Supplementary Material for Perkin Transactions This journal is © The Royal Society of Chemistry 2002 Supplementary data “Conformational Change in the Thiazole and Oxazoline Containing Cyclic Octapeptides, the Patellamides Part II: Solvent Dependent Conformational Change.” Bruce F Milne, Linda A Morris, Marcel Jaspars and Gary S Thompson Calculation of structures NOEs were obtained using a T-ROESY pulse sequence with a 400 ms mixing time NOE’s were classified into weak (1.8 - 5.0 Å), medium (1.8 - 3.5 Å) and strong (1.8 - 2.5 Å) categories Molecular dynamics calculation were carried out using XPLOR 3.8511, modified for swapping of prochiral groups Throughout the calculations a forcefield with purely repulsive non bonded interaction terms (REPEL) was used, with no electrostatic potential being applied The incorporation of the cyclic amino acids Xxx(Thz) and Yyy(Oxn) and the closure of the ring was achieved by a series of patches to the standard XPLOR parameter and topology files for NMR structures These patches included improper terms to keep the thiazole residue flat Circular template structures with good geometry were defined, annealed at 300 K, minimised, and checked for the correct chirality and the geometry of the residues These template structures were then used in the structure calculations Structure calculation used an ab initio simulated annealing strategy (YASAP 3.0 2,3 Sum averaging4 was used for all methyl, methylene, and aromatic ring atom pairs Assignment of prochiral groups was achieved by floating assignment and swapping of prochiral groups5,6 A reduced set of non-bonded interactions and a reduced representation of the amino acid side chains was used during the conformation search phase During all stages of the simulated annealing calculation the temperature was maintained by coupling to a heat bath with a coupling frequency of 10 ps-1 100 initial starting structures were calculated by randomisation of the backbone and angles using an elastic bond between Ser C and Val N to relieve strain The simulated annealing protocol was divided into four stages: 60 ms of annealing at a temperature of 2000 K with a timestep of fs during which the force constants for the distance restraints were increased to their final value in steps This stage of the calculation used the reduced sidechain representation consisting of a 2.25 Å radius C atom cooling from 2000 to 1000 K over 40 ps during which force constants for non bonded interactions were increased to their final value cooling from 1000K to 100 K over 20 ps with a time step of fs 200 steps of minimisation In all cases the lengths of restraints for methyl residues was extended by 0.5 Å to allow for the differential relaxation of methyls A soft potential was used in the ab initio simulated annealing protocol for the distance restraints During the refinement stage the 40 and 52 lowest energy structures from each ensemble of patellamide C and patellamide A respectively were refined by a single round of simulated annealing with slow cooling The high temperature step consisted of 600 ps of cooling from 1500 K to 100 K with a timestep of fs followed 4000 rounds of minimisation A square well potential was used for the distance restraint function A cluster of 22 lowest energy structures with the same backbone were selected to represent the final conformation of patellamide A A cluster of 14 lowest energy structures with the same backbone were selected to calculate statistics for patellamide C Calculations were carried out on a SGI Origin2000 computer The overlay and display of structures was achieved using Molmol.9 Supplementary Material for Perkin Transactions This journal is © The Royal Society of Chemistry 2002 Restraints used for NOE restrained structure calculation of patellamide A in CDCl Structure numbering Patellamide A D-Val 1HG1# L-Ser 8HA 8HB1,2 O H 1HA 2HB1 1HN 7HG2# 7HD1# S NH O L-Ile 1HG2# N Cys N 7HB O 7HA 7HG11,2 NH 7HN 3HN O 3HB 3HA N 5HA S O NH 4HB H O 5HG2# 3HG2# N 5HN 6HB1 Cys 3HG11,2 NH 4HA L-Thr 4HG2# 5HG1# D-Val resid 7 or or or or 1 or or 5 or 1 or 5 or or 1 or or 6 or or 7 or 3 or 7 or or 5 or or 8 or 5 or atom HN HN HN HB# HB HN HN HB HB HB HB HN HN HN HN HB1 HB1 HN HN HN HN HB HB HB# HA HB HB# HA HA HB# resid or 1 or or 5or 7 1 5 7 or 4 or 5 or atom HA HA HB# HN HN HA HA HN HN HB1 HB1 HB HB HG## HG## HG## HG## HG## HG## HG2# HG2# HA HA HB HG2# HG2# HG## HG## HG## HG2# NOE (w, m, s) s m s m m m m m m w w m m m m m m m m w w s s s s s m s s m 3HD1# L-Ile Supplementary Material for Perkin Transactions This journal is © The Royal Society of Chemistry 2002 Energies