De novo protein design: Fully automated sequence selection

Science

Volumn 278, Issue 5335, 1997, Pages 82-87

  Dahiyat, Bassil I ^a,c   Mayo, Stephen L ^b  

^a Mail Code 147 75 ^*  (United States)

^b CALIFORNIA INSTITUTE OF TECHNOLOGY   (United States)

^c Xencor Inc   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ALGORITHM; AMINO ACID SEQUENCE; ARTICLE; AUTOMATION; COMPUTER PROGRAM; MATHEMATICAL ANALYSIS; MATHEMATICAL COMPUTING; PRIORITY JOURNAL; PROTEIN CONFORMATION; PROTEIN SYNTHESIS;

ALGORITHMS; AMINO ACID SEQUENCE; COMPUTER SIMULATION; CRYSTALLOGRAPHY, X-RAY; DNA-BINDING PROTEINS; HYDROGEN BONDING; MAGNETIC RESONANCE SPECTROSCOPY; MODELS, MOLECULAR; MOLECULAR SEQUENCE DATA; PROTEIN CONFORMATION; PROTEIN ENGINEERING; PROTEIN FOLDING; PROTEIN STRUCTURE, SECONDARY; PROTEIN STRUCTURE, TERTIARY; SEQUENCE ALIGNMENT; SOLUTIONS; TRANSCRIPTION FACTORS; ZINC FINGERS;

EID: 0030793767     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.278.5335.82     Document Type: Article

References (62)

