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Methods in Enzymology

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https://www.readbyqxmd.com/read/27793295/preface
#1
EDITORIAL
Maria Spies, Yann R Chemla
No abstract text is available yet for this article.
2016: Methods in Enzymology
https://www.readbyqxmd.com/read/27793294/fluorescent-labeling-of-proteins-in-whole-cell-extracts-for-single-molecule-imaging
#2
S R Hansen, M L Rodgers, A A Hoskins
Cellular machines such as the spliceosome and ribosome can be composed of dozens of individual proteins and nucleic acids. Given this complexity, it is not surprising that many cellular activities have not yet been biochemically reconstituted. Such processes are often studied in vitro in whole cell or fractionated lysates. This presents a challenge for obtaining detailed biochemical information when the components being investigated may be only a minor component of the extract and unrelated processes may interfere with the assay...
2016: Methods in Enzymology
https://www.readbyqxmd.com/read/27793293/enzyme-kinetics-in-femtoliter-arrays
#3
P Mogalisetti, D R Walt
Over the last decade, femtoliter arrays have been used as a simple and robust way to encapsulate and monitor the kinetics of single enzyme molecules. Encapsulating individual enzyme molecules within a femtoliter-sized reaction chamber does not require immobilization of the enzyme molecules or fluorescent tagging of the enzyme molecules, which offers the unique advantage of observing unmodified single enzyme molecules free in solution. Several fascinating details about enzyme kinetics have been revealed using these femtoliter arrays, which were unattainable from traditional ensemble experiments...
2016: Methods in Enzymology
https://www.readbyqxmd.com/read/27793292/measuring-membrane-protein-dimerization-equilibrium-in-lipid-bilayers-by-single-molecule-fluorescence-microscopy
#4
R Chadda, J L Robertson
Dimerization of membrane protein interfaces occurs during membrane protein folding and cell receptor signaling. Here, we summarize a method that allows for measurement of equilibrium dimerization reactions of membrane proteins in lipid bilayers, by measuring the Poisson distribution of subunit capture into liposomes by single-molecule photobleaching analysis. This strategy is grounded in the fact that given a comparable labeling efficiency, monomeric or dimeric forms of a membrane protein will give rise to distinctly different photobleaching probability distributions...
2016: Methods in Enzymology
https://www.readbyqxmd.com/read/27793291/interferometric-scattering-microscopy-for-the-study-of-molecular-motors
#5
J Andrecka, Y Takagi, K J Mickolajczyk, L G Lippert, J R Sellers, W O Hancock, Y E Goldman, P Kukura
Our understanding of molecular motor function has been greatly improved by the development of imaging modalities, which enable real-time observation of their motion at the single-molecule level. Here, we describe the use of a new method, interferometric scattering microscopy, for the investigation of motor protein dynamics by attaching and tracking the motion of metallic nanoparticle labels as small as 20nm diameter. Using myosin-5, kinesin-1, and dynein as examples, we describe the basic assays, labeling strategies, and principles of data analysis...
2016: Methods in Enzymology
https://www.readbyqxmd.com/read/27793290/a-multicolor-single-molecule-fret-approach-to-study-protein-dynamics-and-interactions-simultaneously
#6
M Götz, P Wortmann, S Schmid, T Hugel
Single-molecule Förster resonance energy transfer (smFRET) is a versatile tool for studying biomolecules in a quantitative manner. Multiple conformations within and interactions between biomolecules can be detected and their kinetics can be determined. Thus, smFRET has become an essential tool in enzymology. Ordinary two-color smFRET experiments can provide only limited insight into the function of biological systems, which commonly consist of more than two components. A complete understanding of complex multicomponent biological systems requires correlated information on conformational rearrangements on the one hand and transient interactions with binding partners on the other...
2016: Methods in Enzymology
https://www.readbyqxmd.com/read/27793289/single-molecule-multicolor-fret-assay-for-studying-structural-dynamics-of-biomolecules
#7
S Lee, Y Jang, S-J Lee, S Hohng
Over the last 2 decades, single-molecule Forster resonance energy transfer (FRET) has been widely used to address important questions in molecular biology. However, a conventional approach based on a single donor-acceptor pair is not powerful enough to study complex biological systems. To address this challenge, single-molecule multicolor FRET techniques have been developed. In this chapter, we present practical considerations required for the successful implementation of single-molecule multicolor FRET in the laboratory...
