1. – Introduction Proteins must fold into compact and unique three-dimensional structures to carr... more 1. – Introduction Proteins must fold into compact and unique three-dimensional structures to carry out their specific functions. If folding goes wrong, proteins become useless and often toxic molecules for living cells. Millions of people around the world suffer from diseases caused by protein misfolding, such as Gaucher’s disease, Alzheimer’s disease and Parkinson’s disease [1, 2]. Despite its importance, our understanding of the basic rules that govern how a protein attains its native structure is still incomplete. This lack of information is partly due to the inadequacy of conventional bulk methods to study a process that is highly heterogeneous. During folding, individual molecules are thought to follow different pathways and populate different intermediate structures on their journey to the native state [3]. Such a diversity of behaviors is often blurred in the ensemble average measured
The biological functions of proteins closely depend on their conformational dynamics. This aspect... more The biological functions of proteins closely depend on their conformational dynamics. This aspect is especially relevant for intrinsically disordered proteins (IDP) for which structural ensembles often offer more useful representations than individual conformations. Here we employ extensive enhanced sampling temperature replica-exchange atomistic simulations (TREMD) and deep learning dimensionality reduction to study the conformational ensembles of the human heat shock protein B8 and its pathological mutant K141E, for which no experimental 3D structures are available. First, we combined homology modelling with TREMD to generate high-dimensional data sets of 3D structures. Then, we employed a recently developed machine learning based post-processing algorithm, EncoderMap, to project the large conformational data sets into meaningful two-dimensional maps that helped us interpret the data and extract the most significant conformations adopted by both proteins during TREMD. These studies provide the first 3D structural characterization of HSPB8 and reveal the effects of the pathogenic K141E mutation on its conformational ensembles. In particular, this missense mutation appears to increase the compactness of the protein and its structural variability, at the same time rearranging the hydrophobic patches exposed on the protein surface. These results offer the possibility of rationalizing the pathogenic effects of the K141E mutation in terms of conformational changes.
The Fourth Cell Stress Society International workshop on small heat shock proteins (sHSPs), a fol... more The Fourth Cell Stress Society International workshop on small heat shock proteins (sHSPs), a follow-up to successful workshops held in 2014, 2016 and 2018, took place as a virtual meeting on the 17-18 November 2022. The meeting was designed to provide an opportunity for those working on sHSPs to reconnect and discuss their latest work. The diversity of research in the sHSP field is reflected in the breadth of topics covered in the talks presented at this meeting. Here we summarise the presentations at this meeting and provide some perspectives on exciting future topics to be addressed in the field.
Binding of ligands is often crucial for function yet the effects of ligand binding on the mechani... more Binding of ligands is often crucial for function yet the effects of ligand binding on the mechanical stability and energy landscape of proteins are incompletely understood. Here we use a combination of single-molecule optical tweezers and MD simulations to investigate the effect of ligand binding on the energy landscape of acyl-coenzyme A (CoA) binding protein (ACBP). ACBP is a topologically simple and highly conserved four-α-helix bundle protein that acts as an intracellular transporter and buffer for fatty-acyl CoA and is active in membrane assembly. We have previously described the behavior of ACBP under tension, revealing a highly extended transition state (TS) located almost halfway between the unfolded and native states. Here, we performed force-ramp and force-jump experiments, in combination with advanced statistical analysis, to show that octanoyl-CoA binding increases the activation free energy for the unfolding reaction of ACBP without affecting the position of the transition state along the reaction coordinate. It follows that ligand binding enhances the mechanical resistance and thermodynamic stability of the protein, without changing its mechanical compliance. Steered molecular dynamics simulations allowed us to rationalize the results in terms of key interactions that octanoyl-CoA establishes with the four αhelices of ACBP and showed that the unfolding pathway is marginally affected by the ligand. The results show that ligand-induced mechanical stabilization effects can be complex and may prove useful for the rational design of stabilizing ligands.