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15. s selects the rotamer s on residue j that minimizes the argument of the function. Iterative application of the elimination criterion results in a rapid and substantial reduction in the combinatorial size of the problem and application of similar but higher-order elimination criteria are required to find the ground-state solution.
16. s selects the rotamer s on residue j that minimizes the argument of the function. Iterative application of the elimination criterion results in a rapid and substantial reduction in the combinatorial size of the problem and application of similar but higher-order elimination criteria are required to find the ground-state solution.
17. s selects the rotamer s on residue j that minimizes the argument of the function. Iterative application of the elimination criterion results in a rapid and substantial reduction in the combinatorial size of the problem and application of similar but higher-order elimination criteria are required to find the ground-state solution.
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20. B. I. Dahiyat and S. L. Mayo, unpublished results.
21. Potential functions and parameters for van der Waals interactions, solvation, hydrogen bonding, and secondary structure propensity are described in our previous work (4-6). A secondary structure propensity potential was used for surface β-sheet positions where the i - 1 and i + 1 residues were also in β-sheet conformations (5). Propensity values from Serrano and co-workers were used [V. Munoz and L. Serrano, Proteins Struct Funct. Genet. 20, 301 (1994)].
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23. The coordinates of PDB record 1zaa (9, 13) from residues 33 to 60 were used as the structure template. In our numbering, position 1 corresponds to 1zaa position 33. The program BIOGRAF (Molecular Simulations, Inc., San Diego, CA) was used to generate explicit hydrogens on the structure which was then conjugate-gradient minimized for 50 steps by means of the Dreiding force field (40).
24. -2, and a Cα radius of 1.95 Å. A residue was classified as a core position if the distance from its Cα, along its Cα-Cβ vector, to the solvent-accessible surface was greater than 5.0 Å, and if the distance from its Cβ to the nearest surface point was greater than 2.0 Å. The remaining residues were classified as surface positions if the sum of the distances from their Cα, along their Cα-Cβ vector, to the solvent-accessible surface plus the distance from their Cβ to the closest surface point was less than 2.7 Å. All remaining residues were classified as boundary positions. The classifications for Zif268 were used as computed except that positions 1, 17, and 23 were converted from the boundary to the surface class to account for end effects from the proximity of chain termini to these residues in the tertiary structure and inaccuracies in the assignment.
25. 27 possible amino acid sequences.
26. 7 g = 11.6 metric tons.
27. As in our previous work (5), a backbone-dependent rotamer library was used [R. L. Dunbrack and M. Karplus, J. Mol. Biol. 230, 543 (1993)]. All His rotamers were protonated on both Nδ and Nε.
28. All calculations were performed on a Silicon Graphics Power Challenge server with 10 R10000 processors running in parallel. Peak performance is 3.9 GigaFLOPS (FLOPS = floating point operations per second).
29. The sequence optimization consists of two phases: pairwise rotamer energy calculations and DEE searching. The DEE optimization was initially run with control parameters set for optimal speed followed by a DEE-based, residue-pairwise, round-robin optimization. The energy calculations took 53 CPU (central processing unit) hours and sequence optimizations took 37 CPU hours.
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35. FSD-1 was synthesized by means of standard solid-phase Fmoc chemistry. The peptide was cleaved from the resin with trifluoroacetic acid and purified by reversed-phase high-performance liquid chromatography. Peptide was lyophilized and stored at -20°C. Matrix-assisted laser desorption mass spectrometry yielded a molecular weight of 3489.7 daltons (3489.0 calculated).
36. Protein concentration was 50 μM in 50 mM sodium phosphate at pH 5.0. The spectrum was acquired at 1°C in a 1-mm cuvette and was baseline-corrected with a buffer blank. The spectrum is the average of 3 scans with a 1-s integration time and 1-nm increments. All CD data were acquired on an Aviv 62DS spectrometer equipped with a thermoelectric temperature control unit. Thermal unfolding was monitored at 218 nm in a 1-mm cuvette with 2° increments and an averaging time of 40 s and an equilibration time of 120 s per increment. Reversibility was confirmed by comparison of 1°C CD spectra before and after heating to 99°C. Peptide concentrations were determined by UV spectrophotometry.
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40. Sedimentation equilibrium studies were performed on a Beckman XL-A ultracentrifuge equipped with an An-60 Ti analytical rotor at a speed of 40,000 rpm. Protein concentration was 100 μM, 500 μM, or 1 mM in 50 mM sodium phosphate at pH 5.0 and 7°C. Absorption was monitored at 286 nm (500 μM and 1 mM) or 234 nm (100 μM). Concentration profiles were fit to an ideal single species model which resulted in randomly distributed residuals.
41. 2O with 50 mM sodium phosphate at pH. 5.0 (uncorrected glass electrode). All spectra were collected at 7°C. DQF-COSY [U. Piantini, O. W. Sorensen, R. R. Ernst, J. Am. Chem. Soc. 104, 6800 (1982)], TOCSY [A. Bax and D. G. Davis, J. Magnetic Reson. 65, 355 (1985)], and NOESY [J. Jeener, B. H. Meier, P. Bachmann, R. R. Ernst, J. Chem. Phys. 71, 4546 (1979)] spectra were acquired to accomplish resonance assignments and structure determination. NOESY spectra were recorded with mixing times of 200 ms for use during resonance assignments and 100 ms to derive distance restraints. Water suppression was accomplished either with presaturation during the relaxation delay or pulsed field gradients [M. Piotto, V. Saudek, V. Sklenar, J. Biomol. NMR 2, 661 (1992)]. Spectra were processed with VNMR (Varian Associates, Palo Alto, CA), and spectra were assigned with ANSIG [P. J. Kraulis, J. Magnetic Reson. 24, 627 (1989)].
