Mechanism-Based Strategy for Optimizing HaloTag Protein Labeling

HaloTag labeling technology has introduced unrivaled potential in protein chemistry, molecular and cellular biology. A wide variety of ligands have been developed to meet the specific needs of diverse applications, but only a single protein tag, DhaAHT, is routinely used for their incorporation. Following a systematic kinetic and computational analysis of different reporters, tetramethylrhodamine and three 4-stilbazolium-based fluorescent ligands, we showed that the mechanism of incorporating different ligands depends both on the binding step and the efficiency of the chemical reaction. By studying the different haloalkane dehalogenases DhaA, LinB, and DmmA, we found that the architecture of the access tunnels is critical for the kinetics of both steps and the ligand specificity. We show that highly efficient labelling with specific ligands is achievable with natural dehalogenases. We propose a simple protocol for selecting the optimal protein tag for a specific ligand from a wide pool of available enzymes with diverse access tunnel architectures. The application of this protocol eliminates a need for expensive and laborious protein engineering.

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Simple Protocol to Facilitate Students’ Understanding of the Effects of Enzyme Immobilization on Kinetics of Reaction and Mass Transfer

Journal of Chemical Education ◽

10.1021/acs.jchemed.0c00848 ◽

2020 ◽

Vol 97 (8) ◽

pp. 2308-2313

Author(s):

Manuel Simões ◽

F. Xavier Malcata

Keyword(s):

Mass Transfer ◽

Enzyme Immobilization ◽

Simple Protocol ◽

Kinetics Of Reaction ◽

Kinetics Of

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Kinetics of duplex formation for individual DNA strands within a single protein nanopore

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.231434698 ◽

2001 ◽

Vol 98 (23) ◽

pp. 12996-13001 ◽

Cited By ~ 130

Author(s):

S. Howorka ◽

L. Movileanu ◽

O. Braha ◽

H. Bayley

Keyword(s):

Single Protein ◽

Dna Strands ◽

Duplex Formation ◽

Kinetics Of

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A Protocol for Minimal Single Protein Labeling with CyDye Fluors for Live Cell Internalization Assays

Fluorescence Methods for Investigation of Living Cells and Microorganisms ◽

10.5772/intechopen.92820 ◽

2020 ◽

Author(s):

Antonio J. Castro ◽

Alfonso Clemente ◽

Juan de Dios Alché

Keyword(s):

Live Cell ◽

Protein Labeling ◽

Cell Internalization ◽

Single Protein

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Protein Labeling And Localization In Cells Using A Self Labeling Protein Tag

The FASEB Journal ◽

10.1096/fasebj.20.5.a843-c ◽

2006 ◽

Vol 20 (5) ◽

Author(s):

L. Gedge ◽

K. Muentener ◽

T. Gibbs ◽

A. Brecht

Keyword(s):

Protein Labeling ◽

Protein Tag ◽

In Cells

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Abstract 322: HDL Dynamics in Circulation: Complexity of Protein Distribution and Metabolism Across HDL Size

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.35.suppl_1.322 ◽

2015 ◽

Vol 35 (suppl_1) ◽

Author(s):

Sasha Singh ◽

Allison Andraski ◽

Brett Pieper ◽

Wilson Goh ◽

Frank M Sacks ◽

...

Keyword(s):

Protein Distribution ◽

Size Fractions ◽

Immunoaffinity Purification ◽

Protein Pool ◽

Parallel Reaction Monitoring ◽

Single Protein ◽

Hdl Metabolism ◽

Kinetics Of ◽

Distinct Distribution

Introduction: The composition of specific apolipoproteins may determine HDL functions. We present novel mass spectrometry (MS)-based methods that capture the absolute quantities and kinetics of 7 apolipoproteins in 5 HDL size fractions. Methods and Results: Three participants were recruited, infused with a bolus of D3-Leu tracer, and blood was collected for 70 hrs. ApoA-I-containing HDL was prepared by immunoaffinity purification, separated into 5 size fractions, preβ, α3, α2, α1, and α0 by ND-PAGE, and in-gel trypsinized for MS. We monitored 7 proteins that likely affect HDL metabolism - apoA-I, apoA-II, apoA-IV, apoC-III, apoD, apoE and apoM. Each protein pool size had a distinct distribution across the HDL sizes. ApoE and apoM were enriched in larger HDL, whereas apoC-III and apoA-IV were enriched in smaller HDL sizes. We evaluated the tracer enrichment curves of these 7 proteins in the 5 fractions using high resolution parallel reaction monitoring performed on a quadrupole Orbitrap (Thermo). The enrichment curves for each protein varied from each other by slope and time of peak enrichment (Fig. 1a). In contrast, the enrichment curves across HDL sizes for a single protein showed smaller, but likely meaningful, differences in either slope or time of peak enrichment. Irrespective of the HDL size on which it resides, apoE had the fastest FCR, followed by apoA-IV, and apoC-III. ApoA-I/A-II, apoM, and apoD had slower but similar FCRs (Fig. 1b). Conclusions: This study showed distinct distribution and kinetic behaviors of 7 HDL proteins across 5 HDL size fractions that were conserved in the three participants. These findings may help elucidate the functional role of these proteins and the HDL particles that contain them.

