Structural Insight of the Full-Length Ros Protein: A Prototype of the Prokaryotic Zinc-Finger Family.
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
09 06 2020
09 06 2020
Historique:
received:
14
02
2020
accepted:
15
05
2020
entrez:
11
6
2020
pubmed:
11
6
2020
medline:
15
12
2020
Statut:
epublish
Résumé
Ros/MucR is a widespread family of bacterial zinc-finger (ZF) containing proteins that integrate multiple functions such as virulence, symbiosis and/or cell cycle transcription. NMR solution structure of Ros DNA-binding domain (region 56-142, i.e. Ros87) has been solved by our group and shows that the prokaryotic ZF domain shows interesting structural and functional features that differentiate it from its eukaryotic counterpart as it folds in a significantly larger zinc-binding globular domain. We have recently proposed a novel functional model for this family of proteins suggesting that they may act as H-NS-'like' gene silencers. Indeed, the N-terminal region of this family of proteins appears to be responsible for the formation of functional oligomers. No structural characterization of the Ros N-terminal domain (region 1-55) is available to date, mainly because of serious solubility problems of the full-length protein. Here we report the first structural characterization of the N-terminal domain of the prokaryotic ZF family examining by means of MD and NMR the structural preferences of the full-length Ros protein from Agrobacterium tumefaciens.
Identifiants
pubmed: 32518326
doi: 10.1038/s41598-020-66204-5
pii: 10.1038/s41598-020-66204-5
pmc: PMC7283297
doi:
Substances chimiques
Bacterial Proteins
0
DNA-Binding Proteins
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
9283Références
Chou, A. Y., Archdeacon, J. & Kado, C. I. Agrobacterium transcriptional regulator Ros is a prokaryotic zinc finger protein that regulates the plant oncogene ipt. Proc. Natl Acad. Sci. USA 95, 5293–5298 (1998).
doi: 10.1073/pnas.95.9.5293
Esposito, S. et al. A novel type of zinc finger DNA binding domain in the Agrobacterium tumefaciens transcriptional regulator Ros. Biochemistry 45, 10394–10405, https://doi.org/10.1021/bi060697m (2006).
doi: 10.1021/bi060697m
pubmed: 16922516
Malgieri, G. et al. The prokaryotic zinc-finger: structure, function and comparison with the eukaryotic counterpart. FEBS J. 282, 4480–4496, https://doi.org/10.1111/febs.13503 (2015).
doi: 10.1111/febs.13503
pubmed: 26365095
Bittinger, M. A., Milner, J. L., Saville, B. J. & Handelsman, J. rosR, a determinant of nodulation competitiveness in Rhizobium etli. Mol. Plant. Microbe Interact. 10, 180–186, https://doi.org/10.1094/MPMI.1997.10.2.180 (1997).
doi: 10.1094/MPMI.1997.10.2.180
pubmed: 9057324
Janczarek, M., Kutkowska, J., Piersiak, T. & Skorupska, A. Rhizobium leguminosarum bv. trifolii rosR is required for interaction with clover, biofilm formation and adaptation to the environment. BMC Microbiol. 10, 284, https://doi.org/10.1186/1471-2180-10-284 (2010).
doi: 10.1186/1471-2180-10-284
pubmed: 21070666
pmcid: 2996380
Janczarek, M., Rachwał, K. & Kopcińska, J. Genetic characterization of the Pss region and the role of PssS in exopolysaccharide production and symbiosis of Rhizobium leguminosarum bv. trifolii with clover. Plant. Soil. 396, 257–275, https://doi.org/10.1007/s11104-015-2567-5 (2015).
doi: 10.1007/s11104-015-2567-5
Caswell, C. C. et al. Diverse genetic regulon of the virulence-associated transcriptional regulator MucR in Brucella abortus 2308. Infect. Immun. 81, 1040–1051, https://doi.org/10.1128/IAI.01097-12 (2013).
doi: 10.1128/IAI.01097-12
pubmed: 23319565
pmcid: 3639602
Mirabella, A. et al. Brucella melitensis MucR, an orthologue of Sinorhizobium meliloti MucR, is involved in resistance to oxidative, detergent, and saline stresses and cell envelope modifications. J. Bacteriol. 195, 453–465, https://doi.org/10.1128/JB.01336-12 (2013).