Journal of Vacuum Science & Technology B: Microelectronics and Nanometer Structures Processing, Measurement, and Phenomena, 1997
Hybrid superconductor–semiconductor (S–Sm) nanostructures were fabricated by integrating standard... more Hybrid superconductor–semiconductor (S–Sm) nanostructures were fabricated by integrating standard ultraviolet photolithography and direct patterning of photoresist with an atomic force microscope (AFM). This novel technology was used to fabricate Nb–InAs–Nb weak links comparable in length to the coherence length. These structures exhibit high critical currents up to 10 μA/μm in planar geometry at 0.3 K. The fabrication protocol is based on the modification of photolithographically defined patterns by AFM static ploughing of the photoresist. Wet chemical etching is subsequently used for the definition of nanoscale S–Sm–S bridges. Additionally Lift-off procedures allowed the fabrication of submicron superconducting bridges. Successful fabrication of the nanostructures was verified by electrical characterization and by AFM and scanning electron microscope structural characterization.
data Supplementary http://www.genesdev.org/cgi/content/full/19/20/2466/DC1 "Supplemental Res... more data Supplementary http://www.genesdev.org/cgi/content/full/19/20/2466/DC1 "Supplemental Research Data" References http://www.genesdev.org/cgi/content/full/19/20/2466#References This article cites 41 articles, 14 of which can be accessed free at: service Email alerting click here top right corner of the article or Receive free email alerts when new articles cite this article sign up in the box at the
In the past three decades, the ability to optically manipulate biomolecules has spurred a new era... more In the past three decades, the ability to optically manipulate biomolecules has spurred a new era of medical and biophysical research. Optical tweezers (OT) have enabled experimenters to trap, sort, and probe cells, as well as discern the structural dynamics of proteins and nucleic acids at single molecule level. The steady improvement in OT’s resolving power has progressively pushed the envelope of their applications; there are, however, some inherent limitations that are prompting researchers to look for alternatives to the conventional techniques. To begin with, OT are restricted by their one-dimensional approach, which makes it difficult to conjure an exhaustive three-dimensional picture of biological systems. The high-intensity trapping laser can damage biological samples, a fact that restricts the feasibility of in vivo applications. Finally, direct manipulation of biological matter at nanometer scale remains a significant challenge for conventional OT. A significant amount of...
The unfolding and folding of protein barnase has been extensively investigated in bulk conditions... more The unfolding and folding of protein barnase has been extensively investigated in bulk conditions under the effect of denaturant and temperature. These experiments provided information about structural and kinetic features of both the native and the unfolded states of the protein, and debates about the possible existence of an intermediate state in the folding pathway have arisen. Here, we investigate the folding/unfolding reaction of protein barnase under the action of mechanical force at the single-molecule level using optical tweezers. We measure unfolding and folding force-dependent kinetic rates from pulling and passive experiments, respectively, and using Kramers-based theories (e.g., Bell-Evans and Dudko-Hummer-Szabo models), we extract the position of the transition state and the height of the kinetic barrier mediating unfolding and folding transitions, finding good agreement with previous bulk measurements. Measurements of the force-dependent kinetic barrier using the continuous effective barrier analysis show that protein barnase verifies the Leffler-Hammond postulate under applied force and allow us to extract its free energy of folding, DG 0. The estimated value of DG 0 is in agreement with our predictions obtained using fluctuation relations and previous bulk studies. To address the possible existence of an intermediate state on the folding pathway, we measure the power spectrum of force fluctuations at high temporal resolution (50 kHz) when the protein is either folded or unfolded and, additionally, we study the folding transition-path time at different forces. The finite bandwidth of our experimental setup sets the lifetime of potential intermediate states upon barnase folding/unfolding in the submillisecond timescale.
Protein folding is still a major area of active research. Despite significant progress in underst... more Protein folding is still a major area of active research. Despite significant progress in understanding the underlying principles, we still cannot efficiently predict the folding mechanism for even a moderately sized protein. Proteins are generally thought to fold by diffusion over a three-dimensional energy landscape. Traditional bulk methods have proven to be very powerful in the study of the folding process but they often suffer from inherent ensemble averaging. Single molecule techniques open up new vistas for studying protein folding, allowing direct analysis of the distribution of events that characterize the heterogeneous folding process. Recently it has become possible to directly manipulate individual proteins using optical tweezers. Here we illustrate the experimental strategy and how this approach has provided a fresh perspective on the protein folding problem.
... Dario Alliata and Ciro Cecconi Technobiochip, Via della Marina, 57030 Marciana (LI), Italy Cl... more ... Dario Alliata and Ciro Cecconi Technobiochip, Via della Marina, 57030 Marciana (LI), Italy Claudio Nicolini Technobiochip, Via della Marina, 57030 Marciana (LI ... We wish to thank P. Facci, M. Sartore, and R. Staub for their suggestions and Isabella Zolfino for her keen support. ...
