,68,9956,74,5229,92.02/2.1233,12.62/2.7716,81,96 ASP137116,79,1556,84,2238,92.59/2.74 VAL1381247,4866,13,4312,40.56/1.04 VAL139120,87,53673,3230,82,550.74/1.04 LYS140117,48,1858,14,1330,11.66/1.7624,41.57/1.6626,91.59/1.6841,42.86/2.958,21 LYS141121,37,659,53,9632,71.90/2.1425,51.50/1.73301.76/1.7642,22.90/2.917,61 ILE142121,18,16673,3237,92,06
0.43/1.76/ 1.76
0,1 GLU143117,88,2358,73,5130,51.75/1.7536,82.11/2.11 SER144113,87,4260,94,1562,93.75/3.757,44 PHE145119,57,757,74,9839,32.99/3.557.09/7.287.28/7.281327,28 GLY146105,57,8944,33.58/4.48 SER147109,18,2157,63,7566,13.67/3.873,98 LYS148118,98,8160,53,6629,61.26/1.2723,20.84/1.2529,60.83/1.2642,32.84/2.924,84 SER149109,98,2358,64,3563,93.90/4.217,06
appendix
ResidueNHCAHA*CBHB*CGHG*CDHD*CEHE*CZHZ* GLY150113,38,4645,23.88/4.44 ARG151118,47,4256,44,4930,11.76/1.7626,91.74/1.7443,33.26/3.388,23 THR152115,68,7759,85,571,24,61.28/5.50 SER153116,99,46594,4163,54.06/4.164,17 LYS154117,97,354,24,4537,91.10/1.2525,80.75/0.8229,41.04/1.0542,32.85/2.927,09 LYS155123,48,556,94,2132,41.78/1.88251.21/1.4529,41.66/1.66422.85/2.918,51 ILE156133,29,2959,55,1237,42,3
0.76/1.32/ 1.50
0,7 VAL157128,38,761,24,5535,82,120.75/0.84 ILE158125,78,9760,94,6936,92,11
0.90/0.92/ 1.89
0,72 THR159126,18,8664,33,9569,13,961.24/2.60 ASP160115,18,1353,44,9544,72.94/2.94 CYS161115,48,29554,5230,73.04/3.084,76 GLY162101,56,5645,53.36/3.83 GLN163120,79,0854,85,0630,81.66/1.9633,12.48/2.628.70/9.15 LEU164127,19,155,64,5743,51.66/1.6627,31,660.78/0.89 SER165120,58,1160,14,2864,23.90/3.901,51
appendix
Table 9: Predicted protein parameters .
The listed parameters of the used constructs are predicted using ProtParam (Wilkins 1999)
Construct Number of
amino acids Molecular
weight Theoretical
pI Extinction coefficient
His-CypD43-207 189 20.573 8.32 12950
CypD43-207 165 17.730 9.06 9970
AT-hASIC1a 549 62.535 5.92 51895
His-MSP1-Flag 225 26.304 5.61 26930
His-MSP1E3D1 272 31.962 6.00 28420
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acknowledgemenTs
Acknowledgements
I would like to thank Volker Dötsch for his support during my time as a student in his lab and for the supervision of my PhD thesis. I enjoyed my time in the group, where you managed to create a cooperative and creative working environment. I appreciate the space and the time you gave me for my personal and scientific development. I was able to pursue my own ideas, plan and develop my projects in nearly any way I wanted, work on a whole bouquet of different subjects and had the freedom to establish cooperations. In addition I would like to thank you for your great sense of humor, even in awkward or special situations.
Further I would like to thank Daniel Schwarz for his great support during the whole project.
Thank you for establishing the cooperation that made my thesis possible and for all the organiza-tional work connected with the project. Thank you for your great feedback on my data, my ideas, my presentations and my final thesis, as well as your ideas and help, especially during the critical and difficult times of the thesis.
I would further like to thank Jörg Bombke, Ansgar Wegener, Djordje Musil, Ulrich Graedler and Matthias Frech from the Merck MIB team for scientific input and support during the thesis, as well as Norbert, Ivonne, Eva and Gerlinde for their help in the MIB lab.
Special thanks go to Frank Löhr (Murph) for your enormous efforts with all my NMR samples.
Your support and input saved my PhD thesis.
I would like to thank my cooperation partners, that helped me in a lot of projects even if the projects are not mentioned in this thesis. My thanks go to:
• Oliver Peetz for his MS measurements and for always having time for my samples, even on very short notice.
• Albert Konijnenberg for the cooperation on another MS project.
• Lilia Leisle and Chris Ahern for their big effort in synthesizing modified tRNAs and for their input during the cooperation project.
• Francis Valayaveetil and his lab for the great support on the protein refolding
• Martin Caffrey and Coilin Boland for their work on LCP crystallization of my samples.
• Ekaterina Zaitseva for electrophysiology measurements on a lot of samples.
• Sina Kazemi for all the work concerning the automated assignment and structure calcula-tion as well as the fun during the music festivals and concerts.
I would like to thank Sigrid Oğuzer-Fachinger, who did a great job helping me with all the bu-reaucratic problems; Manfred Strupf, for being better than anyone else in fixing equipment, you kept the lab running and Brigit Schäfer for being the good soul of the lab.
But the most influence on my time had the people I worked with on an everyday basis in the lab. Here I‘d like to thank Susanne Stefer for introducing me to the cell free expression and for the great cooperation during the first six months of my PhD, in which I worked with her on the Get project. I want to thank Sebastian Richers, who was probably the most helpful postdoc in the lab and had an endless amount of great tips, and hands on support. I want to thank Aisha Laguerre for her honest and open conversations and for sharing her postdoc wisdom on what a PhD thesis is about and how it works, as well as her ideas about life, the universe and everything. Further to the former lab members Gergor Deutsch, Robert Hänsel-Hertsch, Peter Tufar, Alena Busche, Sina Reckel, Alexis Rozenknop, Christian Roos and Stefan Haberstock for a warm welcome in the