@article {2149,
	title = {Configurational entropy modulates the mechanical stability of protein GB1},
	journal = {Journal of Molecular Biology},
	volume = {379},
	number = {4},
	year = {2008},
	note = {ISI Document Delivery No.: 314NITimes Cited: 10Cited Reference Count: 39Li, Hongbin Wang, Hui-Chuan Cao, Yi Sharma, Deepak Wang, Meijia},
	month = {Jun},
	pages = {871-880},
	type = {Article},
	abstract = {Configurational entropy plays important roles in defining the thermodynamic stability as well as the folding/unfolding kinetics of proteins. Here we combine single-molecule atomic force microscopy and protein engineering techniques to directly examine the role of configurational entropy in the mechanical unfolding kinetics and mechanical stability of proteins. We used a small protein, GB1, as a model system and constructed four mutants that elongate loop 2 of GB1 by 2, 5, 24 and 46 flexible residues, respectively. These loop elongation mutants fold properly as determined by far-UV circular dichroism spectroscopy, suggesting that loop 2 is well tolerant of loop insertions without affecting GB1{\textquoteright}s native structure. Our single-molecule atomic force microscopy results reveal that loop elongation decreases the mechanical stability of GB1 and accelerates the mechanical unfolding kinetics. These results can be explained by the loss of configurational entropy upon closing an unstructured flexible loop using classical polymer theory, highlighting the important role of loop regions in the mechanical unfolding of proteins. This study not only demonstrates a general approach to investigating the structural deformation of the loop regions in mechanical unfolding transition state, but also provides the foundation to use configurational entropy as an effective means to modulate the mechanical stability of proteins, which is of critical importance towards engineering artificial elastomeric proteins with tailored nanomechanical properties. (C) 2008 Elsevier Ltd. All rights reserved.},
	keywords = {configurational entropy, DISULFIDE BONDS, FORCE, FORCE SPECTROSCOPY, FRAGMENT RECONSTITUTION, IMMUNOGLOBULIN BINDING DOMAIN, length, LOOP, MECHANICAL STABILITY, mechanical unfolding, MODULES, resistance, single molecule atomic force microscopy, SINGLE PROTEIN, SPECTROSCOPY, TITIN, TRANSITION-STATE},
	isbn = {0022-2836},
	url = {<Go to ISI>://000256815700018},
	author = {Li, H. B. and Wang, H. C. and Cao, Y. and Sharma, D. and Wang, M.}
}
@article {2017,
	title = {Recombination of protein fragments: A promising approach toward engineering proteins with novel nanomechanical properties},
	journal = {Protein Science},
	volume = {17},
	number = {10},
	year = {2008},
	note = {ISI Document Delivery No.: 351CKTimes Cited: 4Cited Reference Count: 50Balamurali, M. M. Sharma, Deepak Chang, Anderson Khor, Dingyue Chu, Ricky Li, Hongbin},
	month = {Oct},
	pages = {1815-1826},
	type = {Article},
	abstract = {Combining single molecule atomic force microscopy (AFM) and protein engineering techniques, here we demonstrate that we can use recombination-based techniques to engineer novel elastomeric proteins by recombining protein fragments from structurally homologous parent proteins. Using I27 and I32 domains from the muscle protein titin as parent template proteins, we systematically shuffled the secondary structural elements of the two parent proteins and engineered 13 hybrid daughter proteins. Although I27 and I32 are highly homologous, and homology modeling predicted that the hybrid daughter proteins fold into structures that are similar to that of parent protein, we found that only eight of the 13 daughter proteins showed beta-sheet dominated structures that are similar to parent proteins, and the other five recombined proteins showed signatures of the formation of significant alpha-helical or random coil-like structure. Single molecule AFM revealed that six recombined daughter proteins are mechanically stable and exhibit mechanical properties that are different from the parent proteins. In contrast, another four of the hybrid proteins were found to be mechanically labile and unfold at forces that are lower than the similar to 20 pN, as we could not detect any unfolding force peaks. The last three hybrid proteins showed interesting duality in their mechanical unfolding behaviors. These results demonstrate the great potential of using recombination-based approaches to engineer novel elastomeric protein domains of diverse mechanical properties. Moreover, our results also revealed the challenges and complexity of developing a recombination-based approach into a laboratory-based directed evolution approach to engineer novel elastomeric proteins.},