doi: 10.1128/JB.01336-12
pubmed: 23161025
pmcid: 3554010
Dong, H., Liu, W., Peng, X., Jing, Z. & Wu, Q. The effects of MucR on expression of type IV secretion system, quorum sensing system and stress responses in Brucella melitensis. Vet. Microbiol. 166, 535–542, https://doi.org/10.1016/j.vetmic.2013.06.023 (2013).
doi: 10.1016/j.vetmic.2013.06.023
pubmed: 23932078
Keller, M. et al. Molecular analysis of the Rhizobium meliloti mucR gene regulating the biosynthesis of the exopolysaccharides succinoglycan and galactoglucan. Mol. Plant. Microbe Interact. 8, 267–277 (1995).
doi: 10.1094/MPMI-8-0267
Mueller, K. & González, J. E. Complex regulation of symbiotic functions is coordinated by MucR and quorum sensing in Sinorhizobium meliloti. J. Bacteriol. 193, 485–496, https://doi.org/10.1128/JB.01129-10 (2011).
doi: 10.1128/JB.01129-10
pubmed: 21057009
Jiao, J. et al. MucR Is Required for Transcriptional Activation of Conserved Ion Transporters to Support Nitrogen Fixation of Sinorhizobium fredii in Soybean Nodules. Mol. Plant. Microbe Interact. 29, 352–361, https://doi.org/10.1094/MPMI-01-16-0019-R (2016).
doi: 10.1094/MPMI-01-16-0019-R
pubmed: 26883490
Bertram-Drogatz, P. A., Quester, I., Becker, A. & Pühler, A. The Sinorhizobium meliloti MucR protein, which is essential for the production of high-molecular-weight succinoglycan exopolysaccharide, binds to short DNA regions upstream of exoH and exoY. Mol. Gen. Genet. 257, 433–441 (1998).
doi: 10.1007/s004380050667
Rüberg, S., Pühler, A. & Becker, A. Biosynthesis of the exopolysaccharide galactoglucan in Sinorhizobium meliloti is subject to a complex control by the phosphate-dependent regulator PhoB and the proteins ExpG and MucR. Microbiology 145(Pt 3), 603–611, https://doi.org/10.1099/13500872-145-3-603 (1999).
doi: 10.1099/13500872-145-3-603
pubmed: 10217494
Bahlawane, C., McIntosh, M., Krol, E. & Becker, A. Sinorhizobium meliloti regulator MucR couples exopolysaccharide synthesis and motility. Mol. Plant. Microbe Interact. 21, 1498–1509, https://doi.org/10.1094/MPMI-21-11-1498 (2008).
doi: 10.1094/MPMI-21-11-1498
pubmed: 18842098
McIntosh, M., Krol, E. & Becker, A. Competitive and cooperative effects in quorum-sensing-regulated galactoglucan biosynthesis in Sinorhizobium meliloti. J. Bacteriol. 190, 5308–5317, https://doi.org/10.1128/JB.00063-08 (2008).
doi: 10.1128/JB.00063-08
pubmed: 18515420
pmcid: 2493264
Becker, A. et al. The 32-kilobase exp gene cluster of Rhizobium meliloti directing the biosynthesis of galactoglucan: genetic organization and properties of the encoded gene products. J. Bacteriol. 179, 1375–1384 (1997).
doi: 10.1128/JB.179.4.1375-1384.1997
Martín, M., Lloret, J., Sánchez-Contreras, M., Bonilla, I. & Rivilla, R. MucR is necessary for galactoglucan production in Sinorhizobium meliloti EFB1. Mol. Plant. Microbe Interact. 13, 129–135, https://doi.org/10.1094/MPMI.2000.13.1.129 (2000).
doi: 10.1094/MPMI.2000.13.1.129
pubmed: 10656595
Janczarek, M. & Skorupska, A. The Rhizobium leguminosarum bv. trifolii RosR: transcriptional regulator involved in exopolysaccharide production. Mol. Plant. Microbe Interact. 20, 867–881, https://doi.org/10.1094/MPMI-20-7-0867 (2007).
