Much of what is known about protein dynamics has been studied using bulk biochemical techniques and harsh chemical denaturants. By contrast, we study proteins at the single molecule level and use mechanical forces to denature proteins. Force is a ubiquitous denaturant in biology. Our results are opening new vistas into protein function and challenging some widely accepted views of protein dynamics.
The mechanical architecture of proteins:
One big advantage of our approach is that denaturing forces and extension can be controlled not only in magnitude but also in their direction. For example, we discovered that the mechanical stability and unfolding pathway of ubiquitin strongly depend on the linkage through which the mechanical force is applied to the protein. Hence, a protein that is otherwise very stable may be easily unfolded by a relatively weak mechanical force applied through the right linkage. This may be a widespread mechanism in biological systems. Another surprising finding was the discovery that ankyrin, a protein made of multiple repeats, upon pulling, unfolds in a piecewise manner. The piecewise unfolding of multiple ANK repeats could behave like multiple buffers linked in series; to resist damagingly high forces, ANK repeats can be sacrificed and extended one at a time, without the whole protein losing its tertiary structure. Both of these discoveries are completely novel and could not have been anticipated from solution biochemistry.
Studies of protein folding from highly extended states:
Force-clamp spectroscopy is a novel platform to study protein folding. The coordinate for the unfolding reaction is known (end-to-end length), the unfolded state is well defined and can be controlled over wide ranges, and the folding trajectory can be followed in a single protein over time. We use two force-clamp protocols: force-quench and force-ramp. In contrast to the traditional two-state folding reactions observed in solution biochemistry, our folding trajectories from highly extended unfolded states are continuous and marked by several distinct stages. The time taken to fold is exponentially dependent on the stretching force applied during folding. While chain entropy makes a small contribution to the collapse, we have found that most of the driving force is hydrophobic and varying widely depending on the dihedral space traversed by the folding trajectory. This collapse mechanism is common to highly extended proteins, including non-folding elastomeric proteins like PEVK from titin.
The mechanical design of titin:
The protein titin provides muscle with its passive elasticity. Each titin molecule extends over half a sarcomere, and its extensibility has been studied both in situ6-10 and at the level of single molecules11-14. These studies suggested that titin is not a simple entropic spring but has a complex structure dependent elasticity. We use protein engineering and single molecule atomic force microscopy to examine the mechanical components that form the elastic region of human cardiac titin. We show that when these mechanical elements are combined, they explain the macroscopic behavior of titin in intact muscle. Our studies show the functional reconstitution of a protein from the sum of its parts.
Chemical reactions under a stretching force:
The mechanism by which mechanical forces regulate the kinetics of a chemical reaction is unknown. We use single molecule force-clamp spectroscopy and protein engineering to study the effect of force on the kinetics of thiol/disulfide exchange. Reduction of disulfide bonds via the thiol/disulfide exchange chemical reaction is crucial in regulating protein function and is of common occurrence in mechanically stressed proteins. Our work at the single bond level directly demonstrates that thiol/disulfide exchange in proteins is a force-dependent chemical reaction. Our findings suggest that mechanical force plays a role in disulfide reduction in vivo, a property which has never been explored by traditional biochemistry. Furthermore, our work also suggests that the kinetics of any chemical reaction that results in bond lengthening will be force dependent.
Enzyme catalysis under force:
Thioredoxins are enzymes that catalyze disulfide bond reduction in all living organisms. While catalysis is thought to proceed through a substitution nucleophilic bimolecular (SN2) reaction, the role of the enzyme in modulating this chemical reaction is unknown. Here we use single molecule force-clamp spectroscopy to probe the catalytic mechanism of E. coli thioredoxin (Trx). We apply mechanical force in the range of 25-450 pN to a disulfide bond substrate and monitor the reduction of these bonds by individual enzymes. Our results suggest that substrate conformational changes may be important in the regulation of Trx activity under conditions of oxidative stress and mechanical injury, such as those experienced in cardiovascular disease. Furthermore, single molecule atomic force microscopy (AFM) techniques, as shown here, can probe dynamic rearrangements within an enzyme's active site which cannot be resolved with any other current structural biological technique.
