Performance benchmarking of four cell-free protein expression systems.
Biotechnology and Bioengineering 113(2):292 (2016)
Over the last half century, a range of cell-free protein expression systems based on pro- and eukaryotic organisms have been developed and have found a range of applications, from structural biology to directed protein evolution. While it is generally accepted that significant differences in per...
S-7 : A new cytokine receptor activation paradigm: Activation of JAK2 by the growth hormone receptor
Cytokine 70(1):22 (2014)
The conformational changes required to transmit the GH binding signal from the extracellular domain of the GH receptor to its intracellular domain resulting in activation of JAK2 has been enigmatic. We have recently defined the first complete mechanistic model for JAK2 activation based...
Increased Polyubiquitination and Proteasomal Degradation of a Munc18-1 Disease-Linked Mutant Causes Temperature-Sensitive Defect in Exocytosis
Cell Reports 9(1):206 (2014)
Munc18-1 is a critical component of the core machinery controlling neuroexocytosis. Recently, mutations in Munc18-1 leading to the development of early infantile epileptic encephalopathy have been discovered. However, which degradative pathway controls Munc18-1 levels and how it impact...
Rapid mapping of interactions between Human SNX-BAR proteins measured in vitro by AlphaScreen and single-molecule spectroscopy.
Molecular & Cellular Proteomics 13(9):2233 (2014)
Protein dimerization and oligomerization is commonly used by nature to increase the structural and functional complexity of proteins. Regulated protein assembly is essential to transfer information in signaling, transcriptional, and membrane trafficking events. Here we show that a combination of...
Cortactin scaffolds Arp2/3 and WAVE2 at the epithelial zonula adherens.
Journal of Biological Chemistry 289(11):7764 (2014)
Cadherin junctions arise from the integrated action of cell adhesion, signaling, and the cytoskeleton. At the zonula adherens (ZA), a WAVE2-Arp2/3 actin nucleation apparatus is necessary for junctional tension and integrity. But how this is coordinated with cadherin adhesion is not known. We now...
Single-molecule analysis reveals self assembly and nanoscale segregation of two distinct cavin subcomplexes on caveolae.
eLife 3:e01434 (2014)
In mammalian cells three closely related cavin proteins cooperate with the scaffolding protein caveolin to form membrane invaginations known as caveolae. Here we have developed a novel single-molecule fluorescence approach to directly observe interactions and stoichiometries in protein complexes...
Ultrafast cooling reveals microsecond-scale biomolecular dynamics.
Nature Communications 5:5737 (2014)
The temperature-jump technique, in which the sample is rapidly heated by a powerful laser pulse, has been widely used to probe the fast dynamics of folding of proteins and nucleic acids. However, the existing temperature-jump setups tend to involve sophisticated and expensive instrumentation, wh...
Counteracting chemical chaperone effects on the single-molecule α-synuclein structural landscape.
PNAS 109(44):17826 (2012)
Protein structure and function depend on a close interplay between intrinsic folding energy landscapes and the chemistry of the protein environment. Osmolytes are small-molecule compounds that can act as chemical chaperones by altering the environment in a cellular context. Despite their importa...
Intramolecular three-colour single pair FRET of intrinsically disordered proteins with increased dynamic range.
Molecular BioSystems 8(10):2531 (2012)
Single molecule observation of fluorescence resonance energy transfer can be used to provide insight into the structure and dynamics of proteins. Using a straightforward triple-colour labelling strategy, we present a measurement and analysis scheme that can simultaneously study multiple regions ...
Quantitative analysis of prenylated RhoA interaction with its chaperone, RhoGDI.
Journal of Biological Chemistry 287(32):26549 (2012)
Small GTPases of the Rho family regulate cytoskeleton remodeling, cell polarity, and transcription, as well as the cell cycle, in eukaryotic cells. Membrane delivery and recycling of the Rho GTPases is mediated by Rho GDP dissociation inhibitor (RhoGDI), which forms a stable complex with prenyla...
Visualizing a one-way protein encounter complex by ultrafast single-molecule mixing.
Nature Methods 8(3):239 (2011)
We combined rapid microfluidic mixing with single-molecule fluorescence resonance energy transfer to study the folding kinetics of the intrinsically disordered human protein α-synuclein. The time-resolution of 0.2 ms revealed initial collapse of the unfolded protein induced by binding with lipid...
Multicolor single-molecule FRET to explore protein folding and binding.
Molecular BioSystems 6(9):1540 (2010)
Proper protein function in cells, tissues and organisms depends critically on correct protein folding or interaction with partners. Over the last decade, single-molecule FRET (smFRET) has emerged as a powerful tool to probe complex distributions, dynamics, pathways and landscapes in protein fold...
Variation of the lateral mobility of transmembrane peptides with hydrophobic mismatch.
Journal of Physical Chemistry B 114(10):3559 (2010)
A hydrophobic mismatch between protein length and membrane thickness can lead to a modification of protein conformation, function, and oligomerization. To study the role of hydrophobic mismatch, we have measured the change in mobility of transmembrane peptides possessing a hydrophobic helix of v...
Ultrafast microfluidic mixer with three-dimensional flow focusing for studies of biochemical kinetics.
Lab On a Chip 10(5):598 (2010)
Studies of the kinetics of biochemical reactions, especially of folding of proteins and RNA, are important for understanding the function of biomolecules and processes in live cells. Many biochemical reactions occur rapidly and thus need to be triggered on very short time scales for their kineti...
Single-molecule fluorescence studies of intrinsically disordered proteins.
Methods in Enzymology 472:179 (2010)
Intrinsically disordered proteins (IDPs) (also referred to as natively unfolded proteins) play critical roles in a variety of cellular processes such as transcription and translation and also are linked to several human diseases. Biophysical studies of IDPs present unusual experimental challenge...
Microfluidic device for single-molecule experiments with enhanced photostability.
Journal of the American Chemical Society 131(38):13610 (2009)
A microfluidic device made of polydimethylsiloxane (PDMS) addresses key limitations in single-molecule fluorescence experiments by providing high dye photostability and low sample sticking. Photobleaching is dramatically reduced by deoxygenation via gas diffusion through porous channel walls. Ra...
High-resolution temperature-concentration diagram of alpha-synuclein conformation obtained from a single Förster resonance energy transfer image in a microfluidic device.
Analytical chemistry 81(16):6929 (2009)
We present a microfluidic device for rapid and efficient determination of protein conformations in a range of medium conditions and temperatures. The device generates orthogonal gradients of concentration and temperature in an interrogation area that fits into the field of view of an objective l...
Direct single-molecule observation of a protein living in two opposed native structures.
PNAS 106(25):10153 (2009)
Biological activity in proteins requires them to share the energy landscape for folding and global conformational motions, 2 key determinants of function. Although most structural studies to date have focused on fluctuations around a single structural basin, we directly observe the coexistence o...
Interplay of alpha-synuclein binding and conformational switching probed by single-molecule fluorescence.
PNAS 106(14):5645 (2009)
We studied the coupled binding and folding of alpha-synuclein, an intrinsically disordered protein linked with Parkinson's disease. Using single-molecule fluorescence resonance energy transfer and correlation methods, we directly probed protein membrane association, structural distributions, and...
The Rop-Dimer: A Folded Protein Living Between Two Alternate Structures
Biophysical Journal 96(3):568a (2009)