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Abstract:

Local events that affect specific regions of proteins are of utmost relevance for stability and function. The aim of this study is to quantitatively assess the importance of locally-focused dynamics by means of a simple chemical modification procedure. Taking human Frataxin as a working model, we investigated local fluctuations of the C-terminal region (the last 16 residues of the protein) by means of three L → C replacement mutants: L98C, L200C and L203C. The conformation and thermodynamic stability of each variant was assessed. All the variants exhibited native features and high stabilities: 9.1 (wild type), 8.1 (L198C), 7.0 (L200C) and 10.0 kcal mol −1 (L203C). In addition, kinetic rates of Cys chemical modification by DTNB and DTDPy were measured, conformational dynamics data were extracted and free energy for the local unfolding of the C-terminal region was estimated. The analysis of these results indicates that the conformation of the C-terminal region fluctuates with partial independence from global unfolding events. Additionally, numerical fittings of the kinetic model of the process suggest that the local transition occurs in the seconds to minutes timescale. In fact, standard free energy differences for local unfolding were found to be significantly lower than those of the global unfolding reaction, showing that chemical modification results may not be explained in terms of the global unfolding reaction alone. These results provide unequivocal experimental evidence of local phenomena with global effects and contribute to understanding how global and local stability are linked to protein dynamics. © 2019, The Author(s).

Registro:

Documento: Artículo
Título:Global Implications of Local Unfolding Phenomena, Probed by Cysteine Reactivity in Human Frataxin
Autor:Faraj, S.E.; Noguera, M.E.; Delfino, J.M.; Santos, J.
Filiación:Alejandro Paladini Institute of Biological Chemistry and Chemical Physics (UBA-CONICET), Faculty of Pharmacy and Biochemistry, University of Buenos Aires, Junín 956, (C1113AAD), Buenos Aires, Argentina
Departamento de Fisiología y Biología Molecular y Celular, Facultad de Ciencia Exactas y Naturales, Universidad de Buenos Aires. Instituto de Biociencias, Biotecnología y Biomedicina (iB3). Intendente Güiraldes 2160 - Ciudad Universitaria, C.A.B.A., 1428EGA, Argentina
Año:2019
Volumen:9
Número:1
DOI: http://dx.doi.org/10.1038/s41598-019-39429-2
Título revista:Scientific Reports
Título revista abreviado:Sci. Rep.
ISSN:20452322
Registro:http://digital.bl.fcen.uba.ar/collection/paper/document/paper_20452322_v9_n1_p_Faraj

Referencias:

  • Henzler-Wildman, K., Kern, D., Dynamic personalities of proteins (2007) Nature, 450, pp. 964-972. , COI: 1:CAS:528:DC%2BD2sXhsVaqtr%2FL
  • Campuzano, V., Friedreich’s Ataxia: Autosomal Recessive Disease Caused by an Intronic GAA Triplet Repeat Expansion (1996) Science, 271, pp. 1423-1427. , COI: 1:CAS:528:DyaK28XhsFegtbk%3D
  • Dhe-Paganon, S., Shigeta, R., Chi, Y.I., Ristow, M., Shoelson, S.E., Crystal structure of human frataxin (2000) J Biol Chem, 275, pp. 30753-30756. , COI: 1:CAS:528:DC%2BD3cXntlCjtLs%3D
  • Adinolfi, S., The factors governing the thermal stability of frataxin orthologues: how to increase a protein’s stability (2004) Biochemistry, 43, pp. 6511-6518. , COI: 1:CAS:528:DC%2BD2cXjsF2qt7g%3D
  • Faraj, S.E., Roman, E.A., Aran, M., Gallo, M., Santos, J., The alteration of the C-terminal region of human frataxin distorts its structural dynamics and function (2014) FEBS J, 281, pp. 3397-3419. , COI: 1:CAS:528:DC%2BC2cXht1ajur7K
  • Roman, E.A., Protein stability and dynamics modulation: the case of human frataxin (2012) PLoS One, 7. , COI: 1:CAS:528:DC%2BC38XhsVGhtb3L
  • Bogdanov, M., Mapping of Membrane Protein Topology by Substituted Cysteine Accessibility Method (SCAM) (2017) Methods Mol Biol, 1615, pp. 105-128
  • Jiang, N., Frieden, C., Intestinal fatty acid binding protein: characterization of mutant proteins containing inserted cysteine residues (1993) Biochemistry, 32, pp. 11015-11021. , COI: 1:CAS:528:DyaK3sXmtF2qsbc%3D
  • Santos, J., Folding of an abridged beta-lactamase (2004) Biochemistry, 43, pp. 1715-1723. , COI: 1:CAS:528:DC%2BD2cXltFOitA%3D%3D
  • Santos, J., Risso, V.A., Sica, M.P., Ermacora, M.R., Effects of serine-to-cysteine mutations on beta-lactamase folding (2007) Biophys J, 93, pp. 1707-1718. , COI: 1:CAS:528:DC%2BD2sXpsVKisLg%3D
  • Feng, Z., Ha, J.H., Loh, S.N., Identifying the site of initial tertiary structure disruption during apomyoglobin unfolding (1999) Biochemistry, 38, pp. 14433-14439. , COI: 1:CAS:528:DyaK1MXmsFGgsLY%3D
  • Feng, Z., Butler, M.C., Alam, S.L., Loh, S.N., On the nature of conformational openings: native and unfolded-state hydrogen and thiol-disulfide exchange studies of ferric aquomyoglobin (2001) J Mol Biol, 314, pp. 153-166. , COI: 1:CAS:528:DC%2BD3MXosF2gsbc%3D
  • Stratton, M.M., Cutler, T.A., Ha, J.H., Loh, S.N., Probing local structural fluctuations in myoglobin by size-dependent thiol-disulfide exchange (2010) Protein Sci, 19, pp. 1587-1594. , COI: 1:CAS:528:DC%2BC3cXptlaru7g%3D
  • Hvidt, A., Nielsen, S.O., Hydrogen exchange in proteins (1966) Adv Protein Chem, 21, pp. 287-386. , COI: 1:CAS:528:DyaF28XltVSktb8%3D
  • Baldwin, R.L., Early days of protein hydrogen exchange: 1954–1972 (2011) Proteins, 79, pp. 2021-2026. , COI: 1:CAS:528:DC%2BC3MXnt1Kluro%3D
  • Krishna, M.M., Hoang, L., Lin, Y., Englander, S.W., Hydrogen exchange methods to study protein folding (2004) Methods, 34, pp. 51-64. , COI: 1:CAS:528:DC%2BD2cXmt1Cguro%3D
  • Isom, D.G., Vardy, E., Oas, T.G., Hellinga, H.W., Picomole-scale characterization of protein stability and function by quantitative cysteine reactivity (2010) Proc Natl Acad Sci USA, 107, pp. 4908-4913. , COI: 1:CAS:528:DC%2BC3cXjvFGnsbY%3D
  • Faraj, S.E., Gonzalez-Lebrero, R.M., Roman, E.A., Santos, J., Human Frataxin Folds Via an Intermediate State. Role of the C-Terminal Region (2016) Sci Rep, 6. , COI: 1:CAS:528:DC%2BC28XisVant7c%3D
  • Prischi, F., Giannini, C., Adinolfi, S., Pastore, A., The N-terminus of mature human frataxin is intrinsically unfolded (2009) FEBS J, 276, pp. 6669-6676. , COI: 1:CAS:528:DC%2BD1MXhsVSmsLvM
  • Correia, A.R., Adinolfi, S., Pastore, A., Gomes, C.M., Conformational stability of human frataxin and effect of Friedreich’s ataxia-related mutations on protein folding (2006) Biochem J, 398, pp. 605-611. , COI: 1:CAS:528:DC%2BD28XoslGksrg%3D
  • van Bergen, L.A., Revisiting sulfur H-bonds in proteins: The example of peroxiredoxin AhpE (2016) Sci Rep, 6
  • Bulaj, G., Kortemme, T., Goldenberg, D.P., Ionization-reactivity relationships for cysteine thiols in polypeptides (1998) Biochemistry, 37, pp. 8965-8972. , COI: 1:CAS:528:DyaK1cXjsFehsLk%3D
  • Silvaroli, J.A., Ligand Binding Induces Conformational Changes in Human Cellular Retinol-binding Protein 1 (CRBP1) Revealed by Atomic Resolution Crystal Structures (2016) Journal of Biological Chemistry, 291, pp. 8528-8540. , COI: 1:CAS:528:DC%2BC28XmtVyjsrk%3D
  • Spolaore, B., Local Unfolding Is Required for the Site-Specific Protein Modification by Transglutaminase (2012) Biochemistry, 51, pp. 8679-8689. , COI: 1:CAS:528:DC%2BC38XhsFekur%2FL
  • Perkins, A., The sensitive balance between the fully folded and locally unfolded conformations of a model peroxiredoxin (2013) Biochemistry, 52, pp. 8708-8721. , COI: 1:CAS:528:DC%2BC3sXhslSmtr3K
  • Das, M., Structural Stability and Local Dynamics in Disease-Causing Mutants of Human Apolipoprotein A-I: What Makes the Protein Amyloidogenic? (2016) Journal of Molecular Biology, 428, pp. 449-462. , COI: 1:CAS:528:DC%2BC2MXhvFKktr%2FO
  • Ding, F., Furukawa, Y., Nukina, N., Dokholyan, N.V., Local unfolding of Cu, Zn superoxide dismutase monomer determines the morphology of fibrillar aggregates (2012) Journal of molecular biology, 421, pp. 548-560. , COI: 1:CAS:528:DC%2BC38XhtVOlsLjN
  • Kudryashova, E., Human Defensins Facilitate Local Unfolding of Thermodynamically Unstable Regions of Bacterial Protein Toxins (2014) Immunity, 41, pp. 709-721. , COI: 1:CAS:528:DC%2BC2cXhvFOitrbK
  • Nagano, N., Ota, M., Nishikawa, K., Strong hydrophobic nature of cysteine residues in proteins (1999) FEBS Lett, 458, pp. 69-71. , COI: 1:CAS:528:DyaK1MXmsFWit7k%3D
  • Noguera, M.E., Insights on the conformational dynamics of human frataxin through modifications of loop-1 (2017) Arch Biochem Biophys, 636, pp. 123-137. , COI: 1:CAS:528:DC%2BC2sXhvVKgsrvP
  • Correia, A.R., Pastore, C., Adinolfi, S., Pastore, A., Gomes, C.M., Dynamics, stability and iron-binding activity of frataxin clinical mutants (2008) FEBS J, 275, pp. 3680-3690. , COI: 1:CAS:528:DC%2BD1cXovVSmur8%3D
  • Faggianelli, N., Analyzing the Effects of a G137V Mutation in the FXN Gene (2015) Front Mol Neurosci, 8, p. 66
  • Myers, J.K., Pace, C.N., Scholtz, J.M., Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding (1995) Protein Sci, 4, pp. 2138-2148. , COI: 1:CAS:528:DyaK2MXoslygt7s%3D
  • Hoops, S., COPASI–a COmplex PAthway SImulator (2006) Bioinformatics, 22, pp. 3067-3074. , COI: 1:CAS:528:DC%2BD28Xht1OgsrvK