"Micromechanics of the Extracellular Matrix"
NIH R01 HL66030
The extracellular matrix is a pre-stressed mechanical network composed of a heterogeneous mixture of modular proteins and oligosaccharides that form the mechanical scaffold of living tissues. It plays crucial roles in the development of the cellular architectures that form all living organisms. A characteristic feature of the extracellular matrix is that its constituent molecules function under a stretching force. Our long-term aim is to understand the mechanisms by which these modular proteins respond and equilibrate with a stretching force, at the single molecule level. Studying the mechanical activity of these proteins will provide a mechanistic understanding of their function in healthy and diseased states.
Single molecule studies of the protein modules that compose extracellular matrix proteins such as tenascin and fibronectin have measured their mechanical stability, revealing strong unfolding hierarchies in their mechanical design. However, resistance to unfolding is not sufficient to describe the function of these proteins, which must equilibrate mechanically against a pulling force. This equilibration involves a dynamic balance of folding and unfolding events taking place against the pulling force. The molecular mechanisms underlying this kinetic equilibrium are unknown.
We will take advantage of recent advances in single molecule force spectroscopy that now permit a detailed observation of the folding and unfolding kinetics of a protein, while being pulled by a constant mechanical force. Force-clamp spectroscopy combined with protein engineering will be used to study the dynamics of unfolding of the cell binding module of the type III region of fibronectin, 10FNIII, and other key modules of extracellular matrix proteins. We will use the force-quench mode of this technique to study the mechanisms of protein folding under a stretching force. We will use single molecule techniques to study the mechanical strength of engineered disulphide bonds and their effect on the folding/unfolding pathways of selected modules from the proteins fibrillin and fibronectin. Our studies will reveal the molecular mechanisms underlying the dynamic equilibration of proteins with a pulling force, and more generally, demonstrate a novel approach to study the mechanisms of protein folding
"Molecular Basis of Titin Elasticity"
NIH R01 HL61228
We use single molecule techniques to study the mechanical design of the giant muscle protein titin. Titin spans half the length of a muscle sarcomere and can be over a micrometer long. The region of titin that overlaps with the sarcomeric I band determines muscle elasticity. I band titin has a characteristic modular design composed of tandem repeats of immunoglobulin (Ig) type domains, interrupted by a region rich in P, E, V and K residues and, in the case of cardiac muscle, another region made of a unique sequence named N2B. Our long-term aim is to understand the molecular mechanisms underlying titin elasticity in normal and diseased states.
Detailed single molecule studies of a few modules of the constitutively expressed regions of thin have revealed a complex structure-dependent mechanical design. However, titin elasticity is finely regulated through alternative splicing of its I band by a stunning number of axons, 106 exons coding for Ig modules and 114 exons coding for PEVK sequences. Nothing is known of the mechanical motifs encoded by the alternatively spliced regions of titin or how they govern titin elasticity. Also, a large number of titin lg domains have been shown to have a potential for the formation of mechanically stabilizing disulfide bridges. Moreover, titin molecules have been shown to interact suggesting that each elastic filament maybe be composed of several thin molecules, which may form elastic quaternary structures.
Hence, titin elasticity might be finely modulated by a several novel mechanisms.
We combine single molecule AFM and protein engineering techniques to study the individual building blocks of thin mechanics. We will examine the mechanical features of the different Ig modules and the PEVK sequences in the alternatively spliced regions of titin. We will examine the role of disulfide bridge formation on the mechanical properties of Ig domains. We will examine the mechanical stability of other protein folds such as the helical coiled-coil topology of the muscle protein uthropin and the alpha-betta topology of the highly conserved protein ubiquitin. We will study the origin of their mechanical design and compare it to that of the titin immunoglobulin modules. Through the use of protein engineering and mutagenesis, we will examine the role played by prolines (as well as E,V and K residues) in controlling the elasticity of the PEVK regions. Using a variety of oligomerization domains we will assemble bundles of titin based polyproteins to examine the effect of supra-molecular arrangements on titin mechanics. We expect that in addition to uncovering new mechanisms of regulating titin elasticity, our studies will contribute to further develop the new field of research on single molecule force spectroscopy.
PDFs of recent publications
Perez-Jimenez R, Wiita AP, Rodriguez-Larrea D, Kosuri P, Gavira JA, Sanchez-Ruiz JM, Fernandez JM.
Force-clamp spectroscopy detects residue co-evolution in enzyme catalysis.
J Biol Chem. 2008 Aug 7, In Press
Koti Ainavarapu SR, Wiita AP, Dougan L, Uggerud E, Fernandez JM.
