The translocation of specific polypeptide chains across membranes is an essential activity for al... more The translocation of specific polypeptide chains across membranes is an essential activity for all life forms. The main components of the general secretory (Sec) system of E. coli include integral membrane translocon SecYEG, peripheral ATPase SecA, and SecDF, an ancillary complex that enhances polypeptide secretion by coupling translocation to proton motive force. Atomic force microscopy (AFM), a single-molecule imaging technique, is well suited to unmask complex, asynchronous molecular activities of membrane-associated proteins including those comprising the Sec apparatus. Using AFM, the dynamic structure of membrane-external protein topography of Sec system components can be directly visualized with high spatial-temporal precision. This mini-review is focused on AFM imaging of the Sec system in near-native fluid conditions where activity can be maintained and biochemically verified. Angstrom-scale conformational changes of SecYEG are reported on 100 ms timescales in fluid lipid bi...
Candida albicans causes severe invasive candidiasis. C. albicans infection requires the virulence... more Candida albicans causes severe invasive candidiasis. C. albicans infection requires the virulence factor candidalysin (CL) which damages target cell membranes. However, the mechanism that CL uses to permeabilize membranes is unclear. We reveal that CL forms membrane pores using a unique mechanism. Unexpectedly, CL readily assembled into polymers in solution. We propose that the basic structural unit in polymer formation is a CL oligomer, which is sequentially added into a string configuration that can close into a loop. CL loops appear to spontaneously insert into the membrane to become pores. A CL mutation (G4W) inhibited the formation of polymers in solution and prevented pore formation in synthetic lipid systems. Epithelial cell studies showed that G4W CL failed to activate the danger response pathway, a hallmark of the pathogenic effect of CL. These results indicate that CL polymerization in solution is a necessary step for the damage of cellular membranes. Analysis of CL pores ...
Atomic force microscopy has emerged as a valuable complementary technique in membrane structural ... more Atomic force microscopy has emerged as a valuable complementary technique in membrane structural biology. The apparatus is capable of probing individual membrane proteins in fluid lipid bilayers at room temperature with spatial resolution at the molecular length scale. Protein conformational dynamics are accessible over a range of biologically relevant timescales. This chapter presents methodology our group uses to achieve robust AFM image data of the General Secretory system, the primary pathway of protein export from the cytoplasm to the periplasm of E. coli. Emphasis is given to measuring and maintaining biochemical activity and to objective AFM image processing methods. For example, the biochemical assays can be used to determine chemomechanical coupling efficiency of surface adsorbed translocases. The Hessian blob algorithm and its extension to nonlocalized linear features, the line detection algorithm, provide automated feature delineations. Many of the methods discussed here can be applied to other membrane protein systems of interest.
1) Method for for integration of high resolution biological AFM with other powerful optical techn... more 1) Method for for integration of high resolution biological AFM with other powerful optical techniques 2) Straight-forward cleaning procedure for treatment of glass for Microscopy and Micromachining applications 3) Molecular high resolution imaging of bacteriorhodopsin and Sec translocase on glass supports 4) Direct observation of protein-protein interactions 5) Atomic Force Microscopy measurements of surface roughness of Glass, Mica, Lipid and comparison of several glass chemical treatments for Microscopy <strong>READ THE PEER-REVIEWED PUBLICATION HERE</strong> <strong>ASSOCIATED PEER-REVIEWED PUBLICATION </strong> <strong>Abstract</strong> Since its invention in the mid-1980s, the atomic force microscope (AFM) has become a valuable complementary<br> tool for studying membrane proteins in near-native environments. Historically, mica is the most common<br> substrate utilized for biological AFM. Glass being amorphous, transparent, and o...
In bacteria and archaea the protein conducting channel SecYEG provides a ubiquitous pathway for p... more In bacteria and archaea the protein conducting channel SecYEG provides a ubiquitous pathway for protein transfer across and into membranes. Further, it is known that SecA is the ATPase of the gener...
Intrinsic apoptosis is orchestrated by a group of proteins that mediate the coordinated disruptio... more Intrinsic apoptosis is orchestrated by a group of proteins that mediate the coordinated disruption of mitochondrial membranes. Bax is a multi-domain protein that, upon activation, disrupts the integrity of the...
