Labeling of Proteins for Single-Molecule Fluorescence Spectroscopy

Conformational Dynamics ◽

Control Sample ◽

Resonance Energy ◽

Disordered Proteins ◽

Single Molecule Fluorescence Spectroscopy

Single-molecule fluorescence spectroscopy has become an important technique for studying the conformational dynamics and folding of proteins. A key step for performing such experiments is the availability of high-quality samples. Here we describe the practical details of a simple and widely applicable strategy for preparing proteins that are site-specifically labeled with a donor and an acceptor dye for single-molecule Förster resonance energy transfer (FRET) experiments. The method is based on introducing two cysteine residues that are labeled with maleimide-functionalized fluorophores, combined with high-resolution chromatography. We discuss how to optimize site-specific labeling even in the absence of orthogonal coupling chemistry and present purification strategies that are suitable for samples ranging from intrinsically disordered proteins to large folded proteins. We also discuss common problems in protein labeling, how to avoid them, and how to stringently control sample quality.<br>

Recent Advances in Computational Protocols Addressing Intrinsically Disordered Proteins

Biomolecules ◽

10.3390/biom9040146 ◽

2019 ◽

Vol 9 (4) ◽

pp. 146 ◽

Cited By ~ 9

Author(s):

Supriyo Bhattacharya ◽

Xingcheng Lin

Keyword(s):

Single Molecule ◽

Degrees Of Freedom ◽

Structural Information ◽

Conformational Dynamics ◽

Resonance Energy ◽

Structural Data ◽

Disordered Proteins ◽

Conformational Ensemble ◽

Intrinsically Disordered

Intrinsically disordered proteins (IDP) are abundant in the human genome and have recently emerged as major therapeutic targets for various diseases. Unlike traditional proteins that adopt a definitive structure, IDPs in free solution are disordered and exist as an ensemble of conformations. This enables the IDPs to signal through multiple signaling pathways and serve as scaffolds for multi-protein complexes. The challenge in studying IDPs experimentally stems from their disordered nature. Nuclear magnetic resonance (NMR), circular dichroism, small angle X-ray scattering, and single molecule Förster resonance energy transfer (FRET) can give the local structural information and overall dimension of IDPs, but seldom provide a unified picture of the whole protein. To understand the conformational dynamics of IDPs and how their structural ensembles recognize multiple binding partners and small molecule inhibitors, knowledge-based and physics-based sampling techniques are utilized in-silico, guided by experimental structural data. However, efficient sampling of the IDP conformational ensemble requires traversing the numerous degrees of freedom in the IDP energy landscape, as well as force-fields that accurately model the protein and solvent interactions. In this review, we have provided an overview of the current state of computational methods for studying IDP structure and dynamics and discussed the major challenges faced in this field.

Extreme Fuzziness: Direct Interactions between Two IDPs

Biomolecules ◽

10.3390/biom9030081 ◽

2019 ◽

Vol 9 (3) ◽

pp. 81 ◽

Cited By ~ 8

Author(s):

Wenning Wang ◽

Dongdong Wang

Keyword(s):

Single Molecule ◽

Protein Interaction ◽

Protein Interactions ◽

Conformational Dynamics ◽

Resonance Energy ◽

Disordered Proteins ◽

Full Spectrum ◽

Binding Mechanisms

Protein interactions involving intrinsically disordered proteins (IDPs) greatly extend the range of binding mechanisms available to proteins. In interactions employing coupled folding and binding, IDPs undergo disorder-to-order transitions to form a complex with a well-defined structure. In many other cases, IDPs retain structural plasticity in the final complexes, which have been defined as the fuzzy complexes. While a large number of fuzzy complexes have been characterized with variety of fuzzy patterns, many of the interactions are between an IDP and a structured protein. Thus, whether two IDPs can interact directly to form a fuzzy complex without disorder-to-order transition remains an open question. Recently, two studies of interactions between IDPs (4.1G-CTD/NuMA and H1/ProTα) have found a definite answer to this question. Detailed characterizations combined with nuclear magnetic resonance (NMR), single-molecule Förster resonance energy transfer (smFRET) and molecular dynamics (MD) simulation demonstrate that direct interactions between these two pairs of IDPs do form fuzzy complexes while retaining the conformational dynamics of the isolated proteins, which we name as the extremely fuzzy complexes. Extreme fuzziness completes the full spectrum of protein-protein interaction modes, suggesting that a more generalized model beyond existing binding mechanisms is required. Previous models of protein interaction could be applicable to some aspects of the extremely fuzzy interactions, but in more general sense, the distinction between native and nonnative contacts, which was used to understand protein folding and binding, becomes obscure. Exploring the phenomenon of extreme fuzziness may shed new light on molecular recognition and drug design.