Single-molecule force spectroscopy measurements of bond elongation during a bimolecular reaction.
J Am Chem Soc. 2008 May 21;130(20):6479-87
Dougan L, Feng G, Lu H, Fernandez JM.
Solvent molecules bridge the mechanical unfolding transition state of a protein.
Proc Natl Acad Sci U S A. 2008 Mar 4;105(9):3185-90
Szoszkiewicz R, Ainavarapu SR, Wiita AP, Perez-Jimenez R, Sanchez-Ruiz JM, Fernandez JM.
Dwell time analysis of a single-molecule mechanochemical reaction.
Langmuir. 2008 Feb 19;24(4):1356-64
Ainavarapu SR, Wiita AP, Huang HH, Fernandez JM.
A single-molecule assay to directly identify solvent-accessible disulfide bonds and probe their effect on protein folding.
J Am Chem Soc. 2008 Jan 16;130(2):436-7
Dougan L, Fernandez JM.
Tandem repeating modular proteins avoid aggregation in single molecule force spectroscopy experiments.
J Phys Chem A. 2007 Dec 13;111(49):12402-8
Wiita AP, Perez-Jimenez R, Walther KA, Gräter F, Berne BJ, Holmgren A, Sanchez-Ruiz JM, Fernandez JM.
Probing the chemistry of thioredoxin catalysis with force.
Nature. 2007 Nov 1;450(7166):124-7
Sarkar, Atom, Caamano, Sofia, Fernandez, Julio M.
The Mechanical Fingerprint of a Parallel Polyprotein Dimer
Biophys. J. 2007 92: L36-38
Brujic, Jasna, Hermans, Rodolfo I. Z., Garcia-Manyes, Sergi, Walther, Kirstin A., Fernandez, Julio M.
Dwell-Time Distribution Analysis of Polyprotein Unfolding Using Force-Clamp Spectroscopy
Biophys. J. 2007 92: 2896-2903
Garcia-Manyes, Sergi, Brujic, Jasna, Badilla, Carmen L., Fernandez, Julio M.
Force-clamp spectroscopy of single protein monomers reveal the individual unfolding and folding pathways of I27 and ubiquitin
Biophys. J. 2007 0: biophysj.107.104422
Raul Perez-Jimenez, Sergi Garcia-Manyes, Sri Rama Koti Ainavarapu, and Julio M. Fernandez
Mechanical Unfolding Pathways of the Enhanced Yellow
Fluorescent Protein Revealed by Single Molecule
Force Spectroscopy. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 52, pp. 40010–40014, December 29, 2006
Arun P. Wiita, Sri Rama Koti Ainavarapu, Hector H. Huang, and Julio M. Fernandez
Force-dependent chemical kinetics of disulfide bond
reduction observed with single-molecule techniques. PNAS May 9, 2006 vol. 103 no. 19
Jasna Brujic, Rodolfo I. Hermans Z., Kirstin A. Walther, and Julio M. Fernanez (2006)
Single-molecule force spectroscopy reveals signatures of glassy dynamics in the energy landscape of ubiquitin. Nature Physics, 2:282-286.
Kirstin A. Walther, Jasna Brujic, Hongbin Li, and Julio M. Fernanez (2006)
Sub-Angstrom Conformational Changes of a Single Molecule Captured by AFM Variance Analysis. Biophys. J., 90:3806-3812.
Lewyn Li, Hector Han-Li Huang, Carmen L. Badilla, and Julio M. Fernandez (2005)
Mechanical Unfolding Intermediates Observed by Single-molecule Force Spectroscopy in a Fibronectin Type III Module. J. Mol. Biol., 345:817-826.
Sri Rama Koti Ainavarapu, Lewyn Li, Carmen L. Badilla, and Julio M. Fernandez (2005)
Ligand Binding Modulates the Mechanical Stability of Dihydrofolate Reductase. Biophys. J., 89:3337-3344.
Lewyn Li, Svava Wetzel, Andreas Plueckthun, and Julio M. Fernandez (2006)
Stepwise Unfolding of Ankyrin Repeats in a Single Protein Revealed by Atomic Force Microscopy. Biophys. J.: Biophys. Lett.
A, Caamano S, Fernandez JM. The elasticity of individual
titin PEVK exons measured by single molecule atomic force
microscopy. J Biol Chem. 2005 Feb 25;280(8):6261-4.
