Pulling geometry defines the mechanical resistance of a β-sheet protein

Proteins show diverse responses when placed under mechanical stress. The molecular origins of their differing mechanical resistance are still unclear, although the orientation of secondary structural elements relative to the applied force vector is thought to have an important function. Here, by using a method of protein immobilization that allows force to be applied to the same all-β protein, E2lip3, in two different directions, we show that the energy landscape for mechanical unfolding is markedly anisotropic. These results, in combination with molecular dynamics (MD) simulations, reveal that the unfolding pathway depends on the pulling geometry and is associated with unfolding forces that differ by an order of magnitude. Thus, the mechanical resistance of a protein is not dictated solely by amino acid sequence, topology or unfolding rate constant, but depends critically on the direction of the applied extension.

[1]  J Kolberg,et al.  A branched DNA signal amplification assay for quantification of nucleic acid targets below 100 molecules/ml. , 1997, Nucleic acids research.

[2]  K. Flaherty,et al.  Three-dimensional structure of a hammerhead ribozyme , 1994, Nature.

[3]  O. Uhlenbeck,et al.  A covalent crosslink converts the hammerhead ribozyme from a ribonuclease to an RNA ligase , 2001, Nature Structural Biology.

[4]  Sheena E Radford,et al.  Mechanically unfolding proteins: The effect of unfolding history and the supramolecular scaffold , 2002, Protein science : a publication of the Protein Society.

[5]  A. E. Sauer-Eriksson,et al.  Structure of the SRP19–RNA complex and implications for signal recognition particle assembly , 2002, Nature.

[6]  D. Speicher,et al.  Forced unfolding modulated by disulfide bonds in the Ig domains of a cell adhesion molecule. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[7]  A. Oberhauser,et al.  Mechanical design of proteins studied by single-molecule force spectroscopy and protein engineering. , 2000, Progress in biophysics and molecular biology.

[8]  J. M. Buzayan,et al.  Nucleic Acids Research Nucleotide sequence and newly formed phosphodJester bond of spontaneously Ugated satellite tobacco ringspot virus RNA , 2005 .

[9]  Vijay S. Pande,et al.  Mechanical Unfolding of a β-Hairpin Using Molecular Dynamics , 2000 .

[10]  D. Lilley,et al.  Dissection of the ion-induced folding of the hammerhead ribozyme using 19F NMR , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[11]  C Massire,et al.  MANIP: an interactive tool for modelling RNA. , 1998, Journal of molecular graphics & modelling.

[12]  R. Perham,et al.  Swinging arms and swinging domains in multifunctional enzymes: catalytic machines for multistep reactions. , 2000, Annual review of biochemistry.

[13]  O. Uhlenbeck,et al.  Divalent metal ions and the internal equilibrium of the hammerhead ribozyme. , 1995, Biochemistry.

[14]  I R Vetter,et al.  Solid-state synthesis and mechanical unfolding of polymers of T4 lysozyme. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[15]  J. Esteban,et al.  Kinetic Mechanism of the Hairpin Ribozyme , 1997, The Journal of Biological Chemistry.

[16]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[17]  W. Neupert,et al.  The protein import motor of mitochondria , 2002, Nature Reviews Molecular Cell Biology.

[18]  R Cedergren,et al.  Numbering system for the hammerhead. , 1992, Nucleic acids research.

[19]  N. Walter,et al.  Stability of hairpin ribozyme tertiary structure is governed by the interdomain junction , 1999, Nature Structural Biology.

[20]  K. Schulten,et al.  Steered molecular dynamics simulations of force‐induced protein domain unfolding , 1999, Proteins.

[21]  G D Stormo,et al.  Sequence requirements of the hammerhead RNA self-cleavage reaction. , 1990, Biochemistry.

[22]  Anthony C. Forster,et al.  Self-cleavage of virusoid RNA is performed by the proposed 55-nucleotide active site , 1987, Cell.

[23]  M. Karplus,et al.  Effective energy function for proteins in solution , 1999, Proteins.

[24]  M Karplus,et al.  Unfolding proteins by external forces and temperature: the importance of topology and energetics. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[25]  C Massire,et al.  DRAWNA: a program for drawing schematic views of nucleic acids. , 1994, Journal of molecular graphics.

[26]  S. Woodson,et al.  The effect of long-range loop-loop interactions on folding of the Tetrahymena self-splicing RNA. , 1999, Journal of molecular biology.

[27]  K. Schulten,et al.  Unfolding of titin immunoglobulin domains by steered molecular dynamics simulation. , 1998, Biophysical journal.

[28]  J. Clarke,et al.  Mechanical and chemical unfolding of a single protein: a comparison. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[29]  M. Rief,et al.  Reversible unfolding of individual titin immunoglobulin domains by AFM. , 1997, Science.

[30]  C. Tanford.  Macromolecules , 1994, Nature.

[31]  O. Uhlenbeck,et al.  Kinetic characterization of intramolecular and intermolecular hammerhead RNAs with stem II deletions. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[32]  P. Reche,et al.  Recognition of the lipoyl domain is the ultimate determinant of substrate channelling in the pyruvate dehydrogenase multienzyme complex. , 2001, Journal of molecular biology.

[33]  D. Thirumalai,et al.  Native topology determines force-induced unfolding pathways in globular proteins. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[34]  A. Matouschek,et al.  ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal. , 2001, Molecular cell.