Journal of Photochemistry and Photobiology B: Biology, 1996
This paper reports a systematic study of the step-up photophobic responses exhibited by the unice... more This paper reports a systematic study of the step-up photophobic responses exhibited by the unicellular alga Haematococcus pluvialis when s:imulated unidirectionally or bidirectionally. The stimulus-response curves, obtained at four different wavelengths, are interpreted in terms ;~ the structure of the photoreceptive apparatus. In addition, an indirect method to obtain information about the stigma and photoreceptor(s) i~ reported. This method is based on the interpolation of experimental with simulated data. Our results confirm the widely accepted view that tie photoreceptors are located in the stigma region, and suggest the presence of two photoreceptors.
Leech AP neurons react to axotomy by increasing excitability and resting potential of the cell bo... more Leech AP neurons react to axotomy by increasing excitability and resting potential of the cell body membrane. In a previous report we described single potassium channels contributing to the leak conductance in the soma membrane of AP cells. Here we compare both properties and density of single potassium leak channels in cell-free patches from normal and axotomized AP neurons. We show that properties such as single channel conductance, outward rectification, time constants of open and shut interval distributions and absence of inactivation do not significantly differ between normal and axotomized cells. On the other hand, we find that the number of channels per patch progressively increases with time after axotomy. We conclude that changes in density rather than alterations in properties of single channels can account for the increase in the resting potential, observed after axotomy.
The mechanical properties of proteins and their force-induced structural changes play key roles i... more The mechanical properties of proteins and their force-induced structural changes play key roles in many biological processes. Previous studies have shown that natively folded proteins are brittle under tension, unfolding after small mechanical deformations, while partially folded intermediate states, such as molten globules, are compliant and can deform elastically a great amount before crossing the transition state barrier. Moreover, under tension proteins appear to unfold through a different sequence of events than during spontaneous unfolding. Here, we describe the response to force of the four-α-helix acyl-CoA binding protein (ACBP) in the low-force regime using optical tweezers and ratcheted molecular dynamics simulations. The results of our studies reveal an unprecedented mechanical behavior of a natively folded protein. ACBP displays an atypical compliance along two nearly orthogonal pulling axes, with transition states located almost halfway between the unfolded and folded states. Surprisingly, the deformability of ACBP is greater than that observed for the highly pliant molten globule intermediate states. Furthermore, when manipulated from the N-and C-termini, ACBP unfolds by populating a transition state that resembles that observed during chemical denaturation, both for structure and position along the reaction coordinate. Our data provide the first experimental evidence of a spontaneous-like mechanical unfolding pathway of a protein. The mechanical behavior of ACBP is discussed in terms of topology and helix propensity.
Integrins are cell adhesion receptors that link to the cytoskeleton and the extracellular matrix,... more Integrins are cell adhesion receptors that link to the cytoskeleton and the extracellular matrix, and transmit signals bidirectionally across the cell membrane. Signal transduction between the cytoplasmic domain and the ligand-binding domain (the "Inserted" or I-domain) occurs by a series of conformational changes that lead to allosteric transitions in the I-domain. Different allosteries of the I-domain have markedly different binding affinities for ligands and are critical for cell motility and adhesion. In addition, the I-domain bears the Rossmann fold, a common motif found in nucleic-acid binding proteins and von Willebrand factor A domains, which has a characteristic of inner beta strands being connected by peripheral alpha helices in an alternating matter. We have performed single molecule pulling experiments on the integrin LFA-1 I-domain using mini optical tweezers. In pulling experiments, mechanical unfolding and refolding revealed 2 or more transitions for the nat...
Proceedings of the National Academy of Sciences, 2000
Recent advances in single molecule manipulation methods offer a novel approach to investigating t... more Recent advances in single molecule manipulation methods offer a novel approach to investigating the protein folding problem. These studies usually are done on molecules that are naturally organized as linear arrays of globular domains. To extend these techniques to study proteins that normally exist as monomers, we have developed a method of synthesizing polymers of protein molecules in the solid state. By introducing cysteines at locations where bacteriophage T4 lysozyme molecules contact each other in a crystal and taking advantage of the alignment provided by the lattice, we have obtained polymers of defined polarity up to 25 molecules long that retain enzymatic activity. These polymers then were manipulated mechanically by using a modified scanning force microscope to characterize the force-induced reversible unfolding of the individual lysozyme molecules. This approach should be general and adaptable to many other proteins with known crystal structures. For T4 lysozyme, the for...