	keywords = {ATOMIC-FORCE MICROSCOPY, BIOLOGICAL ROLES, COMPUTATIONAL DESIGN, ELASTICITY, elastomeric protein, EVOLUTION, IMMUNOGLOBULIN DOMAINS, MECHANICAL STABILITY, mechanical unfolding, MOLECULAR-DYNAMICS SIMULATION, recombination, SEQUENCE, SINGLE PROTEIN, single-molecule force spectroscopy, TITIN},
	isbn = {0961-8368},
	url = {<Go to ISI>://000259401900019},
	author = {Balamurali, M. M. and Sharma, D. and Chang, A. and Khor, D. and Chu, R. and Li, H. B.}
}
@article {2034,
	title = {Single molecule force spectroscopy reveals engineered metal chelation is a general approach to enhance mechanical stability of proteins},
	journal = {Proceedings of the National Academy of Sciences of the United States of America},
	volume = {105},
	number = {32},
	year = {2008},
	note = {ISI Document Delivery No.: 339EKTimes Cited: 16Cited Reference Count: 44Cao, Yi Yoo, Teri Li, Hongbin},
	month = {Aug},
	pages = {11152-11157},
	type = {Article},
	abstract = {Significant mechanical stability is an essential feature shared by many elastomeric proteins, which function as molecular springs in a wide variety of biological machinery and biomaterials of superb mechanical properties. Despite the progress in understanding molecular determinants of mechanical stability, it remains challenging to rationally enhance the mechanical stability of proteins. Using single molecule force spectroscopy and protein engineering techniques, we demonstrate that engineered bi-histidine metal chelation can enhance the mechanical stability of proteins significantly and reversibly. Based on simple thermodynamic cycle analysis, we engineered a bi-histidine metal chelation site into various locations of the small protein, GB1, to achieve preferential stabilization of the native state over the mechanical unfolding transition state of GB1 through the binding of metal ions. Our results demonstrate that the metal chelation can enhance the mechanical stability of GB1 by as much as 100 pN. Since bi-histidine metal chelation sites can be easily implemented, engineered metal chelation provides a general methodology to enhance the mechanical stability of a wide variety of proteins. This general approach in protein mechanics will enable the rational tuning of the mechanical stability of proteins. It will not only open new avenues toward engineering proteins of tailored nanomechanical properties, but also provide new approaches to systematically map the mechanical unfolding pathway of proteins.},
	keywords = {BINDING SITES, charge, DESIGN, DOMAINS, ELASTICITY, engineering, EXTRACELLULAR-MATRIX PROTEIN, mechanical unfolding, PROTEIN, protein mechanics, rational design, SIMULATIONS, STABILIZATION, STRENGTH, THERMOSTABILITY, TITIN},
	isbn = {0027-8424},
	url = {<Go to ISI>://000258560700024},
	author = {Cao, Y. and Yoo, T. and Li, H. B.}
}
@article {2247,
	title = {Stabilization provided by neighboring strands is critical for the mechanical stability of proteins},
	journal = {Biophysical Journal},
	volume = {95},
	number = {8},
	year = {2008},
	note = {ISI Document Delivery No.: 352NMTimes Cited: 5Cited Reference Count: 37Sharma, Deepak Feng, Gang Khor, Dingyue Genchev, Georgi Z. Lu, Hui Li, Hongbin},
	month = {Oct},
	pages = {3935-3942},
	type = {Article},