Citas:

---------- APA ----------
Faraj, S.E., Noguera, M.E., Delfino, J.M. & Santos, J. (2019) . Global Implications of Local Unfolding Phenomena, Probed by Cysteine Reactivity in Human Frataxin. Scientific Reports, 9(1).
http://dx.doi.org/10.1038/s41598-019-39429-2
---------- CHICAGO ----------
Faraj, S.E., Noguera, M.E., Delfino, J.M., Santos, J. "Global Implications of Local Unfolding Phenomena, Probed by Cysteine Reactivity in Human Frataxin" . Scientific Reports 9, no. 1 (2019).
http://dx.doi.org/10.1038/s41598-019-39429-2
---------- MLA ----------
Faraj, S.E., Noguera, M.E., Delfino, J.M., Santos, J. "Global Implications of Local Unfolding Phenomena, Probed by Cysteine Reactivity in Human Frataxin" . Scientific Reports, vol. 9, no. 1, 2019.
http://dx.doi.org/10.1038/s41598-019-39429-2
---------- VANCOUVER ----------
Faraj, S.E., Noguera, M.E., Delfino, J.M., Santos, J. Global Implications of Local Unfolding Phenomena, Probed by Cysteine Reactivity in Human Frataxin. Sci. Rep. 2019;9(1).
http://dx.doi.org/10.1038/s41598-019-39429-2