2016: Methods in Enzymology
https://www.readbyqxmd.com/read/27793288/single-molecule-fluorescence-studies-of-fast-protein-folding
#8
Z Wang, L A Campos, V Muñoz
Watching the conformational wanderings of protein molecules during their search for their native structure is a holy grail for protein folding experimentalists. Such capability is essential to provide reality checks to the complex mechanistic heterogeneity that theory and molecular simulations predict. Single-molecule fluorescence resonance energy transfer (SM-FRET) is an attractive technique to meet that end, but its time resolution was insufficient for the microsecond motions of folding proteins. The temporal resolution of SM-FRET pivots on how quickly photons emitted by an individual molecule can be collected in sufficient numbers as to minimize statistical shot noise...
2016: Methods in Enzymology
https://www.readbyqxmd.com/read/27793287/methods-for-investigating-dna-accessibility-with-single-nucleosomes
#9
M D Gibson, M Brehove, Y Luo, J North, M G Poirier
Nucleosomes are the fundamental organizing unit of all eukaryotic genomes. Understanding how proteins gain access to DNA-binding sites located within nucleosomes is important for understanding DNA processing including transcription, replication, and repair. Single-molecule total internal reflection fluorescence (smTIRF) microscopy measurements can provide key insight into how proteins gain and maintain access to DNA sites within nucleosomes. Here, we describe methods for smTIRF experiments including the preparation of fluorophore-labeled nucleosomes, the smTIRF system, data acquisition, analysis, and controls...
2016: Methods in Enzymology
https://www.readbyqxmd.com/read/27793286/probing-the-conformational-landscape-of-dna-polymerases-using-diffusion-based-single-molecule-fret
#10
J Hohlbein, A N Kapanidis
Monitoring conformational changes in DNA polymerases using single-molecule Förster resonance energy transfer (smFRET) has provided new tools for studying fidelity-related mechanisms that promote the rejection of incorrect nucleotides before DNA synthesis. In addition to the previously known open and closed conformations of DNA polymerases, our smFRET assays utilizing doubly labeled variants of Escherichia coli DNA polymerase I were pivotal in identifying and characterizing a partially closed conformation as a primary checkpoint for nucleotide selection...
2016: Methods in Enzymology
https://www.readbyqxmd.com/read/27793285/single-molecule-imaging-with-one-color-fluorescence
#11
Y Qiu, S Myong
Single-molecule fluorescence imaging is a powerful tool that enables real-time observation of DNA-protein or RNA-protein interactions with a nanometer precision. Here, we provide a detailed procedure for a previously developed single-molecule fluorescence method, termed "single-molecule protein-induced fluorescence enhancement" (smPIFE). While smFRET (Förster resonance energy transfer) requires both donor and acceptor, protein-induced fluorescence enhancement (PIFE) employs a single dye and measures the increase in fluorescence intensity induced by protein binding near the dye...
2016: Methods in Enzymology
https://www.readbyqxmd.com/read/27793284/single-molecule-confocal-fret-microscopy-to-dissect-conformational-changes-in-the-catalytic-cycle-of-dna-topoisomerases
#12
S Hartmann, D Weidlich, D Klostermeier
Molecular machines undergo large-scale conformational changes during their catalytic cycles that are linked to their biological functions. DNA topoisomerases are molecular machines that interconvert different DNA topoisomers and resolve torsional stress that is introduced during cellular processes that involve local DNA unwinding. DNA gyrase catalyzes the introduction of negative supercoils into DNA in an ATP-dependent reaction. During its catalytic cycle, gyrase undergoes large-scale conformational changes that drive the supercoiling reaction...
2016: Methods in Enzymology
https://www.readbyqxmd.com/read/27793283/single-molecule-fret-to-measure-conformational-dynamics-of-dna-mismatch-repair-proteins
#13
J W Gauer, S LeBlanc, P Hao, R Qiu, B C Case, M Sakato, M M Hingorani, D A Erie, K R Weninger
Single-molecule FRET measurements have a unique sensitivity to protein conformational dynamics. The FRET signals can either be interpreted quantitatively to provide estimates of absolute distance in a molecule configuration or can be qualitatively interpreted as distinct states, from which quantitative kinetic schemes for conformational transitions can be deduced. Here we describe methods utilizing single-molecule FRET to reveal the conformational dynamics of the proteins responsible for DNA mismatch repair...