for patellamide A minimum energy conformation: ETotal EBonds 11.82 EAngles 1.69 EImproper 9.22 Evan der Waals 0.87 6.15 x 10 -5 ENOE 4.19 x 10-2 No NOE violations Statistics for patellamide A (22 lowest energy structures) Mean global backbone RMSD: 0.22 ± 0.14 Å Mean global heavy atom RMSD: 0.55 ± 0.22 Å Average global displacements Res # Name Val Cys Ile Thr Val Cys Ile Ser BB Heavy Heavysc Average local RMSDs BB 0.12 0.16 0.22 0.25 0.11 0.12 0.15 0.13 0.18 0.20 0.70 0.41 0.15 0.15 0.60 0.18 0.22 0.22 0.90 0.50 0.19 0.16 0.78 0.26 0.00 0.07 0.11 0.09 0.09 0.04 0.08 0.00 Average local displacements BB Heavy Heavysc 0.00 0.04 0.08 0.07 0.05 0.02 0.06 0.00 0.00 0.07 0.45 0.15 0.07 0.04 0.49 0.00 0.00 0.09 0.58 0.19 0.08 0.06 0.65 0.00 Restraints used for NOE restrained structure calculation of patellamide C in CDCl Structure numbering 1HE2 1HD2 Patellamide C 1HZ 1HB2 H L-Thr 8HG2# 8HA H 8HB O H D-Phe 1HD1 1HA S NH O 2HB1 1HN 7HG2# L-Ile 1HE1 1HB1 N Cys N 7HB O 7HA 7HD1# 7HG11 H NH 7HN 3HN H O NH 3HG2# 3HB 3HA 7HG12 N Cys N 5HN 6HB1 S 5HA NH 5HB# 3HG1# O L-Val 4HB O H 4HA L-Thr 4HG2# D-Ala resid atom resid atom NOE (w, m, s) Supplementary Material for Perkin Transactions This journal is © The Royal Society of Chemistry 2002 3 1 3 7 5 7 6 1 1 1 1 3 7 7 4 8 3 7 7 7 7 3 HN HN HN HN HN HN HN HN HN HN HN HN HN HN HN HN HN HB1 HB1 HB1 HD# HD# HD# HD# HD# HE# HE# HA HA HA HA HB1 HB HB HB HB HA HG11 HG11 HG2# HG2# HG2# HG2# HG12 HG2# HG2# HG2# HG2# HG2# HD1# HD1# HB HB HB HB HB HB HG11 8 1 3 7 1 5 1 1 1 7 4 8 8 7 7 7 8 HA HB HA HA HA HB1 HB2 HB HG2# HA HB HA HA HA HB HB# HG12 HB1 HB2 HB# HA HA HA HB1 HB2 HA HB2 HB HB HB1 HB2 HB2 HA HB HA HB HB# HA HB HA HB HA HB HA HA HA HB HA HA HA HB HG11 HG12 HG2# HD1# HG2# HG2# HG2# m w w w w m w m w m w m w w m m w m m m w w w w w w w m m m m s m w m w s m w s s s s m w s m w s m w m m m m m m w Supplementary Material for Perkin Transactions This journal is © The Royal Society of Chemistry 2002 7 HG11 HG11 HG2# HG12 HG2# 7 7 HG2# HD1# HG2# HG2# HD1# m m m m m Energies for patellamide C minimum energy conformation: ETotal EBonds EAngles EImproper Evan der Waals ENOE 37.3311 3.23041 19.0393 4.9251 3.78779 6.3485 NOE Violations: Restraint CYS HB1 - PHE HB1 PHE HD# - ILE HA PHE HE# - PHE HA Actual distance (Å) 3.649 5.278 5.114 Restraint distance (Å) 3.50 5.00 5.00 ENOE 1.110 3.852 0.646 Statistics for patellamide C (14 lowest energy structures) Mean global backbone RMSD: 0.01 ± 0.00 Å Mean global heavy atom RMSD: 0.40 ± 0.26 Å Average global displacements Res # Name PHE CYS VAL THR ALA CYS ILE THR BB Heavy Heavysc Average local RMSDs BB 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.00 0.70 0.00 0.00 0.01 0.00 0.00 0.27 0.00 0.84 0.00 0.01 0.01 0.00 0.00 0.36 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Average local displacements BB Heavy Heavysc 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.27 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.35 0.00 References (1) Brünger, A T X-PLOR A system for X-ray crystallography and NMR 851.; Yale University: New Haven, 1993 (2) Nilges, M.; Gronenborn, A M.; Brünger, A T.; Clore, G M Prot Engng 1988, 2, 27-38 (3) Nilges, M.; Kuszewski, J.; Brünger, A T In Computational Aspects of the Study of Biological Macromolecules by Nuclear Magnetic Resonance; Hoch, J C., Poulsen, F M., Redfield, C., Eds.; Plenum Press: New York, 1991; Vol Vol 225, pp 451-455 (4) Brünger, A T.; Clore, G M.; Gronenborn, A M.; Karplus, M Proc Natl Acad Sci U.S.A 1986, 83, 38013805 (5) Holak, T A.; Nilges, M.; Oschkinat, H FEBS Lett 1989, 242, 218-224 (6) Folmer, R H A.; Hilbers, C W.; Konings, R N H.; Nilges, M J Biomol NMR 1997, 9, 245-258 (7) Nilges, M Proteins: Struct Funct and Genet 1993, 17, 297-309 (8) Berendsen, H J C.; Postma, J P M.; van Gunsteren, W F.; DiNicola, A.; J.R., H J Chem Phys 1984, 81, 3684-3690 Supplementary Material for Perkin Transactions This journal is © The Royal Society of Chemistry 2002 (9) Koradi, R M B.; Wuthrich, K J Mol Graph 1994, 14, 51-59 ... Material for Perkin Transactions This journal is © The Royal Society of Chemistry 2002 Restraints used for NOE restrained structure calculation of patellamide A in CDCl Structure numbering Patellamide... 0.00 0.00 0.09 0.58 0.19 0.08 0.06 0.65 0.00 Restraints used for NOE restrained structure calculation of patellamide C in CDCl Structure numbering 1HE2 1HD2 Patellamide C 1HZ 1HB2 H L-Thr 8HG2#... Nilges, M.; Oschkinat, H FEBS Lett 1989, 242, 218-224 (6) Folmer, R H A.; Hilbers, C W.; Konings, R N H.; Nilges, M J Biomol NMR 1997, 9, 245-258 (7) Nilges, M Proteins: Struct Funct and Genet 1993,