Characterization of the structural forces governing the reversibility of the thermal unfolding of the human acidic fibroblast growth factor

Agrawal, Shilpi; Govind Kumar, Vivek; Gundampati, Ravi Kumar; Moradi, Mahmoud; Kumar, Thallapuranam Krishnaswamy Suresh

doi:10.1038/s41598-021-95050-2

Title: Characterization of the structural forces governing the reversibility of the thermal unfolding of the human acidic fibroblast growth factor

Journal Article · 01 August 2021 · Scientific Reports

DOI: https://doi.org/10.1038/s41598-021-95050-2 · OSTI ID:1816669

Agrawal, Shilpi ^[1]; Govind Kumar, Vivek ^[2]; Gundampati, Ravi Kumar ^[2]; Moradi, Mahmoud ^[2]; Kumar, Thallapuranam Krishnaswamy Suresh ^[2]

Univ. of Arkansas, Fayetteville, AR (United States). Dept. of Chemistry and Biochemistry; OSTI
Univ. of Arkansas, Fayetteville, AR (United States). Dept. of Chemistry and Biochemistry

Human acidic fibroblast growth factor (hFGF1) is an all beta-sheet protein that is involved in the regulation of key cellular processes including cell proliferation and wound healing. hFGF1 is known to aggregate when subjected to thermal unfolding. In this study, we investigate the equilibrium unfolding of hFGF1 using a wide array of biophysical and biochemical techniques. Systematic analyses of the thermal and chemical denaturation data on hFGF1 variants (Q54P, K126N, R136E, K126N/R136E, Q54P/K126N, Q54P/R136E, and Q54P/K126N/R136E) indicate that nullification of charges in the heparin-binding pocket can significantly increase the stability of wtFGF1. Triple variant (Q54P/K126N/R136E) was found to be the most stable of all the hFGF1 variants studied. With the exception of triple variant, thermal unfolding of wtFGF1 and the other variants is irreversible. Thermally unfolded triple variant refolds completely to its biologically native conformation. Microsecond-level molecular dynamic simulations reveal that a network of hydrogen bonds and salt bridges linked to Q54P, K126N, and R136E mutations, are responsible for the high stability and reversibility of thermal unfolding of the triple variant. In our opinion, the findings of the study provide valuable clues for the rational design of a stable hFGF1 variant that exhibits potent wound healing properties.

View Accepted Manuscript (DOE)

Cite

Export

Save

Research Organization:: Univ. of Arkansas, Fayetteville, AR (United States)

Sponsoring Organization:: USDOE

Grant/Contract Number:: FG02-01ER15161

OSTI ID:: 1816669

Journal Information:: Scientific Reports, Journal Name: Scientific Reports Journal Issue: 1 Vol. 11; ISSN 2045-2322

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

References (50)