SecA is the critical adenosine triphosphatase that drives preprotein transport through the transl... more SecA is the critical adenosine triphosphatase that drives preprotein transport through the translocon, SecYEG, in Escherichia coli. This process is thought to be regulated by conformational changes of specific domains of SecA, but real-time, real-space measurement of these changes is lacking. We use single-molecule atomic force microscopy (AFM) to visualize nucleotide-dependent conformations and conformational dynamics of SecA. Distinct topographical populations were observed in the presence of specific nucleotides. AFM investigations during basal adenosine triphosphate (ATP) hydrolysis revealed rapid, reversible transitions between a compact and an extended state at the ~100-ms time scale. A SecA mutant lacking the precursor-binding domain (PBD) aided interpretation. Further, the biochemical activity of SecA prepared for AFM was confirmed by tracking inorganic phosphate release. We conclude that ATP-driven dynamics are largely due to PBD motion but that other segments of SecA contr...
Methods in molecular biology (Clifton, N.J.), 2018
Atomic force microscopy (AFM)-based force spectroscopy is a powerful technique which has seen sig... more Atomic force microscopy (AFM)-based force spectroscopy is a powerful technique which has seen significant enhancements in both force and time resolution in recent years. This chapter details two AFM cantilever modification procedures that yield high force precision over different temporal bandwidths. Specifically, it explains a fairly straightforward method to achieve sub-pN force precision and stability at low frequencies (<50 Hz) by removing the metal coatings from a commercially available cantilever. A more involved procedure utilizing a focused ion beam milling machine is required to maintain high force precision at enhanced bandwidths. Both modification methods allow site-specific attachment of biomolecules onto the apex area of the tips for force spectroscopy. The chapter concludes with a comparative demonstration using the two cantilever modification methods to study a lipid-protein interaction.
Imaging by atomic force microscopy (AFM) offers high-resolution descriptions of many biological s... more Imaging by atomic force microscopy (AFM) offers high-resolution descriptions of many biological systems; however, regardless of resolution, conclusions drawn from AFM images are only as robust as the analysis leading to those conclusions. Vital to the analysis of biomolecules in AFM imagery is the initial detection of individual particles from large-scale images. Threshold and watershed algorithms are conventional for automatic particle detection but demand manual image preprocessing and produce particle boundaries which deform as a function of user-defined parameters, producing imprecise results subject to bias. Here, we introduce the Hessian blob to address these shortcomings. Combining a scale-space framework with measures of local image curvature, the Hessian blob formally defines particle centers and their boundaries, both to subpixel precision. Resulting particle boundaries are independent of user defined parameters, with no image preprocessing required. We demonstrate through...
The translocation of specific polypeptide chains across membranes is an essential activity for al... more The translocation of specific polypeptide chains across membranes is an essential activity for all life forms. The main components of the general secretory (Sec) system of E. coli include integral membrane translocon SecYEG, peripheral ATPase SecA, and SecDF, an ancillary complex that enhances polypeptide secretion by coupling translocation to proton motive force. Atomic force microscopy (AFM), a single-molecule imaging technique, is well suited to unmask complex, asynchronous molecular activities of membrane-associated proteins including those comprising the Sec apparatus. Using AFM, the dynamic structure of membrane-external protein topography of Sec system components can be directly visualized with high spatial-temporal precision. This mini-review is focused on AFM imaging of the Sec system in near-native fluid conditions where activity can be maintained and biochemically verified. Angstrom-scale conformational changes of SecYEG are reported on 100 ms timescales in fluid lipid bi...
Candida albicans causes severe invasive candidiasis. C. albicans infection requires the virulence... more Candida albicans causes severe invasive candidiasis. C. albicans infection requires the virulence factor candidalysin (CL) which damages target cell membranes. However, the mechanism that CL uses to permeabilize membranes is unclear. We reveal that CL forms membrane pores using a unique mechanism. Unexpectedly, CL readily assembled into polymers in solution. We propose that the basic structural unit in polymer formation is a CL oligomer, which is sequentially added into a string configuration that can close into a loop. CL loops appear to spontaneously insert into the membrane to become pores. A CL mutation (G4W) inhibited the formation of polymers in solution and prevented pore formation in synthetic lipid systems. Epithelial cell studies showed that G4W CL failed to activate the danger response pathway, a hallmark of the pathogenic effect of CL. These results indicate that CL polymerization in solution is a necessary step for the damage of cellular membranes. Analysis of CL pores ...
Atomic force microscopy has emerged as a valuable complementary technique in membrane structural ... more Atomic force microscopy has emerged as a valuable complementary technique in membrane structural biology. The apparatus is capable of probing individual membrane proteins in fluid lipid bilayers at room temperature with spatial resolution at the molecular length scale. Protein conformational dynamics are accessible over a range of biologically relevant timescales. This chapter presents methodology our group uses to achieve robust AFM image data of the General Secretory system, the primary pathway of protein export from the cytoplasm to the periplasm of E. coli. Emphasis is given to measuring and maintaining biochemical activity and to objective AFM image processing methods. For example, the biochemical assays can be used to determine chemomechanical coupling efficiency of surface adsorbed translocases. The Hessian blob algorithm and its extension to nonlocalized linear features, the line detection algorithm, provide automated feature delineations. Many of the methods discussed here can be applied to other membrane protein systems of interest.