Hector Han-Li Huang, Carmen L. Badilla and Julio M. Fernandez
Mechanical Unfolding Intermediates Observed by Single-molecule
Force Spectroscopy in a Fibronectin Type III Module (In
JM, Li H, Brujic J. (2004) Response to Comment on "Force-Clamp
Spectroscopy Monitors the Folding Trajectory of a Single
Protein" Science. 306(5695):411c.
A, Robertson RB, Fernandez JM. (2004) Simultaneous atomic
force microscope and fluorescence measurements of protein
unfolding using a calibrated evanescent wave. Proc Natl
Acad Sci U S A. 31;101(35):12882-6.
M, Li H, Fernandez JM. (2004) The unfolding kinetics of
ubiquitin captured with single-molecule force-clamp techniques.
Proc Natl Acad Sci U S A. 101(19):7299-304.
JM, Li H. (2004) Force-clamp spectroscopy monitors the folding
trajectory of a single protein. Science 303(5664):1674-8.
H. and J.M. Fernandez. (2003) Mechanical design of the first
proximal Ig domain of human cardiac titin revealed by single
molecule force spectroscopy. J. Mol. Biol., 334(1):75-86
PE, Oberhauser AF, Li H, Fernandez JM. (2003) The force-driven
conformations of heparin studied with single molecule force
microscopy. Biophys J. 85(4):2696-704.
M, Li H, Lu H, Marszalek PE, Oberhauser AF, Fernandez JM.
(2003) The mechanical stability of ubiquitin is linkage
dependent. Nat Struct Biol. 10(9):738-43.
JA, Baker TA, Fernandez JM, Sauer RT. (2003) Linkage between
ATP consumption and mechanical unfolding during the protein
processing reactions of an AAA+ degradation machine. Cell.
P.E., Li, H., Oberhauser, A.F., and Fernandez, J.M. (2002)
Chair-boat transitions in single polysaccharide molecules
observed with force-clamp AFM. Proc. Nat'l. Acad. Sci ,
Linke, W. A.; Kulke,
M.; Li, H. B.; Fujita-Becker, S.; Neagoe, C.; Manstein,
D. J.; Gautel, M.; Fernandez, J. M. (2002) PEVK domain of
titin: an entropic spring with actin-binding properties.
Journal of Structural Biology, 137: 194-205.
Li, H. B.; Linke,
W. A.; Oberhauser, A. F.; Carrion-Vazquez, M.; Kerkvliet,
J. G.; Lu, H.; Marszalek, P. E.; Fernandez, J. M. (2002)
Reverse engineering of the giant muscle protein titin. Nature,
A. F.; Badilla-Fernandez, C.; Carrion-Vazquez M.; Fernandez,
J. M. (2002) The Mechanical Hierarchies of Fibronectin Observed
with Single-molecule AFM. J. Mol. Biol., 319: 433-447.
J.M., Chu, S. and A.F. Oberhauser, (2001) Pulling on hairpins
(perspective), Science, 292:653-654.
T, Nie S, Fernandez JM (2001) Single molecules. Proc Natl
Acad Sci. 98(19):10527-8.
P.E., Li, H., & Fernandez, J.M., (2001) Fingerprinting
polysaccharides with single molecule atomic force microscopy,
Nature Biotechnology, 19:258-262.
M., Oberhauser,AF., Fisher,TE., Marszalek, PM., Li
H., & Fernandez, J.M. (2001) Mechanical design
of proteins studied by single-molecule force spectroscopy
and protein engineering.
Progress in Biophysics and Molecular Biology, 74:63-91.
Minajeva, Marc Ivemeyer, Julio M. Fernandez & Wolfgang
A. Linke (2001) Unfolding of titin domains explains
behavior of skeletal myofibrils. Biophys. J., 2001 Mar;80(3):1442-1451.
Hansma, P.K., Carrion-Vazquez, M., and Fernandez, J.M. (2001).
Stepwise unfolding of titin under force-clamp AFM. Proc.
Nat'l. Acad. Sci., 98:468-472.
H., Oberhauser, A.F., Redick, S. D., Carrion-Vazquez, M.,
Erickson, H.P., and Fernandez, J.M. (2001) Multiple conformations
of PEVK proteins detected by single molecule techniques.
Proc. Nat'l. Acad. Sci, 98:10682-10686.