1. – Introduction Proteins must fold into compact and unique three-dimensional structures to carr... more 1. – Introduction Proteins must fold into compact and unique three-dimensional structures to carry out their specific functions. If folding goes wrong, proteins become useless and often toxic molecules for living cells. Millions of people around the world suffer from diseases caused by protein misfolding, such as Gaucher’s disease, Alzheimer’s disease and Parkinson’s disease [1, 2]. Despite its importance, our understanding of the basic rules that govern how a protein attains its native structure is still incomplete. This lack of information is partly due to the inadequacy of conventional bulk methods to study a process that is highly heterogeneous. During folding, individual molecules are thought to follow different pathways and populate different intermediate structures on their journey to the native state [3]. Such a diversity of behaviors is often blurred in the ensemble average measured
The biological functions of proteins closely depend on their conformational dynamics. This aspect... more The biological functions of proteins closely depend on their conformational dynamics. This aspect is especially relevant for intrinsically disordered proteins (IDP) for which structural ensembles often offer more useful representations than individual conformations. Here we employ extensive enhanced sampling temperature replica-exchange atomistic simulations (TREMD) and deep learning dimensionality reduction to study the conformational ensembles of the human heat shock protein B8 and its pathological mutant K141E, for which no experimental 3D structures are available. First, we combined homology modelling with TREMD to generate high-dimensional data sets of 3D structures. Then, we employed a recently developed machine learning based post-processing algorithm, EncoderMap, to project the large conformational data sets into meaningful two-dimensional maps that helped us interpret the data and extract the most significant conformations adopted by both proteins during TREMD. These studies provide the first 3D structural characterization of HSPB8 and reveal the effects of the pathogenic K141E mutation on its conformational ensembles. In particular, this missense mutation appears to increase the compactness of the protein and its structural variability, at the same time rearranging the hydrophobic patches exposed on the protein surface. These results offer the possibility of rationalizing the pathogenic effects of the K141E mutation in terms of conformational changes.
The Fourth Cell Stress Society International workshop on small heat shock proteins (sHSPs), a fol... more The Fourth Cell Stress Society International workshop on small heat shock proteins (sHSPs), a follow-up to successful workshops held in 2014, 2016 and 2018, took place as a virtual meeting on the 17-18 November 2022. The meeting was designed to provide an opportunity for those working on sHSPs to reconnect and discuss their latest work. The diversity of research in the sHSP field is reflected in the breadth of topics covered in the talks presented at this meeting. Here we summarise the presentations at this meeting and provide some perspectives on exciting future topics to be addressed in the field.
Binding of ligands is often crucial for function yet the effects of ligand binding on the mechani... more Binding of ligands is often crucial for function yet the effects of ligand binding on the mechanical stability and energy landscape of proteins are incompletely understood. Here we use a combination of single-molecule optical tweezers and MD simulations to investigate the effect of ligand binding on the energy landscape of acyl-coenzyme A (CoA) binding protein (ACBP). ACBP is a topologically simple and highly conserved four-α-helix bundle protein that acts as an intracellular transporter and buffer for fatty-acyl CoA and is active in membrane assembly. We have previously described the behavior of ACBP under tension, revealing a highly extended transition state (TS) located almost halfway between the unfolded and native states. Here, we performed force-ramp and force-jump experiments, in combination with advanced statistical analysis, to show that octanoyl-CoA binding increases the activation free energy for the unfolding reaction of ACBP without affecting the position of the transition state along the reaction coordinate. It follows that ligand binding enhances the mechanical resistance and thermodynamic stability of the protein, without changing its mechanical compliance. Steered molecular dynamics simulations allowed us to rationalize the results in terms of key interactions that octanoyl-CoA establishes with the four αhelices of ACBP and showed that the unfolding pathway is marginally affected by the ligand. The results show that ligand-induced mechanical stabilization effects can be complex and may prove useful for the rational design of stabilizing ligands.