42. 2O with 50 mM sodium phosphate at pH. 5.0 (uncorrected glass electrode). All spectra were collected at 7°C. DQF-COSY [U. Piantini, O. W. Sorensen, R. R. Ernst, J. Am. Chem. Soc. 104, 6800 (1982)], TOCSY [A. Bax and D. G. Davis, J. Magnetic Reson. 65, 355 (1985)], and NOESY [J. Jeener, B. H. Meier, P. Bachmann, R. R. Ernst, J. Chem. Phys. 71, 4546 (1979)] spectra were acquired to accomplish resonance assignments and structure determination. NOESY spectra were recorded with mixing times of 200 ms for use during resonance assignments and 100 ms to derive distance restraints. Water suppression was accomplished either with presaturation during the relaxation delay or pulsed field gradients [M. Piotto, V. Saudek, V. Sklenar, J. Biomol. NMR 2, 661 (1992)]. Spectra were processed with VNMR (Varian Associates, Palo Alto, CA), and spectra were assigned with ANSIG [P. J. Kraulis, J. Magnetic Reson. 24, 627 (1989)].
43. 2O with 50 mM sodium phosphate at pH. 5.0 (uncorrected glass electrode). All spectra were collected at 7°C. DQF-COSY [U. Piantini, O. W. Sorensen, R. R. Ernst, J. Am. Chem. Soc. 104, 6800 (1982)], TOCSY [A. Bax and D. G. Davis, J. Magnetic Reson. 65, 355 (1985)], and NOESY [J. Jeener, B. H. Meier, P. Bachmann, R. R. Ernst, J. Chem. Phys. 71, 4546 (1979)] spectra were acquired to accomplish resonance assignments and structure determination. NOESY spectra were recorded with mixing times of 200 ms for use during resonance assignments and 100 ms to derive distance restraints. Water suppression was accomplished either with presaturation during the relaxation delay or pulsed field gradients [M. Piotto, V. Saudek, V. Sklenar, J. Biomol. NMR 2, 661 (1992)]. Spectra were processed with VNMR (Varian Associates, Palo Alto, CA), and spectra were assigned with ANSIG [P. J. Kraulis, J. Magnetic Reson. 24, 627 (1989)].
44. 2O with 50 mM sodium phosphate at pH. 5.0 (uncorrected glass electrode). All spectra were collected at 7°C. DQF-COSY [U. Piantini, O. W. Sorensen, R. R. Ernst, J. Am. Chem. Soc. 104, 6800 (1982)], TOCSY [A. Bax and D. G. Davis, J. Magnetic Reson. 65, 355 (1985)], and NOESY [J. Jeener, B. H. Meier, P. Bachmann, R. R. Ernst, J. Chem. Phys. 71, 4546 (1979)] spectra were acquired to accomplish resonance assignments and structure determination. NOESY spectra were recorded with mixing times of 200 ms for use during resonance assignments and 100 ms to derive distance restraints. Water suppression was accomplished either with presaturation during the relaxation delay or pulsed field gradients [M. Piotto, V. Saudek, V. Sklenar, J. Biomol. NMR 2, 661 (1992)]. Spectra were processed with VNMR (Varian Associates, Palo Alto, CA), and spectra were assigned with ANSIG [P. J. Kraulis, J. Magnetic Reson. 24, 627 (1989)].
45. 2O with 50 mM sodium phosphate at pH. 5.0 (uncorrected glass electrode). All spectra were collected at 7°C. DQF-COSY [U. Piantini, O. W. Sorensen, R. R. Ernst, J. Am. Chem. Soc. 104, 6800 (1982)], TOCSY [A. Bax and D. G. Davis, J. Magnetic Reson. 65, 355 (1985)], and NOESY [J. Jeener, B. H. Meier, P. Bachmann, R. R. Ernst, J. Chem. Phys. 71, 4546 (1979)] spectra were acquired to accomplish resonance assignments and structure determination. NOESY spectra were recorded with mixing times of 200 ms for use during resonance assignments and 100 ms to derive distance restraints. Water suppression was accomplished either with presaturation during the relaxation delay or pulsed field gradients [M. Piotto, V. Saudek, V. Sklenar, J. Biomol. NMR 2, 661 (1992)]. Spectra were processed with VNMR (Varian Associates, Palo Alto, CA), and spectra were assigned with ANSIG [P. J. Kraulis, J. Magnetic Reson. 24, 627 (1989)].
46. K. Wuthrich, NMR of Proteins and Nucleic Acids (Wiley, New York, 1986).
47. 25 were protected from exchange at 7°C, pH 5.0. Hydrogen bond restraints (two per hydrogen bond) were only included at the late stages of structure refinement when initial calculations indicated the donor-acceptor pairings.
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49. Standard hybrid distance geometry-simulated annealing protocols were followed [M. Nilges, G. M. Clore, A. M. Gronenborn, FEBS Lett. 229, 317 (1988); M. Nilges, J. Kuszewski, A. T. Brünger, in Computational Aspects of the Study of Biological Macromolecules by NMR J. C. Hoch, Ed. (Plenum, New York, 1991); J. Kuszewski, M. Nilges, A. T. Brünger, J. Biomol. NMR 2, 33 (1992)]. Distance geometry structures (100) were generated, regularized, and refined, resulting in an ensemble, called 〈SA〉, of 41 structures with no restraint violations greater than 0.3 Å, rms deviations from idealized bond lengths less than 0.01 Å, and rms deviations from idealized bond angles and impropers less than 1°. An average structure was generated by superimposing and then averaging the coordinates of the ensemble, followed by refinement and restrained minimization.
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62. We thank P. Poon and T. Laue for sedimentation equilibrium measurements and discussions, A. Su for assistance calculating super-secondary structure parameters, S. Ross for assistance with NMR measurements, G. Hathaway for mass spectrometry, J. Abelson and P. Bjorkman for critical reading of the manuscript, and R. A. Olofson for helpful discussions. Supported by the Howard Hughes Medical Institute (S.L.M.), the Rita Allen Foundation, the Chandler Family Trust, the Booth Ferris Foundation, the David and Lucile Packard Foundation, the Searle Scholars Program and The Chicago Community Trust, and grant GM08346 from the National Institutes of Health (B.I.D.). Coordinates and NMR restraints have been deposited in the Brookhaven Protein Data Bank with accession numbers 1FSD and R1FSDMR, respectively.