T. E., Carrion-Vazquez, M., Oberhauser, A.F., Hongbin Li,
Marszalek, P.E., & Fernandez, J.M. (2000). Single molecule
force spectroscopy of modular proteins in the nervous system.
T. E., Marszalek, P.E., and Fernandez, J.M. (2000). Stretching
single molecules into novel conformations using the atomic
force microscope. Nature Struct. Biol., 7(9) 719-724.
P.E., Greenleaf, W.J., Li, H., Oberhauser, A.F., and Fernandez,
J.M. (2000) Atomic force microscopy captures quantized plastic
deformation in gold nanowires. Proc. Nat'l. Acad. Sci.,
Li, H., Oberhauser,
A.F., Fowler, S.B., Clarke, J., and Fernandez, J.M. (2000)
Atomic force microscopy reveals the mechanical design of
a modular protein. Proc. Nat'l. Acad. Sci., 97:6527-6531.
Fisher T. E.,
Carrion-Vazquez, M and Fernandez, J.M. (2000). Intracellular
Ca++ channel immunoreactivity in neuroendocrine axon terminals.
FEBS letters 24153:1-8.
H., Carrion-Vazquez, M., Oberhauser, A.F., Marszalek, P.E.
and Fernandez, J.M. (2000) Point mutations alter the mechanical
stability of immunoglobulin modules. Nature Struct. Biol.,
T.E., and Fernandez, J.M. (1999). Pulsed laser imaging of
Ca2+ influx in a neuroendocrine terminal. J. Neurosci. 19:7450-7457.
TE, Marszalek, PE, Oberhauser, AF, Carrion-Vazquez, M, and
Fernandez, JM (1999). The micro-mechanics of single molecules
studied with atomic force microscopy. Journal of Physiology,
P.E., Pang, Y.P., Li, H., Yazal, J.E., Oberhauser, A.F.,
and Fernandez, J.M. (1999). Atomic levers control pyranose
ring conformations. Proc. Natl. Acad. Sci. 96:7894-7898.
M., Oberhauser, A.F., Fowler, S.B., Marszalek, P.E., Broedel,
S.E., Clarke, J., and Fernandez, J.M. (1999). Mechanical
and chemical unfolding of a single protein: a comparison.
Proc. Natl. Acad. Sci. 96:3694-3699.
TE, Oberhauser, AF, Carrion-Vazquez, M, Marszalek, PE, and
Fernandez, JM (1999). Protein mechanics studied with atomic
force microscopy. Trends in Biochemical Sciences,24:369-410.
A.F., Marszalek, P.E., Carrion-Vazquez, M., and Fernandez,
J.M. (1999). Single protein misfolding events captured by
atomic force microscopy. Nature Struct. Biol. 6:1025-1028.
M., Marszalek, P.E., Oberhauser, A.F., and Fernandez, J.M.
(1999). Atomic force microscopy captures length phenotypes
in single proteins. Proc. Natl. Acad. Sci. USA 96:11288-11292.
P.E., Lu, H., Li, H., Carrion-Vazquez, M., Oberhauser, A.F.,
Schulten, K., and Fernandez, J.M. (1999). Mechanical unfolding
intermediates in titin modules. Nature 402:100-103.
C., Innocenti, B., and Fernandez, J.M. (1998). Regulation
of exocytotic fusion by cell inflation. Biophys. J., 74:1061-1073.
P.M., Oberhauser, A.F., Pang, Y.-P., and Fernandez, J.M.
(1998) Polysaccharide elasticity governed by chair-boat
transitions of the glucopyranose ring. Nature, 396:661-664.
A.F., Marszalek, P.E., Erickson, H.P., and Fernandez, J.M.
(1998). The molecular elasticity of tenascin, an extracellular
matrix protein. Nature, 393:181-185.
Fernandez, J.M., and Gaub, H.E. (1998). Elastically coupled
two-level systems as a model for biopolymer extensibility.
Phys. Rev. Lett., 81:4764-4767.
H.E., and Fernandez, J.M. (1998). The molecular elasticity
of individual proteins studied by AFM-related techniques.
M., Gautel, M., Oesterhelt, F., Fernandez, J.M. and Gaub,
H.E. (1997). Reversible unfolding of individual titin immunoglobulin
domains by AFM. Science, 276:1109-1112.
J.M. (1997). Cellular and molecular mechanics by atomic
force microscopy: capturing the exocytotic fusion pore in
vivo? Proc. Natl. Acad. Sci. 94:9-10.