Journal of Vacuum Science & Technology B: Microelectronics and Nanometer Structures Processing, Measurement, and Phenomena, 1997
Hybrid superconductor–semiconductor (S–Sm) nanostructures were fabricated by integrating standard... more Hybrid superconductor–semiconductor (S–Sm) nanostructures were fabricated by integrating standard ultraviolet photolithography and direct patterning of photoresist with an atomic force microscope (AFM). This novel technology was used to fabricate Nb–InAs–Nb weak links comparable in length to the coherence length. These structures exhibit high critical currents up to 10 μA/μm in planar geometry at 0.3 K. The fabrication protocol is based on the modification of photolithographically defined patterns by AFM static ploughing of the photoresist. Wet chemical etching is subsequently used for the definition of nanoscale S–Sm–S bridges. Additionally Lift-off procedures allowed the fabrication of submicron superconducting bridges. Successful fabrication of the nanostructures was verified by electrical characterization and by AFM and scanning electron microscope structural characterization.
data Supplementary http://www.genesdev.org/cgi/content/full/19/20/2466/DC1 "Supplemental Res... more data Supplementary http://www.genesdev.org/cgi/content/full/19/20/2466/DC1 "Supplemental Research Data" References http://www.genesdev.org/cgi/content/full/19/20/2466#References This article cites 41 articles, 14 of which can be accessed free at: service Email alerting click here top right corner of the article or Receive free email alerts when new articles cite this article sign up in the box at the
In the past three decades, the ability to optically manipulate biomolecules has spurred a new era... more In the past three decades, the ability to optically manipulate biomolecules has spurred a new era of medical and biophysical research. Optical tweezers (OT) have enabled experimenters to trap, sort, and probe cells, as well as discern the structural dynamics of proteins and nucleic acids at single molecule level. The steady improvement in OT’s resolving power has progressively pushed the envelope of their applications; there are, however, some inherent limitations that are prompting researchers to look for alternatives to the conventional techniques. To begin with, OT are restricted by their one-dimensional approach, which makes it difficult to conjure an exhaustive three-dimensional picture of biological systems. The high-intensity trapping laser can damage biological samples, a fact that restricts the feasibility of in vivo applications. Finally, direct manipulation of biological matter at nanometer scale remains a significant challenge for conventional OT. A significant amount of...
The unfolding and folding of protein barnase has been extensively investigated in bulk conditions... more The unfolding and folding of protein barnase has been extensively investigated in bulk conditions under the effect of denaturant and temperature. These experiments provided information about structural and kinetic features of both the native and the unfolded states of the protein, and debates about the possible existence of an intermediate state in the folding pathway have arisen. Here, we investigate the folding/unfolding reaction of protein barnase under the action of mechanical force at the single-molecule level using optical tweezers. We measure unfolding and folding force-dependent kinetic rates from pulling and passive experiments, respectively, and using Kramers-based theories (e.g., Bell-Evans and Dudko-Hummer-Szabo models), we extract the position of the transition state and the height of the kinetic barrier mediating unfolding and folding transitions, finding good agreement with previous bulk measurements. Measurements of the force-dependent kinetic barrier using the continuous effective barrier analysis show that protein barnase verifies the Leffler-Hammond postulate under applied force and allow us to extract its free energy of folding, DG 0. The estimated value of DG 0 is in agreement with our predictions obtained using fluctuation relations and previous bulk studies. To address the possible existence of an intermediate state on the folding pathway, we measure the power spectrum of force fluctuations at high temporal resolution (50 kHz) when the protein is either folded or unfolded and, additionally, we study the folding transition-path time at different forces. The finite bandwidth of our experimental setup sets the lifetime of potential intermediate states upon barnase folding/unfolding in the submillisecond timescale.
Protein folding is still a major area of active research. Despite significant progress in underst... more Protein folding is still a major area of active research. Despite significant progress in understanding the underlying principles, we still cannot efficiently predict the folding mechanism for even a moderately sized protein. Proteins are generally thought to fold by diffusion over a three-dimensional energy landscape. Traditional bulk methods have proven to be very powerful in the study of the folding process but they often suffer from inherent ensemble averaging. Single molecule techniques open up new vistas for studying protein folding, allowing direct analysis of the distribution of events that characterize the heterogeneous folding process. Recently it has become possible to directly manipulate individual proteins using optical tweezers. Here we illustrate the experimental strategy and how this approach has provided a fresh perspective on the protein folding problem.
... Dario Alliata and Ciro Cecconi Technobiochip, Via della Marina, 57030 Marciana (LI), Italy Cl... more ... Dario Alliata and Ciro Cecconi Technobiochip, Via della Marina, 57030 Marciana (LI), Italy Claudio Nicolini Technobiochip, Via della Marina, 57030 Marciana (LI ... We wish to thank P. Facci, M. Sartore, and R. Staub for their suggestions and Isabella Zolfino for her keen support. ...