1) Method for for integration of high resolution biological AFM with other powerful optical techn... more 1) Method for for integration of high resolution biological AFM with other powerful optical techniques 2) Straight-forward cleaning procedure for treatment of glass for Microscopy and Micromachining applications 3) Molecular high resolution imaging of bacteriorhodopsin and Sec translocase on glass supports 4) Direct observation of protein-protein interactions 5) Atomic Force Microscopy measurements of surface roughness of Glass, Mica, Lipid and comparison of several glass chemical treatments for Microscopy <strong>READ THE PEER-REVIEWED PUBLICATION HERE</strong> <strong>ASSOCIATED PEER-REVIEWED PUBLICATION </strong> <strong>Abstract</strong> Since its invention in the mid-1980s, the atomic force microscope (AFM) has become a valuable complementary<br> tool for studying membrane proteins in near-native environments. Historically, mica is the most common<br> substrate utilized for biological AFM. Glass being amorphous, transparent, and o...
In bacteria and archaea the protein conducting channel SecYEG provides a ubiquitous pathway for p... more In bacteria and archaea the protein conducting channel SecYEG provides a ubiquitous pathway for protein transfer across and into membranes. Further, it is known that SecA is the ATPase of the gener...
Intrinsic apoptosis is orchestrated by a group of proteins that mediate the coordinated disruptio... more Intrinsic apoptosis is orchestrated by a group of proteins that mediate the coordinated disruption of mitochondrial membranes. Bax is a multi-domain protein that, upon activation, disrupts the integrity of the...
SecA is the critical adenosine triphosphatase that drives preprotein transport through the transl... more SecA is the critical adenosine triphosphatase that drives preprotein transport through the translocon, SecYEG, in Escherichia coli. This process is thought to be regulated by conformational changes of specific domains of SecA, but real-time, real-space measurement of these changes is lacking. We use single-molecule atomic force microscopy (AFM) to visualize nucleotide-dependent conformations and conformational dynamics of SecA. Distinct topographical populations were observed in the presence of specific nucleotides. AFM investigations during basal adenosine triphosphate (ATP) hydrolysis revealed rapid, reversible transitions between a compact and an extended state at the ~100-ms time scale. A SecA mutant lacking the precursor-binding domain (PBD) aided interpretation. Further, the biochemical activity of SecA prepared for AFM was confirmed by tracking inorganic phosphate release. We conclude that ATP-driven dynamics are largely due to PBD motion but that other segments of SecA contr...
Methods in molecular biology (Clifton, N.J.), 2018
Atomic force microscopy (AFM)-based force spectroscopy is a powerful technique which has seen sig... more Atomic force microscopy (AFM)-based force spectroscopy is a powerful technique which has seen significant enhancements in both force and time resolution in recent years. This chapter details two AFM cantilever modification procedures that yield high force precision over different temporal bandwidths. Specifically, it explains a fairly straightforward method to achieve sub-pN force precision and stability at low frequencies (<50 Hz) by removing the metal coatings from a commercially available cantilever. A more involved procedure utilizing a focused ion beam milling machine is required to maintain high force precision at enhanced bandwidths. Both modification methods allow site-specific attachment of biomolecules onto the apex area of the tips for force spectroscopy. The chapter concludes with a comparative demonstration using the two cantilever modification methods to study a lipid-protein interaction.
Imaging by atomic force microscopy (AFM) offers high-resolution descriptions of many biological s... more Imaging by atomic force microscopy (AFM) offers high-resolution descriptions of many biological systems; however, regardless of resolution, conclusions drawn from AFM images are only as robust as the analysis leading to those conclusions. Vital to the analysis of biomolecules in AFM imagery is the initial detection of individual particles from large-scale images. Threshold and watershed algorithms are conventional for automatic particle detection but demand manual image preprocessing and produce particle boundaries which deform as a function of user-defined parameters, producing imprecise results subject to bias. Here, we introduce the Hessian blob to address these shortcomings. Combining a scale-space framework with measures of local image curvature, the Hessian blob formally defines particle centers and their boundaries, both to subpixel precision. Resulting particle boundaries are independent of user defined parameters, with no image preprocessing required. We demonstrate through...
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