Simulating freely-diffusing single-molecule FRET data with consideration of protein conformational dynamics

10.1101/2021.01.19.427359 ◽

2021 ◽

Author(s):

James Losey ◽

Michael Jauch ◽

David S. Matteson ◽

Mahmoud Moradi

Keyword(s):

Single Molecule ◽

Conformational Dynamics ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Disordered Proteins ◽

Analysis Techniques ◽

Single Molecule Fret ◽

Great Progress

AbstractSingle molecule Förster resonance energy transfer experiments have added a great deal to the under-standing of conformational states of biologically important molecules. While great progress has been made, much is still unknown in systems that are highly flexible such as intrinsically disordered proteins because of the high degeneracy of distance states, particularly when freely diffusing smFRET experiments are used. Simulated smFRET data allows for the control of underlying process that generates the data to examine if analytic techniques can detect these underlying differences. We have extended the PyBroMo software that simulates the freely diffusing smFRET data to include a distribution of inter-dye distances generated using Langevin dynamics in order to model proteins with greater flexibility or disorder in structure. Standard analysis techniques for smFRET data compared highlighted the differences observed between data generated with the base software and data that included the distribution of inter-dye distance.

Synergies of Single Molecule Fluorescence and NMR for the Study of Intrinsically Disordered Proteins

Biomolecules ◽

10.3390/biom12010027 ◽

2021 ◽

Vol 12 (1) ◽

pp. 27

Author(s):

Samuel Naudi-Fabra ◽

Martin Blackledge ◽

Sigrid Milles

Keyword(s):

Single Molecule ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Disordered Proteins ◽

Resonance Spectroscopy ◽

Multidomain Proteins ◽

Shed Light

Single molecule fluorescence and nuclear magnetic resonance spectroscopy (NMR) are two very powerful techniques for the analysis of intrinsically disordered proteins (IDPs). Both techniques have individually made major contributions to deciphering the complex properties of IDPs and their interactions, and it has become evident that they can provide very complementary views on the distance-dynamics relationships of IDP systems. We now review the first approaches using both NMR and single molecule fluorescence to decipher the molecular properties of IDPs and their interactions. We shed light on how these two techniques were employed synergistically for multidomain proteins harboring intrinsically disordered linkers, for veritable IDPs, but also for liquid–liquid phase separated systems. Additionally, we provide insights into the first approaches to use single molecule Förster resonance energy transfer (FRET) and NMR for the description of multiconformational models of IDPs.

From folding to function: complex macromolecular reactions unraveled one-by-one with optical tweezers

Essays in Biochemistry ◽

10.1042/ebc20200024 ◽

2021 ◽

Author(s):

Pétur O. Heidarsson ◽

Ciro Cecconi

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Structural Changes ◽

Conformational Dynamics ◽

Disordered Proteins ◽

Kinase Activation ◽

Complex Protein ◽

Methodological Principles

Abstract Single-molecule manipulation with optical tweezers has uncovered macromolecular behaviour hidden to other experimental techniques. Recent instrumental improvements have made it possible to expand the range of systems accessible to optical tweezers. Beyond focusing on the folding and structural changes of isolated single molecules, optical tweezers studies have evolved into unraveling the basic principles of complex molecular processes such as co-translational folding on the ribosome, kinase activation dynamics, ligand–receptor binding, chaperone-assisted protein folding, and even dynamics of intrinsically disordered proteins (IDPs). In this mini-review, we illustrate the methodological principles of optical tweezers before highlighting recent advances in studying complex protein conformational dynamics – from protein synthesis to physiological function – as well as emerging future issues that are beginning to be addressed with novel approaches.