doi: 10.1094/MPMI-20-7-0867
pubmed: 17601173
Bahlawane, C., Baumgarth, B., Serrania, J., Rüberg, S. & Becker, A. Fine-tuning of galactoglucan biosynthesis in Sinorhizobium meliloti by differential WggR (ExpG)-, PhoB-, and MucR-dependent regulation of two promoters. J. Bacteriol. 190, 3456–3466, https://doi.org/10.1128/JB.00062-08 (2008).
doi: 10.1128/JB.00062-08
pubmed: 18344362
pmcid: 2394980
Fumeaux, C. et al. Cell cycle transition from S-phase to G1 in Caulobacter is mediated by ancestral virulence regulators. Nat. Commun. 5, 4081, https://doi.org/10.1038/ncomms5081 (2014).
doi: 10.1038/ncomms5081
pubmed: 24939058
pmcid: 4083442
Panis, G., Murray, S. R. & Viollier, P. H. Versatility of global transcriptional regulators in alpha-Proteobacteria: from essential cell cycle control to ancillary functions. FEMS Microbiol. Rev. 39, 120–133, https://doi.org/10.1093/femsre/fuu002 (2015).
doi: 10.1093/femsre/fuu002
pubmed: 25793963
Baglivo, I. et al. Ml proteins from Mesorhizobium loti and MucR from Brucella abortus: an AT-rich core DNA-target site and oligomerization ability. Sci. Rep. 7, 15805, https://doi.org/10.1038/s41598-017-16127-5 (2017).
doi: 10.1038/s41598-017-16127-5
pubmed: 29150637
pmcid: 5693944
Pirone, L. et al. Identifying the region responsible for Brucella abortus MucR higher-order oligomer formation and examining its role in gene regulation. Sci. Rep. 8, 17238, https://doi.org/10.1038/s41598-018-35432-1 (2018).
doi: 10.1038/s41598-018-35432-1
pubmed: 30467359
pmcid: 6250670
Baglivo, I. et al. MucR binds multiple target sites in the promoter of its own gene and is a heat-stable protein: Is MucR a H-NS-like protein? FEBS Open. Bio 8, 711–718, https://doi.org/10.1002/2211-5463.12411 (2018).
doi: 10.1002/2211-5463.12411
pubmed: 29632823
pmcid: 5881533
Lang, B. et al. High-affinity DNA binding sites for H-NS provide a molecular basis for selective silencing within proteobacterial genomes. Nucleic Acids Res. 35, 6330–6337, https://doi.org/10.1093/nar/gkm712 (2007).
doi: 10.1093/nar/gkm712
pubmed: 17881364
pmcid: 2094087
Castang, S. & Dove, S. L. High-order oligomerization is required for the function of the H-NS family member MvaT in Pseudomonas aeruginosa. Mol. Microbiol. 78, 916–931, https://doi.org/10.1111/j.1365-2958.2010.07378.x (2010).
doi: 10.1111/j.1365-2958.2010.07378.x
pubmed: 20815825
pmcid: 2978250
Netti, F. et al. An experimentally tested scenario for the structural evolution of eukaryotic Cys2His2 zinc fingers from eubacterial ros homologs. Mol. Biol. Evol. 30, 1504–1513, https://doi.org/10.1093/molbev/mst068 (2013).
doi: 10.1093/molbev/mst068
pubmed: 23576569
Palmieri, M. et al. Structural Zn(II) implies a switch from fully cooperative to partly downhill folding in highly homologous proteins. J. Am. Chem. Soc. 135, 5220–5228, https://doi.org/10.1021/ja4009562 (2013).
doi: 10.1021/ja4009562
pubmed: 23484956
Malgieri, G. et al. The prokaryotic Cys2His2 zinc-finger adopts a novel fold as revealed by the NMR structure of Agrobacterium tumefaciens Ros DNA-binding domain. Proc. Natl Acad. Sci. USA 104, 17341–17346, https://doi.org/10.1073/pnas.0706659104 (2007).
doi: 10.1073/pnas.0706659104
pubmed: 17956987
Malgieri, G. et al. Zinc to cadmium replacement in the A. thaliana SUPERMAN Cys
doi: 10.1002/bip.21680
pubmed: 21618209
Malgieri, G. et al. Zinc to cadmium replacement in the prokaryotic zinc-finger domain. Metallomics 6, 96–104, https://doi.org/10.1039/c3mt00208j (2014).
