Exploring the energy landscape of GFP by single-molecule mechanical experiments.

We use single-molecule force spectroscopy to drive single GFP molecules from the native state through their complex energy landscape into the completely unfolded state. Unlike many smaller proteins, mechanical GFP unfolding proceeds by means of two subsequent intermediate states. The transition from the native state to the first intermediate state occurs near thermal equilibrium at approximately 35 pN and is characterized by detachment of a seven-residue N-terminal alpha-helix from the beta barrel. We measure the equilibrium free energy cost associated with this transition as 22 k(B)T. Detachment of this small alpha-helix completely destabilizes GFP thermodynamically even though the beta-barrel is still intact and can bear load. Mechanical stability of the protein on the millisecond timescale, however, is determined by the activation barrier of unfolding the beta-barrel out of this thermodynamically unstable intermediate state. High bandwidth, time-resolved measurements of the cantilever relaxation phase upon unfolding of the beta-barrel revealed a second metastable mechanical intermediate with one complete beta-strand detached from the barrel. Quantitative analysis of force distributions and lifetimes lead to a detailed picture of the complex mechanical unfolding pathway through a rough energy landscape.

[1]  Hongbin Li,et al.  The unfolding kinetics of ubiquitin captured with single-molecule force-clamp techniques. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[2]  Angelika A Noegel,et al.  A mechanical unfolding intermediate in an actin-crosslinking protein , 2004, Nature Structural &Molecular Biology.

[3]  Jane Clarke,et al.  Hidden complexity in the mechanical properties of titin , 2003, Nature.

[4]  Dieter Oesterhelt,et al.  Stability of Bacteriorhodopsin α-Helices and Loops Analyzed by Single-Molecule Force Spectroscopy , 2002 .

[5]  Matthias Rief,et al.  The myosin coiled-coil is a truly elastic protein structure , 2002, Nature materials.

[6]  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.

[7]  Wolfgang A. Linke,et al.  Reverse engineering of the giant muscle protein titin , 2002, Nature.

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

[9]  M. Zimmer,et al.  Green fluorescent protein (GFP): applications, structure, and related photophysical behavior. , 2002, Chemical reviews.

[10]  J. Gilman,et al.  Nanotechnology , 2001 .

[11]  A. Negro,et al.  Crystal structure and refolding properties of the mutant F99S/M153T/V163A of the green fluorescent protein , 2000, Proteins.

[12]  H. Weiner,et al.  Basis for half-of-the-site reactivity and the dominance of the K487 oriental subunit over the E487 subunit in heterotetrameric human liver mitochondrial aldehyde dehydrogenase. , 2000, Biochemistry.

[13]  H. Gaub,et al.  Force spectroscopy with single bio-molecules. , 2000, Current opinion in chemical biology.

[14]  Hongbin Li,et al.  Single Molecule Force Spectroscopy of Modular Proteins in the Nervous System , 2000, Neuron.

[15]  Piotr E. Marszalek,et al.  Stretching single molecules into novel conformations using the atomic force microscope , 2000, Nature Structural Biology.

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

[17]  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.

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

[19]  A. Oberhauser,et al.  Atomic force microscopy captures length phenotypes in single proteins. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[20]  T. Holak,et al.  Molecular architecture of the rod domain of the Dictyostelium gelation factor (ABP120). , 1999, Journal of molecular biology.

[21]  T. Kiefhaber,et al.  The speed limit for protein folding measured by triplet-triplet energy transfer. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[22]  M. Viani,et al.  Small cantilevers for force spectroscopy of single molecules , 1999 .

[23]  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.

[24]  Matthias Rief,et al.  Elastically Coupled Two-Level Systems as a Model for Biopolymer Extensibility , 1998 .

[25]  R. Tsien,et al.  green fluorescent protein , 2020, Catalysis from A to Z.

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

[27]  S. Kain,et al.  Deletions of the Aequorea victoria Green Fluorescent Protein Define the Minimal Domain Required for Fluorescence* , 1997, The Journal of Biological Chemistry.

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

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

[30]  C. Herberhold,et al.  The repeating segments of the F-actin cross-linking gelation factor (ABP-120) have an immunoglobulin-like fold , 1997, Nature Structural Biology.

[31]  Eric J. Brown,et al.  Decreased Resistance to Bacterial Infection and Granulocyte Defects in IAP-Deficient Mice , 1996, Science.

[32]  W. Stemmer,et al.  Improved Green Fluorescent Protein by Molecular Evolution Using DNA Shuffling , 1996, Nature Biotechnology.

[33]  C. Bustamante,et al.  Overstretching B-DNA: The Elastic Response of Individual Double-Stranded and Single-Stranded DNA Molecules , 1996, Science.

[34]  Matthias Rief,et al.  Sensing specific molecular interactions with the atomic force microscope , 1995 .

[35]  E. Siggia,et al.  Entropic elasticity of lambda-phage DNA. , 1994, Science.

[36]  W. Ward,et al.  Renaturation of Aequorea green-fluorescent protein , 1981 .

[37]  G. I. Bell.  Models for the specific adhesion of cells to cells. , 1978, Science.