Single-molecule fluorescence studies of intrinsically disordered proteins and liquid phase separation

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics ◽

10.1016/j.bbapap.2019.04.007 ◽

2019 ◽

Vol 1867 (10) ◽

pp. 980-987 ◽

Cited By ~ 7

Author(s):

Irem Nasir ◽

Paulo L. Onuchic ◽

Sergio R. Labra ◽

Ashok A. Deniz

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Single Molecule ◽

Liquid Phase Separation ◽

Disordered Proteins ◽

Fluorescence Studies ◽

Intrinsically Disordered

Faculty Opinions recommendation of Single-molecule fluorescence studies of intrinsically disordered proteins and liquid phase separation.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.735696315.793570596 ◽

2020 ◽

Author(s):

Vladimir Uversky

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Single Molecule ◽

Liquid Phase Separation ◽

Disordered Proteins ◽

Fluorescence Studies ◽

Intrinsically Disordered

Phosphorylation-induced conformational dynamics in an intrinsically disordered protein and potential role in phenotypic heterogeneity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1700082114 ◽

2017 ◽

Vol 114 (13) ◽

pp. E2644-E2653 ◽

Cited By ~ 43

Author(s):

Prakash Kulkarni ◽

Mohit Kumar Jolly ◽

Dongya Jia ◽

Steven M. Mooney ◽

Ajay Bhargava ◽

...

Keyword(s):

Single Molecule ◽

Large Fraction ◽

Conformational Dynamics ◽

3D Structure ◽

Random Coil ◽

Disordered Proteins ◽

Conformational Ensemble ◽

Androgen Independent

Intrinsically disordered proteins (IDPs) that lack a unique 3D structure and comprise a large fraction of the human proteome play important roles in numerous cellular functions. Prostate-Associated Gene 4 (PAGE4) is an IDP that acts as a potentiator of the Activator Protein-1 (AP-1) transcription factor. Homeodomain-Interacting Protein Kinase 1 (HIPK1) phosphorylates PAGE4 at S9 and T51, but only T51 is critical for its activity. Here, we identify a second kinase, CDC-Like Kinase 2 (CLK2), which acts on PAGE4 and hyperphosphorylates it at multiple S/T residues, including S9 and T51. We demonstrate that HIPK1 is expressed in both androgen-dependent and androgen-independent prostate cancer (PCa) cells, whereas CLK2 and PAGE4 are expressed only in androgen-dependent cells. Cell-based studies indicate that PAGE4 interaction with the two kinases leads to opposing functions. HIPK1-phosphorylated PAGE4 (HIPK1-PAGE4) potentiates c-Jun, whereas CLK2-phosphorylated PAGE4 (CLK2-PAGE4) attenuates c-Jun activity. Consistent with the cellular data, biophysical measurements (small-angle X-ray scattering, single-molecule fluorescence resonance energy transfer, and NMR) indicate that HIPK1-PAGE4 exhibits a relatively compact conformational ensemble that binds AP-1, whereas CLK2-PAGE4 is more expanded and resembles a random coil with diminished affinity for AP-1. Taken together, the results suggest that the phosphorylation-induced conformational dynamics of PAGE4 may play a role in modulating changes between PCa cell phenotypes. A mathematical model based on our experimental data demonstrates how differential phosphorylation of PAGE4 can lead to transitions between androgen-dependent and androgen-independent phenotypes by altering the AP-1/androgen receptor regulatory circuit in PCa cells.

Single Molecule FRET: A Powerful Tool to Study Intrinsically Disordered Proteins

Biomolecules ◽

10.3390/biom8040140 ◽

2018 ◽

Vol 8 (4) ◽

pp. 140 ◽

Cited By ~ 17

Author(s):

Sharonda LeBlanc ◽

Prakash Kulkarni ◽

Keith Weninger

Keyword(s):

Single Molecule ◽

Biological Networks ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Polymer Physics ◽

Disordered Proteins ◽

Single Molecule Fret ◽

Random Fluctuations

Intrinsically disordered proteins (IDPs) are often modeled using ideas from polymer physics that suggest they smoothly explore all corners of configuration space. Experimental verification of this random, dynamic behavior is difficult as random fluctuations of IDPs cannot be synchronized across an ensemble. Single molecule fluorescence (or Förster) resonance energy transfer (smFRET) is one of the few approaches that are sensitive to transient populations of sub-states within molecular ensembles. In some implementations, smFRET has sufficient time resolution to resolve transitions in IDP behaviors. Here we present experimental issues to consider when applying smFRET to study IDP configuration. We illustrate the power of applying smFRET to IDPs by discussing two cases in the literature of protein systems for which smFRET has successfully reported phosphorylation-induced modification (but not elimination) of the disordered properties that have been connected to impacts on the related biological function. The examples we discuss, PAGE4 and a disordered segment of the GluN2B subunit of the NMDA receptor, illustrate the great potential of smFRET to inform how IDP function can be regulated by controlling the detailed ensemble of disordered states within biological networks.