doi: 10.1039/c3mt00208j
pubmed: 24287553
Malgieri, G. et al. Folding mechanisms steer the amyloid fibril formation propensity of highly homologous proteins. Chem. Sci. 9, 3290–3298, https://doi.org/10.1039/c8sc00166a (2018).
doi: 10.1039/c8sc00166a
pubmed: 29780459
pmcid: 5933289
Sivo, V. et al. Ni(II), Hg(II), and Pb(II) Coordination in the Prokaryotic Zinc-Finger Ros87. Inorg. Chem. 58, 1067–1080, https://doi.org/10.1021/acs.inorgchem.8b02201 (2019).
doi: 10.1021/acs.inorgchem.8b02201
pubmed: 30596504
Baglivo, I. et al. Genetic and epigenetic mutations affect the DNA binding capability of human ZFP57 in transient neonatal diabetes type 1. FEBS Lett. 587, 1474–1481, https://doi.org/10.1016/j.febslet.2013.02.045 (2013).
doi: 10.1016/j.febslet.2013.02.045
pubmed: 23499433
pmcid: 3655262
Kochańczyk, T., Drozd, A. & Krężel, A. Relationship between the architecture of zinc coordination and zinc binding affinity in proteins–insights into zinc regulation. Metallomics 7, 244–257, https://doi.org/10.1039/c4mt00094c (2015).
doi: 10.1039/c4mt00094c
pubmed: 25255078
Padjasek, M. et al. Structural zinc binding sites shaped for greater works: Structure-function relations in classical zinc finger, hook and clasp domains. J. Inorg. Biochem. 204, 110955, https://doi.org/10.1016/j.jinorgbio.2019.110955 (2019).
doi: 10.1016/j.jinorgbio.2019.110955
pubmed: 31841759
Kluska, K., Adamczyk, J. & Krężel, A. Metal binding properties, stability and reactivity of zinc fingers. Coord. Chem. Rev. 367, 18–64 (2018).
doi: 10.1016/j.ccr.2018.04.009
Baglivo, I. et al. The structural role of the zinc ion can be dispensable in prokaryotic zinc-finger domains. Proc. Natl Acad. Sci. USA 106, 6933–6938, https://doi.org/10.1073/pnas.0810003106 (2009).
doi: 10.1073/pnas.0810003106
pubmed: 19369210
Baglivo, I. et al. Molecular strategies to replace the structural metal site in the prokaryotic zinc finger domain. Biochim. Biophys. Acta 1844, 497–504, https://doi.org/10.1016/j.bbapap.2013.12.019 (2014).
doi: 10.1016/j.bbapap.2013.12.019
pubmed: 24389235
Palmieri, M. et al. Deciphering the zinc coordination properties of the prokaryotic zinc finger domain: The solution structure characterization of Ros87 H42A functional mutant. J. Inorg. Biochem. 131, 30–36, https://doi.org/10.1016/j.jinorgbio.2013.10.016 (2014).
doi: 10.1016/j.jinorgbio.2013.10.016
pubmed: 24239910
D’Abrosca, G. et al. The (unusual) aspartic acid in the metal coordination sphere of the prokaryotic zinc finger domain. J. Inorg. Biochem. 161, 91–98, https://doi.org/10.1016/j.jinorgbio.2016.05.006 (2016).
doi: 10.1016/j.jinorgbio.2016.05.006
pubmed: 27238756
Sivo, V. et al. Co(II) Coordination in Prokaryotic Zinc Finger Domains as Revealed by UV-Vis Spectroscopy. Bioinorg. Chem. Appl. 2017, 1527247, https://doi.org/10.1155/2017/1527247 (2017).
doi: 10.1155/2017/1527247
pubmed: 29386985
pmcid: 5745721
Simons, K. T., Kooperberg, C., Huang, E. & Baker, D. Assembly of protein tertiary structures from fragments with similar local sequences using simulated annealing and Bayesian scoring functions. J. Mol. Biol. 268, 209–225, https://doi.org/10.1006/jmbi.1997.0959 (1997).
doi: 10.1006/jmbi.1997.0959
pubmed: 9149153
Drozdetskiy, A., Cole, C., Procter, J. & Barton, G. J. JPred4: a protein secondary structure prediction server. Nucleic Acids Res. 43, W389–394, https://doi.org/10.1093/nar/gkv332 (2015).
doi: 10.1093/nar/gkv332
pubmed: 25883141
pmcid: 4489285
D’Abrosca, G. et al. Structural Characterization of the Lactobacillus Plantarum FlmC Protein Involved in Biofilm Formation. Molecules 23, https://doi.org/10.3390/molecules23092252 (2018).