2016: Methods in Enzymology
https://www.readbyqxmd.com/read/27793282/putting-humpty-dumpty-together-clustering-the-functional-dynamics-of-single-biomolecular-machines-such-as-the-spliceosome
#14
C E Rohlman, M R Blanco, N G Walter
The spliceosome is a biomolecular machine that, in all eukaryotes, accomplishes site-specific splicing of introns from precursor messenger RNAs (pre-mRNAs) with high fidelity. Operating at the nanometer scale, where inertia and friction have lost the dominant role they play in the macroscopic realm, the spliceosome is highly dynamic and assembles its active site around each pre-mRNA anew. To understand the structural dynamics underlying the molecular motors, clocks, and ratchets that achieve functional accuracy in the yeast spliceosome (a long-standing model system), we have developed single-molecule fluorescence resonance energy transfer (smFRET) approaches that report changes in intra- and intermolecular interactions in real time...
2016: Methods in Enzymology
https://www.readbyqxmd.com/read/27793281/quantification-of-functional-dynamics-of-membrane-proteins-reconstituted-in-nanodiscs-membranes-by-single-turnover-functional-readout
#15
M E Moses, P Hedegård, N S Hatzakis
Single-molecule measurements are emerging as a powerful tool to study the individual behavior of biomolecules, revolutionizing our understanding of biological processes. Their ability to measure the distribution of behaviors, instead of the average behavior, allows the direct observation and quantification of the activity, abundance, and lifetime of multiple states and transient intermediates in the energy landscape that are typically averaged out in nonsynchronized ensemble measurements. Studying the function of membrane proteins at the single-molecule level remains a formidable challenge, and to date there is limited number of available functional assays...
2016: Methods in Enzymology
https://www.readbyqxmd.com/read/27793280/precisely-and-accurately-inferring-single-molecule-rate-constants
#16
C D Kinz-Thompson, N A Bailey, R L Gonzalez
The kinetics of biomolecular systems can be quantified by calculating the stochastic rate constants that govern the biomolecular state vs time trajectories (i.e., state trajectories) of individual biomolecules. To do so, the experimental signal vs time trajectories (i.e., signal trajectories) obtained from observing individual biomolecules are often idealized to generate state trajectories by methods such as thresholding or hidden Markov modeling. Here, we discuss approaches for idealizing signal trajectories and calculating stochastic rate constants from the resulting state trajectories...
2016: Methods in Enzymology
https://www.readbyqxmd.com/read/27793279/how-to-measure-separations-and-angles-between-intramolecular-fluorescent-markers
#17
K I Mortensen, J Sung, J A Spudich, H Flyvbjerg
Structure and function of an individual biomolecule can be explored with minimum two fluorescent markers of different colors. Since the light of such markers can be spectrally separated and imaged simultaneously, the markers can be colocalized. Here, we describe the method used for such two-color colocalization microscopy. Then we extend it to fluorescent markers with fixed orientations and in intramolecular proximity. Our benchmarking of this extension produced two extra results: (a) we established short double-labeled DNA molecules as probes of 3D orientation of anything to which one can attach them firmly; (b) we established how to map with super-resolution between color-separated channels, which should be useful for all dual-color colocalization measurements with either fixed or freely rotating fluorescent molecules...
2016: Methods in Enzymology
https://www.readbyqxmd.com/read/27793278/quantifying-the-assembly-of-multicomponent-molecular-machines-by-single-molecule-total-internal-reflection-fluorescence-microscopy
#18
E M Boehm, S Subramanyam, M Ghoneim, M Todd Washington, M Spies
Large, dynamic macromolecular complexes play essential roles in many cellular processes. Knowing how the components of these complexes associate with one another and undergo structural rearrangements is critical to understanding how they function. Single-molecule total internal reflection fluorescence (TIRF) microscopy is a powerful approach for addressing these fundamental issues. In this article, we first discuss single-molecule TIRF microscopes and strategies to immobilize and fluorescently label macromolecules...
2016: Methods in Enzymology
https://www.readbyqxmd.com/read/27793277/direct-fluorescent-imaging-of-translocation-and-unwinding-by-individual-dna-helicases
#19
T L Pavankumar, J C Exell, S C Kowalczykowski
The unique translocation and DNA unwinding properties of DNA helicases can be concealed by the stochastic behavior of enzyme molecules within the necessarily large populations used in ensemble experiments. With recent technological advances, the direct visualization of helicases acting on individual DNA molecules has contributed significantly to the current understanding of their mechanisms of action and biological functions. The combination of single-molecule techniques that enable both manipulation of individual protein or DNA molecules and visualization of their actions has made it possible to literally see novel and unique biochemical characteristics that were previously masked...
2016: Methods in Enzymology
https://www.readbyqxmd.com/read/27586351/preface
#20
EDITORIAL
V L Pecoraro
No abstract text is available yet for this article.
2016: Methods in Enzymology
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