Scalable molecular dynamics with NAMD Phillips, James C.; Braun, Rosemary; Wang, Wei Journal of Computational Chemistry, Vol. 26, Issue 16, p. 1781-1802 https://doi.org/10.1002/jcc.20289	journal	January 2005
Folding nucleus structure persists in thermally-aggregated FGF-1: Folding Nucleus in FGF-1 Aggregate Longo, Liam M.; Gao, Yuan; Tenorio, Connie A. Protein Science, Vol. 27, Issue 2 https://doi.org/10.1002/pro.3332	journal	November 2017
An atomic resolution structure for human fibroblast growth factor 1 Bernett, Matthew J.; Somasundaram, Thayumanasamy; Blaber, Michael Proteins: Structure, Function, and Bioinformatics, Vol. 57, Issue 3 https://doi.org/10.1002/prot.20239	journal	July 2004
Identification of "Molten Globule"-like State in an All β-Sheet Protein Kumar, T. K. S.; Jayaraman, G.; Lee, C. S. Biochemical and Biophysical Research Communications, Vol. 207, Issue 2 https://doi.org/10.1006/bbrc.1995.1221	journal	February 1995
Complete Unfolding of the Titin Molecule under External Force Kellermayer, Miklós S. Z.; Smith, Steven B.; Bustamante, Carlos Journal of Structural Biology, Vol. 122, Issue 1-2 https://doi.org/10.1006/jsbi.1998.3988	journal	January 1998
VMD: Visual molecular dynamics Humphrey, William; Dalke, Andrew; Schulten, Klaus Journal of Molecular Graphics, Vol. 14, Issue 1 https://doi.org/10.1016/0263-7855(96)00018-5	journal	February 1996
Unfolding of Titin Domains Explains the Viscoelastic Behavior of Skeletal Myofibrils Minajeva, Ave; Kulke, Michael; Fernandez, Julio M. Biophysical Journal, Vol. 80, Issue 3 https://doi.org/10.1016/S0006-3495(01)76116-4	journal	March 2001
Structurally Homologous All β-Barrel Proteins Adopt Different Mechanisms of Folding Srimathi, Thiagarajan; Kumar, Thallampuranam Krishnaswamy S.; Kathir, Karuppanan Muthusamy Biophysical Journal, Vol. 85, Issue 1 https://doi.org/10.1016/S0006-3495(03)74491-9	journal	July 2003
Reversible Thermal Denaturation of Human FGF-1 Induced by Low Concentrations of Guanidine Hydrochloride Blaber, Sachiko I.; Culajay, Juan F.; Khurana, Archana Biophysical Journal, Vol. 77, Issue 1 https://doi.org/10.1016/S0006-3495(99)76904-3	journal	July 1999
Hysteresis in the triple helix-coil transition of type III collagen. Davis, J. M.; Bächinger, H. P. Journal of Biological Chemistry, Vol. 268, Issue 34 https://doi.org/10.1016/S0021-9258(19)74481-0	journal	December 1993
Probing the role of proline −135 on the structure, stability, and cell proliferation activity of human acidic fibroblast growth factor Davis, Julie Eberle; Alghanmi, Arwa; Gundampati, Ravi Kumar Archives of Biochemistry and Biophysics, Vol. 654 https://doi.org/10.1016/j.abb.2018.07.017	journal	September 2018
Equilibrium unfolding of an oligomeric protein involves formation of a multimeric intermediate state(s) Hsieh, Hui-Chu; Kumar, Thallapuranam Krishnaswamy S.; Chiu, Chi-Cheng Biochemical and Biophysical Research Communications, Vol. 326, Issue 1 https://doi.org/10.1016/j.bbrc.2004.10.211	journal	December 2004
Design of a thrombin resistant human acidic fibroblast growth factor (hFGF1) variant that exhibits enhanced cell proliferation activity Kerr, Rebecca; Agrawal, Shilpi; Maity, Sanhita Biochemical and Biophysical Research Communications, Vol. 518, Issue 2 https://doi.org/10.1016/j.bbrc.2019.08.029	journal	October 2019
Uncoupling the Mitogenic and Metabolic Functions of FGF1 by Tuning FGF1-FGF Receptor Dimer Stability Huang, Zhifeng; Tan, Yi; Gu, Junlian Cell Reports, Vol. 20, Issue 7 https://doi.org/10.1016/j.celrep.2017.06.063	journal	August 2017
Protein aggregation – Mechanisms, detection, and control Wang, Wei; Roberts, Christopher J. International Journal of Pharmaceutics, Vol. 550, Issue 1-2 https://doi.org/10.1016/j.ijpharm.2018.08.043	journal	October 2018
Highly Stable Mutants of Human Fibroblast Growth Factor-1 Exhibit Prolonged Biological Action Zakrzewska, Malgorzata; Krowarsch, Daniel; Wiedlocha, Antoni Journal of Molecular Biology, Vol. 352, Issue 4 https://doi.org/10.1016/j.jmb.2005.07.066	journal	September 2005
The Ambivalent Role of Proline Residues in an Intrinsically Disordered Protein: From Disorder Promoters to Compaction Facilitators Mateos, Borja; Conrad-Billroth, Clara; Schiavina, Marco Journal of Molecular Biology, Vol. 432, Issue 9 https://doi.org/10.1016/j.jmb.2019.11.015	journal	April 2020