	abstract = {Single-molecule force spectroscopy studies and steered molecular dynamics simulations have revealed that protein topology and pulling geometry play important roles in determining the mechanical stability of proteins. Most studies have focused on local interactions that are associated with the force-bearing beta-strands. Interactions mediated by neighboring strands are often overlooked. Here we use Top7 and barstar as model systems to illustrate the critical importance of the stabilization effect provided by neighboring beta-strands on the mechanical stability. Using single-molecule atomic force microscopy, we showed that Top7 and barstar, which have similar topology in their force-bearing region, exhibit vastly different mechanical-stability characteristics. Top7 is mechanically stable and unfolds at similar to 150 pN, whereas barstar is mechanically labile and unfolds largely below 50 pN. Steered molecular dynamics simulations revealed that stretching force peels one force-bearing strand away from barstar to trigger unfolding, whereas Top7 unfolds via a substructure-sliding mechanism. This previously overlooked stabilization effect from neighboring beta-strands is likely to be a general mechanism in protein mechanics and can serve as a guideline for the de novo design of proteins with significant mechanical stability and novel protein topology.},
	keywords = {BARSTAR, DYNAMICS SIMULATIONS, IMMUNOGLOBULIN DOMAINS, MICROSCOPE, MOLECULE FORCE SPECTROSCOPY, resistance, SINGLE PROTEIN, TITIN, TOPOLOGY, UBIQUITIN},
	isbn = {0006-3495},
	url = {<Go to ISI>://000259503900036},
	author = {Sharma, D. and Feng, G. and Khor, D. and Genchev, G. Z. and Lu, H. and Li, H. B.}
}
@article {1555,
	title = {Engineering proteins with novel mechanical properties by recombination of protein fragments},
	journal = {Angewandte Chemie-International Edition},
	volume = {45},
	number = {34},
	year = {2006},
	note = {ISI Document Delivery No.: 082MRTimes Cited: 20Cited Reference Count: 51Sharma, Deepak Cao, Yi Li, Hongbin},
	pages = {5633-5638},
	type = {Article},
	keywords = {AFM, ATOMIC-FORCE MICROSCOPY, ELASTICITY, EVOLUTION, IG DOMAIN, IMMUNOGLOBULIN DOMAINS, MOLECULAR-DYNAMICS SIMULATIONS, protein engineering, protein structures, scanning probe microscopy, SINGLE PROTEIN, single-molecule studies, SPECTROSCOPY, STABILITY, TITIN},
	isbn = {1433-7851},
	url = {<Go to ISI>://000240391400016},
	author = {Sharma, D. and Cao, Y. and Li, H. B.}
}
@article {1350,
	title = {Single molecule force spectroscopy reveals a weakly populated microstate of the FnIII domains of tenascin},
	journal = {Journal of Molecular Biology},
	volume = {361},
	number = {2},
	year = {2006},
	note = {ISI Document Delivery No.: 074WPTimes Cited: 8Cited Reference Count: 48Cao, Y. Li, Hongbin},
	month = {Aug},
	pages = {372-381},
	type = {Article},
	abstract = {The native states of proteins exist as an ensemble of conformationally similar microstates. The fluctuations among different microstates are of great importance for the functions and structural stability of proteins. Here, we demonstrate that single molecule atomic force microscopy (AFM) can be used to directly probe the existence of multiple folded microstates. We used the AFM to repeatedly stretch and relax a recombinant tenascin fragment TNfnALL to allow the fibronectin type III (FnIII) domains to undergo repeated unfolding/refolding cycles. In addition to the native state, we discovered that some FnIII domains can refold from the unfolded state into a previously unrecognized microstate, N* state. This novel state is conformationally similar to the native state, but mechanically less stable. The native state unfolds at similar to 120 pN, while the N* state unfolds at similar to 50 pN. These two distinct populations of microstates constitute the ensemble of the folded states for some FnIII domains. An unfolded FnIII domain can fold into either one of the two microstates via two distinct folding routes. These results reveal the dynamic and heterogeneous picture of the folded ensemble for some FnIII domains of tenascin, which may carry important implications for the mechanical functions of tenascins in vivo. (c) 2006 Elsevier Ltd. All rights reserved.},
	keywords = {DYNAMICS, fluctuations, FnIII domains, HYDROGEN-EXCHANGE, IMMUNOGLOBULIN, MECHANICAL STABILITY, mechanical unfolding, microscopy, MODULES, PROTEIN-STRUCTURE, scanning probe, single-molecule force spectroscopy, tenascin, TITIN, UNFOLDING PATHWAYS},
	isbn = {0022-2836},
	url = {<Go to ISI>://000239842800014},
	author = {Cao, Y. and Li, H. B.}
}