[35]  A. Oberhauser,et al.  Atomic levers control pyranose ring conformations. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[36]  Restricted motion of the lipoyl-lysine swinging arm in the pyruvate dehydrogenase complex of Escherichia coli. , 2000, Biochemistry.

[37]  Mariano Carrion-Vazquez,et al.  The mechanical hierarchies of fibronectin observed with single-molecule AFM. , 2002, Journal of molecular biology.

[38]  W. Kabsch,et al.  Dictionary of protein secondary structure: Pattern recognition of hydrogen‐bonded and geometrical features , 1983, Biopolymers.

[39]  A. Klug,et al.  The crystal structure of an AII-RNAhammerhead ribozyme: A proposed mechanism for RNA catalytic cleavage , 1995, Cell.

[40]  M. Nilges,et al.  Pathways and intermediates in forced unfolding of spectrin repeats. , 2002, Structure.

[41]  Klaus Schulten,et al.  Mechanical unfolding intermediates in titin modules , 1999, Nature.

[42]  J. Rossi,et al.  Ribozymes expressed within the loop of a natural antisense RNA form functional transcription terminators. , 1995, Gene.

[43]  R. Lavery,et al.  Unraveling proteins: a molecular mechanics study. , 1999, Biophysical journal.

[44]  E. Evans,et al.  Dynamic strength of molecular adhesion bonds. , 1997, Biophysical journal.

[45]  V. Daggett,et al.  Can non-mechanical proteins withstand force? Stretching barnase by atomic force microscopy and molecular dynamics simulation. , 2001, Biophysical journal.

[46]  D. Thirumalai,et al.  Magnesium-dependent folding of self-splicing RNA: exploring the link between cooperativity, thermodynamics, and kinetics. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[47]  S. Radford,et al.  The effect of core destabilization on the mechanical resistance of I27. , 2002, Biophysical journal.

[48]  E A Merritt,et al.  Raster3D Version 2.0. A program for photorealistic molecular graphics. , 1994, Acta crystallographica. Section D, Biological crystallography.

[49]  M. Rief,et al.  Single molecule force spectroscopy of spectrin repeats: low unfolding forces in helix bundles. , 1999, Journal of molecular biology.

[50]  M. Fedor.  Tertiary structure stabilization promotes hairpin ribozyme ligation. , 1999, Biochemistry.

[51]  C. Hernández,et al.  Plus and minus RNAs of peach latent mosaic viroid self-cleave in vitro via hammerhead structures. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[52]  E Westhof,et al.  New loop-loop tertiary interactions in self-splicing introns of subgroup IC and ID: a complete 3D model of the Tetrahymena thermophila ribozyme. , 1996, Chemistry & biology.

[53]  M Karplus,et al.  Forced unfolding of fibronectin type 3 modules: an analysis by biased molecular dynamics simulations. , 1999, Journal of molecular biology.

[54]  Jane Clarke,et al.  Mechanical unfolding of a titin Ig domain: structure of unfolding intermediate revealed by combining AFM, molecular dynamics simulations, NMR and protein engineering. , 2002, Journal of molecular biology.

[55]  F. John,et al.  Stretching DNA , 2022 .

[56]  A. R. Srinivasan,et al.  The nucleic acid database. A comprehensive relational database of three-dimensional structures of nucleic acids. , 1992, Biophysical journal.

[57]  M. Rief,et al.  How strong is a covalent bond? , 1999, Science.

[58]  C. Mou,et al.  Mechanical unfolding and refolding of proteins: an off-lattice model study. , 2001, Physical review. E, Statistical, nonlinear, and soft matter physics.

[59]  D. Lilley,et al.  The folding of the hairpin ribozyme: dependence on the loops and the junction. , 2000, RNA.

[60]  O. Uhlenbeck,et al.  Synthesis of small RNAs using T7 RNA polymerase. , 1989, Methods in enzymology.

[61]  A. Matouschek,et al.  Mitochondria unfold precursor proteins by unraveling them from their N-termini , 1999, Nature Structural Biology.

[62]  O. Uhlenbeck,et al.  Internal equilibrium of the hammerhead ribozyme is altered by the length of certain covalent cross-links. , 2002, Biochemistry.

[63]  Christian Zwieb,et al.  SRPDB (Signal Recognition Particle Database) , 2000, Nucleic Acids Res..

[64]  H Li,et al.  Atomic force microscopy reveals the mechanical design of a modular protein. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[65]  Andres F. Oberhauser,et al.  The molecular elasticity of the extracellular matrix protein tenascin , 1998, Nature.

[66]  Hui Lu,et al.  The mechanical stability of ubiquitin is linkage dependent , 2003, Nature Structural Biology.

[67]  N. Pfanner,et al.  The mitochondrial Hsp70‐dependent import system actively unfolds preproteins and shortens the lag phase of translocation , 2001, The EMBO journal.

[68]  O. Uhlenbeck,et al.  Hammerhead ribozyme kinetics. , 1998, RNA.

[69]  Koji Okamoto,et al.  The protein import motor of mitochondria: a targeted molecular ratchet driving unfolding and translocation , 2002, The EMBO journal.

[70]  M. Karplus,et al.  CHARMM: A program for macromolecular energy, minimization, and dynamics calculations , 1983 .

[71]  A. Oberhauser,et al.  Chair-boat transitions in single polysaccharide molecules observed with force-ramp AFM , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[72]  D. Mckay,et al.  Structure and function of the hammerhead ribozyme: an unfinished story. , 1996, RNA.