Using protein engineering to understand and modulate aggregation Ebo, Jessica S.; Guthertz, Nicolas; Radford, Sheena E. Current Opinion in Structural Biology, Vol. 60 https://doi.org/10.1016/j.sbi.2020.01.005	journal	February 2020
CHARMM-GUI Input Generator for NAMD, GROMACS, AMBER, OpenMM, and CHARMM/OpenMM Simulations Using the CHARMM36 Additive Force Field Lee, Jumin; Cheng, Xi; Swails, Jason M. Journal of Chemical Theory and Computation, Vol. 12, Issue 1 https://doi.org/10.1021/acs.jctc.5b00935	journal	December 2015
Manipulating the Folding Landscape of a Multidomain Protein Kantaev, Raisa; Riven, Inbal; Goldenzweig, Adi The Journal of Physical Chemistry B, Vol. 122, Issue 49 https://doi.org/10.1021/acs.jpcb.8b04834	journal	August 2018
Protein Folding Cooperativity and Thermodynamic Barriers of the Simplest β-Sheet Fold: A Survey of WW Domains Iglesias-Bexiga, Manuel; Szczepaniak, Malwina; Sánchez de Medina, Celia The Journal of Physical Chemistry B, Vol. 122, Issue 49 https://doi.org/10.1021/acs.jpcb.8b05198	journal	July 2018
Cleavage, Downregulation, and Aggregation of Serum Amyloid A Wang, Wenhua; Khatua, Prabir; Hansmann, Ulrich H. E. The Journal of Physical Chemistry B, Vol. 124, Issue 6 https://doi.org/10.1021/acs.jpcb.9b10843	journal	January 2020
Exploring β-Sheet Structure and Interactions with Chemical Model Systems Nowick, James S. Accounts of Chemical Research, Vol. 41, Issue 10 https://doi.org/10.1021/ar800064f	journal	September 2008
Temperature-Induced Denaturation and Renaturation of Triosephosphate Isomerase from Saccharomyces cerevisiae : Evidence of Dimerization Coupled to Refolding of the Thermally Unfolded Protein ^† Benítez-Cardoza, Claudia G.; Rojo-Domínguez, Arturo; Hernández-Arana, Andrés Biochemistry, Vol. 40, Issue 30 https://doi.org/10.1021/bi010528w	journal	July 2001
Investigation of the Structural Stability of the Human Acidic Fibroblast Growth Factor by Hydrogen−Deuterium Exchange ^† Chi, Ya-Hui; Kumar, Thallampuranam Krishnaswamy S.; Kathir, Karuppanan Muthusamy Biochemistry, Vol. 41, Issue 51 https://doi.org/10.1021/bi026218a	journal	December 2002
Observation of Multistate Kinetics during the Slow Folding and Unfolding of Barstar Bhuyan, Abani K.; Udgaonkar, Jayant B. Biochemistry, Vol. 38, Issue 28 https://doi.org/10.1021/bi990285w	journal	June 1999
Optimization of the Additive CHARMM All-Atom Protein Force Field Targeting Improved Sampling of the Backbone ϕ, ψ and Side-Chain χ ₁ and χ ₂ Dihedral Angles Best, Robert B.; Zhu, Xiao; Shim, Jihyun Journal of Chemical Theory and Computation, Vol. 8, Issue 9 https://doi.org/10.1021/ct300400x	journal	August 2012
Hysteresis as a Marker for Complex, Overlapping Landscapes in Proteins Andrews, Benjamin T.; Capraro, Dominique T.; Sulkowska, Joanna I. The Journal of Physical Chemistry Letters, Vol. 4, Issue 1 https://doi.org/10.1021/jz301893w	journal	December 2012
Protein misfolding, aggregation, and conformational strains in neurodegenerative diseases Soto, Claudio; Pritzkow, Sandra Nature Neuroscience, Vol. 21, Issue 10 https://doi.org/10.1038/s41593-018-0235-9	journal	September 2018
Ror2 signaling regulates Golgi structure and transport through IFT20 for tumor invasiveness Nishita, Michiru; Park, Seung-Yeol; Nishio, Tadashi Scientific Reports, Vol. 7, Issue 1 https://doi.org/10.1038/s41598-016-0028-x	journal	January 2017
Particle mesh Ewald: An N ⋅log( N ) method for Ewald sums in large systems Darden, Tom; York, Darrin; Pedersen, Lee The Journal of Chemical Physics, Vol. 98, Issue 12 https://doi.org/10.1063/1.464397	journal	June 1993
Constant pressure molecular dynamics algorithms Martyna, Glenn J.; Tobias, Douglas J.; Klein, Michael L. The Journal of Chemical Physics, Vol. 101, Issue 5 https://doi.org/10.1063/1.467468	journal	September 1994
The case for defined protein folding pathways Englander, S. Walter; Mayne, Leland Proceedings of the National Academy of Sciences, Vol. 114, Issue 31 https://doi.org/10.1073/pnas.1706196114	journal	June 2017