Journal of Photochemistry and Photobiology B: Biology, 1996
This paper reports a systematic study of the step-up photophobic responses exhibited by the unice... more This paper reports a systematic study of the step-up photophobic responses exhibited by the unicellular alga Haematococcus pluvialis when s:imulated unidirectionally or bidirectionally. The stimulus-response curves, obtained at four different wavelengths, are interpreted in terms ;~ the structure of the photoreceptive apparatus. In addition, an indirect method to obtain information about the stigma and photoreceptor(s) i~ reported. This method is based on the interpolation of experimental with simulated data. Our results confirm the widely accepted view that tie photoreceptors are located in the stigma region, and suggest the presence of two photoreceptors.
Leech AP neurons react to axotomy by increasing excitability and resting potential of the cell bo... more Leech AP neurons react to axotomy by increasing excitability and resting potential of the cell body membrane. In a previous report we described single potassium channels contributing to the leak conductance in the soma membrane of AP cells. Here we compare both properties and density of single potassium leak channels in cell-free patches from normal and axotomized AP neurons. We show that properties such as single channel conductance, outward rectification, time constants of open and shut interval distributions and absence of inactivation do not significantly differ between normal and axotomized cells. On the other hand, we find that the number of channels per patch progressively increases with time after axotomy. We conclude that changes in density rather than alterations in properties of single channels can account for the increase in the resting potential, observed after axotomy.
The mechanical properties of proteins and their force-induced structural changes play key roles i... more The mechanical properties of proteins and their force-induced structural changes play key roles in many biological processes. Previous studies have shown that natively folded proteins are brittle under tension, unfolding after small mechanical deformations, while partially folded intermediate states, such as molten globules, are compliant and can deform elastically a great amount before crossing the transition state barrier. Moreover, under tension proteins appear to unfold through a different sequence of events than during spontaneous unfolding. Here, we describe the response to force of the four-α-helix acyl-CoA binding protein (ACBP) in the low-force regime using optical tweezers and ratcheted molecular dynamics simulations. The results of our studies reveal an unprecedented mechanical behavior of a natively folded protein. ACBP displays an atypical compliance along two nearly orthogonal pulling axes, with transition states located almost halfway between the unfolded and folded states. Surprisingly, the deformability of ACBP is greater than that observed for the highly pliant molten globule intermediate states. Furthermore, when manipulated from the N-and C-termini, ACBP unfolds by populating a transition state that resembles that observed during chemical denaturation, both for structure and position along the reaction coordinate. Our data provide the first experimental evidence of a spontaneous-like mechanical unfolding pathway of a protein. The mechanical behavior of ACBP is discussed in terms of topology and helix propensity.
Integrins are cell adhesion receptors that link to the cytoskeleton and the extracellular matrix,... more Integrins are cell adhesion receptors that link to the cytoskeleton and the extracellular matrix, and transmit signals bidirectionally across the cell membrane. Signal transduction between the cytoplasmic domain and the ligand-binding domain (the "Inserted" or I-domain) occurs by a series of conformational changes that lead to allosteric transitions in the I-domain. Different allosteries of the I-domain have markedly different binding affinities for ligands and are critical for cell motility and adhesion. In addition, the I-domain bears the Rossmann fold, a common motif found in nucleic-acid binding proteins and von Willebrand factor A domains, which has a characteristic of inner beta strands being connected by peripheral alpha helices in an alternating matter. We have performed single molecule pulling experiments on the integrin LFA-1 I-domain using mini optical tweezers. In pulling experiments, mechanical unfolding and refolding revealed 2 or more transitions for the nat...
Proceedings of the National Academy of Sciences, 2000
Recent advances in single molecule manipulation methods offer a novel approach to investigating t... more Recent advances in single molecule manipulation methods offer a novel approach to investigating the protein folding problem. These studies usually are done on molecules that are naturally organized as linear arrays of globular domains. To extend these techniques to study proteins that normally exist as monomers, we have developed a method of synthesizing polymers of protein molecules in the solid state. By introducing cysteines at locations where bacteriophage T4 lysozyme molecules contact each other in a crystal and taking advantage of the alignment provided by the lattice, we have obtained polymers of defined polarity up to 25 molecules long that retain enzymatic activity. These polymers then were manipulated mechanically by using a modified scanning force microscope to characterize the force-induced reversible unfolding of the individual lysozyme molecules. This approach should be general and adaptable to many other proteins with known crystal structures. For T4 lysozyme, the for...
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