Raman, S. et al. Structure prediction for CASP8 with all-atom refinement using Rosetta. Proteins 77(Suppl 9), 89–99, https://doi.org/10.1002/prot.22540 (2009).
doi: 10.1002/prot.22540
pubmed: 19701941
pmcid: 3688471
Maestro, Schrödinger, LLC, New York, NY (2016).
Russo, L. et al. Towards understanding the molecular recognition process in prokaryotic zinc-finger domain. Eur J Med Chem, https://doi.org/10.1016/j.ejmech.2014.09.040 (2014).
Malgieri, G. et al. Structural basis of a temporin 1b analogue antimicrobial activity against Gram negative bacteria determined by CD and NMR techniques in cellular environment. ACS Chem. Biol. 10, 965–969, https://doi.org/10.1021/cb501057d (2015).
doi: 10.1021/cb501057d
pubmed: 25622128
Arena, G. et al. Zinc(II) complexes of ubiquitin: speciation, affinity and binding features. Chemistry 17, 11596–11603, https://doi.org/10.1002/chem.201101364 (2011).
doi: 10.1002/chem.201101364
pubmed: 21953931
Wishart, D. S., Sykes, B. D. & Richards, F. M. The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy. Biochemistry 31, 1647–1651 (1992).
doi: 10.1021/bi00121a010
Esposito, D. et al. H-NS oligomerization domain structure reveals the mechanism for high order self-association of the intact protein. J. Mol. Biol. 324, 841–850, https://doi.org/10.1016/s0022-2836(02)01141-5 (2002).
doi: 10.1016/s0022-2836(02)01141-5
pubmed: 12460581
Shindo, H. et al. Solution structure of the DNA binding domain of a nucleoid-associated protein, H-NS, from Escherichia coli. FEBS Lett. 360, 125–131, https://doi.org/10.1016/0014-5793(95)00079-o (1995).
doi: 10.1016/0014-5793(95)00079-o
pubmed: 7875316
Hess, B., Kutzner, C., van der Spoel, D. & Lindahl, E. GROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation. J. Chem. Theory Comput. 4, 435–447, https://doi.org/10.1021/ct700301q (2008).
doi: 10.1021/ct700301q
pubmed: 26620784
Lindorff-Larssen, K. et al. Improved side-chain torsions potentials for the Amber ff99SB protein force field. Proteins 78, 1950–1958 (2010).
Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W. & Klein, M. L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926–935, https://doi.org/10.1063/1.445869 (1983).
doi: 10.1063/1.445869
Paladino, A., Marchetti, F., Ponzoni, L. & Colombo, G. The Interplay between Structural Stability and Plasticity Determines Mutation Profiles and Chaperone Dependence in Protein Kinases. J. Chem. Theory Comput. 14, 1059–1070, https://doi.org/10.1021/acs.jctc.7b00997 (2018).
doi: 10.1021/acs.jctc.7b00997
pubmed: 29262682
Paladino, A. & Zangi, R. Ribose 2’-Hydroxyl Groups Stabilize RNA Hairpin Structures Containing GCUAA Pentaloop. J. Chem. Theory Comput. 9, 1214–1221, https://doi.org/10.1021/ct3006216 (2013).
doi: 10.1021/ct3006216
pubmed: 26588764
Bussi, G., Donadio, D. & Parrinello, M. Canonical sampling through velocity rescaling. J. Chem. Phys. 126, 014101, https://doi.org/10.1063/1.2408420 (2007).
doi: 10.1063/1.2408420
pubmed: 17212484
Berendsen, H. J. C., Postma, J. P. M., van Gunsteren, W. F., DiNola, A. & Haak, J. R. Molecular dynamics with coupling to an external bath. J. Chem. Phys. 81, 3684–3690, https://doi.org/10.1063/1.448118 (1984).