A small single-domain protein folds through the same pathway on and off the ribosome Guinn, Emily J.; Tian, Pengfei; Shin, Mia Proceedings of the National Academy of Sciences, Vol. 115, Issue 48 https://doi.org/10.1073/pnas.1810517115	journal	November 2018
Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding. Matthews, B. W.; Nicholson, H.; Becktel, W. J. Proceedings of the National Academy of Sciences, Vol. 84, Issue 19, p. 6663-6667 https://doi.org/10.1073/pnas.84.19.6663	journal	October 1987
Structural Events during the Refolding of an All β-Sheet Protein Samuel, Dharmaraj; Kumar, Thallampuranam Krishnaswamy Suresh; Balamurugan, Krishnaswamy Journal of Biological Chemistry, Vol. 276, Issue 6 https://doi.org/10.1074/jbc.M005921200	journal	February 2001
Increased Protein Stability of FGF1 Can Compensate for Its Reduced Affinity for Heparin Zakrzewska, Malgorzata; Wiedlocha, Antoni; Szlachcic, Anna Journal of Biological Chemistry, Vol. 284, Issue 37 https://doi.org/10.1074/jbc.M109.001289	journal	September 2009
Observed Hysteresis of Virus Capsid Disassembly Is Implicit in Kinetic Models of Assembly Singh, Sushmita; Zlotnick, Adam Journal of Biological Chemistry, Vol. 278, Issue 20 https://doi.org/10.1074/jbc.M211408200	journal	May 2003
Signaling switches and bistability arising from multisite phosphorylation in protein kinase cascades Markevich, Nick I.; Hoek, Jan B.; Kholodenko, Boris N. Journal of Cell Biology, Vol. 164, Issue 3 https://doi.org/10.1083/jcb.200308060	journal	January 2004
Prion-Like Protein Aggregates and Type 2 Diabetes Mukherjee, Abhisek; Soto, Claudio Cold Spring Harbor Perspectives in Medicine, Vol. 7, Issue 5 https://doi.org/10.1101/cshperspect.a024315	journal	February 2017
In vivo aspects of protein folding and quality control Balchin, David; Hayer-Hartl, Manajit; Hartl, F. Ulrich Science, Vol. 353, Issue 6294 https://doi.org/10.1126/science.aac4354	journal	June 2016
Large Sparse Sets of Linear Equations (J. K. Reid, ed.); Sparse Matrices and Their Applications (Donald J. Rose and Ralph A. Willoughby, ed.) Parlett, B. N. SIAM Review, Vol. 16, Issue 3 https://doi.org/10.1137/1016066	journal	July 1974
A 32x32x32, spatially distributed 3D FFT in four microseconds on Anton Young, Cliff; Bank, Joseph A.; Dror, Ron O. Proceedings of the Conference on High Performance Computing Networking, Storage and Analysis - SC '09 https://doi.org/10.1145/1654059.1654083	conference	January 2009
Automated Builder and Database of Protein/Membrane Complexes for Molecular Dynamics Simulations Jo, Sunhwan; Kim, Taehoon; Im, Wonpil PLoS ONE, Vol. 2, Issue 9 https://doi.org/10.1371/journal.pone.0000880	journal	September 2007
Protein folding, misfolding and aggregation: The importance of two-electron stabilizing interactions Cieplak, Andrzej Stanisław PLOS ONE, Vol. 12, Issue 9 https://doi.org/10.1371/journal.pone.0180905	journal	September 2017
Targeting Drugs Against Fibroblast Growth Factor(s)-Induced Cell Signaling Agrawal, Shilpi; Maity, Sanhita; AlRaawi, Zeina Current Drug Targets, Vol. 22, Issue 2 https://doi.org/10.2174/1389450121999201012201926	journal	February 2021
Elucidating Protein-protein Interactions Through Computational Approaches and Designing Small Molecule Inhibitors Against them for Various Diseases Sarkar, Sharanya; Gulati, Khushboo; Kairamkonda, Manikyaprabhu Current Topics in Medicinal Chemistry, Vol. 18, Issue 20 https://doi.org/10.2174/1568026618666181025114903	journal	December 2018
Protein comparability assessments and potential applicability of high throughput biophysical methods and data visualization tools to compare physical stability profiles Alsenaidy, Mohammad A.; Jain, Nishant K.; Kim, Jae H. Frontiers in Pharmacology, Vol. 5 https://doi.org/10.3389/fphar.2014.00039	journal	March 2014
Low Stability of Integrin-Binding Deficient Mutant of FGF1 Restricts Its Biological Activity Szlachcic, Anna; Sochacka, Martyna; Czyrek, Aleksandra Cells, Vol. 8, Issue 8 https://doi.org/10.3390/cells8080899	journal	August 2019
Protein Folding and Mechanisms of Proteostasis Díaz-Villanueva, José; Díaz-Molina, Raúl; García-González, Victor International Journal of Molecular Sciences, Vol. 16, Issue 8 https://doi.org/10.3390/ijms160817193	journal	July 2015