doi: 10.1063/1.448118
Darden, T., York, D. & Pedersen, L. Particle mesh Ewald: An N⋅log(N) method for Ewald sums in large systems. J. Chem. Phys. 98, 10089–10092, https://doi.org/10.1063/1.464397 (1993).
doi: 10.1063/1.464397
Hess, B., Bekker, H., Berendsen, H. & Fraaije, J. LINCS: A Linear Constraint Solver for molecular simulations. Journal of Computational Chemistry 18, https://doi.org/10.1002/(SICI)1096-987X(199709)18:123.0.CO;2-H (1998).
Daura, X., Jaun, B., Seebach, D., van Gunsteren, W. F. & Mark, A. E. Reversible peptide folding in solution by molecular dynamics simulation. J. Mol. Biol. 280, 925–932, https://doi.org/10.1006/jmbi.1998.1885 (1998).
doi: 10.1006/jmbi.1998.1885
pubmed: 9671560
Muhandiram, D. R. & Kay, L. E. Gradient-Enhanced Triple-Resonance Three-Dimensional NMR Experiments with Improved Sensitivity. J. Magnetic Resonance, Ser. B 103, 203–216, https://doi.org/10.1006/jmrb.1994.1032 (1994).
doi: 10.1006/jmrb.1994.1032
Kay, L. E., Xu, G. Y. & Yamazaki, T. Enhanced-Sensitivity Triple-Resonance Spectroscopy with Minimal H2O Saturation. J. Magnetic Resonance, Ser. A 109, 129–133, https://doi.org/10.1006/jmra.1994.1145 (1994).
doi: 10.1006/jmra.1994.1145
Pelta, M. D., Barjat, H., Morris, G. A., Davis, A. L. & Hammond, S. J. Pulse sequences for high-resolution diffusion-ordered spectroscopy (HR-DOSY). Magnetic Resonance in Chemistry 36, 706–714, 10.1002/(SICI)1097-458X(199810)36:10<706::AID-OMR363>3.0.CO;2-W (1998).
Travaglia, A. et al. Zinc(II) interactions with brain-derived neurotrophic factor N-terminal peptide fragments: inorganic features and biological perspectives. Inorg. Chem. 52, 11075–11083, https://doi.org/10.1021/ic401318t (2013).
doi: 10.1021/ic401318t
pubmed: 24070197
Keller, R. The Computer Aided Resonance Assignment Tutorial (Cantina-Verlag, Switzerland (2004).
Urbani, A. et al. The metal binding site of the hepatitis C virus NS3 protease. A spectroscopic investigation. J. Biol. Chem. 273, 18760–18769 (1998).
doi: 10.1074/jbc.273.30.18760
Simpson, R. J. Y. et al. CCHX Zinc Finger Derivatives Retain the Ability to Bind Zn(II) and Mediate Protein-DNA Interactions. J. Biol. Chem. 278, 28011–28018 (2003).
doi: 10.1074/jbc.M211146200
Malgieri, G. & Grasso, G. The clearance of misfolded proteins in neurodegenerative diseases by zinc metalloproteases: An inorganic perspective. Coord. Chem. Rev. 260, 139–155, https://doi.org/10.1016/j.ccr.2013.10.008 (2014).
doi: 10.1016/j.ccr.2013.10.008
De Tommaso, G. et al. fac-[Re(H2O)3(CO)3]+ Complexed with Histidine and Imidazole in Aqueous Solution: Speciation, Affinity and Binding Features. ChemistrySelect 1, 3739–3744, https://doi.org/10.1002/slct.201600817 (2016).
doi: 10.1002/slct.201600817
Garrett, D. S., Seok, Y. J., Peterkofsky, A., Clore, G. M. & Gronenborn, A. M. Identification by NMR of the binding surface for the histidine-containing phosphocarrier protein HPr on the N-terminal domain of enzyme I of the Escherichia coli phosphotransferase system. Biochemistry 36, 4393–4398, https://doi.org/10.1021/bi970221q (1997).
doi: 10.1021/bi970221q
pubmed: 9109646