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Self-perpetuating changes in Sup35 protein conformation as a mechanism of heredity in yeast

Biochemical Society Symposium ◽

10.1042/bss0680035 ◽

2001 ◽

Vol 68 ◽

pp. 35-43 ◽

Cited By ~ 3

Author(s):

Tricia R. Serio ◽

Anil G. Cashikar ◽

Anthony S. Kowal ◽

George J. Sawicki ◽

Susan L. Lindquist

Keyword(s):

Protein Conformation ◽

Physical State ◽

Yeast Saccharomyces Cerevisiae ◽

Single Protein ◽

Alternative Conformation ◽

Amyloid Assembly ◽

Kinetics Of ◽

Prion Hypothesis

Recently, a novel mode of inheritance has been described in the yeast Saccharomyces cerevisiae. The mechanism is based on the prion hypothesis, which posits that self-perpetuating changes in the conformation of single protein, PrP, underlie the severe neurodegeneration associated with the transmissible spongiform enchephalopathies in mammals. In yeast, two prions, [URE3] and [PSI+], have been identified, but these factors confer unique phenotypes rather than disease to the organism. In each case, the prion-associated phenotype has been linked to alternative conformations of the Ure2 and Sup35 proteins. Remarkably, Ure2 and Sup35 proteins existing in the alternative conformations have the unique capacity to transmit this physical state to the newly synthesized protein in vivo. Thus, a mechanism exists to ensure replication of the conformational information that underlies protein-only inheritance. We have characterized the mechanism by which Sup35 conformational information is replicated in vitro. The assembly of amyloid fibres by a region of Sup35 encompassing the N-terminal 254 amino acids faithfully recapitulates the in vivo propagation of [PSI+]. Mutations that alter [PSI+] inheritance in vivo change the kinetics of amyloid assembly in vitro in a complementary fashion, and lysates from [PSI+] cells, but not [psi-] cells, accelerate assembly in vitro. Using this system we propose a mechanism by which the alternative conformation of Sup35 is adopted by an unstructured oilgomeric intermediate at the time of assembly.

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In VivoStable Tumor-Specific Painting in Various Colors Using Dehalogenase-Based Protein-Tag Fluorescent Ligands

Bioconjugate Chemistry ◽

10.1021/bc9001344 ◽

2009 ◽

Vol 20 (7) ◽

pp. 1367-1374 ◽

Cited By ~ 29

Author(s):

Nobuyuki Kosaka ◽

Mikako Ogawa ◽

Peter L. Choyke ◽

Natasha Karassina ◽

Cesear Corona ◽

...

Keyword(s):

Protein Tag ◽

Fluorescent Ligands

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In-cell thermodynamics and a new role for protein surfaces

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1518620113 ◽

2016 ◽

Vol 113 (7) ◽

pp. 1725-1730 ◽

Cited By ~ 92

Author(s):

Austin E. Smith ◽

Larry Z. Zhou ◽

Annelise H. Gorensek ◽

Michael Senske ◽

Gary J. Pielak

Keyword(s):

Relevant Information ◽

Protein Chemistry ◽

Folding Kinetics ◽

Protein Surfaces ◽

Fluorine Nmr ◽

Signal Transduction Protein ◽

Nmr Data ◽

Kinetics Of ◽

In Cells

There is abundant, physiologically relevant knowledge about protein cores; they are hydrophobic, exquisitely well packed, and nearly all hydrogen bonds are satisfied. An equivalent understanding of protein surfaces has remained elusive because proteins are almost exclusively studied in vitro in simple aqueous solutions. Here, we establish the essential physiological roles played by protein surfaces by measuring the equilibrium thermodynamics and kinetics of protein folding in the complex environment of living Escherichia coli cells, and under physiologically relevant in vitro conditions. Fluorine NMR data on the 7-kDa globular N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) show that charge–charge interactions are fundamental to protein stability and folding kinetics in cells. Our results contradict predictions from accepted theories of macromolecular crowding and show that cosolutes commonly used to mimic the cellular interior do not yield physiologically relevant information. As such, we provide the foundation for a complete picture of protein chemistry in cells.

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Kinetics of the Reaction of Nickel(II) and 2,2′-Bipyridine in Ethanol

Canadian Journal of Chemistry ◽

10.1139/v73-593 ◽

1973 ◽

Vol 51 (23) ◽

pp. 3975-3977 ◽

Cited By ~ 1

Author(s):

M. L. Sanduja ◽

W. MacF. Smith

Keyword(s):

Kinetic Parameters ◽

Rate Constant ◽

Order Rate Constant ◽

Second Order ◽

Stopped Flow ◽

Ligand Specificity ◽

Temperature Range ◽

Kinetics Of Formation ◽

The Difference ◽

Kinetics Of

The kinetics of formation of the monobipyridine complex of nickel(II) in ethanol has been studied with stopped-flow methods over the temperature range 7 to 35 °C. The value of the second order rate constant kf at 25 °C of 6.6 × 10−3M−1 s1 and the values of ΔH≠ (10.1 ± 1.0 kcal mol−1) and of ΔS≠ (−7.3 ± 3.4 cal deg−1 mol−1) are close to the corresponding values for ethanol exchange on nickel(II) and suggest that the mechanism is dissociative interchange. However the difference in the values of the kinetic parameters of this reaction and those previously reported for the reactions involving the chemically similar phenanthroline imply a degree of ligand specificity for the reactions in ethanol which is considerably larger than is the case for reactions in water and methanol and that a common Id mechanism with monodentate formation being rate controlling is not applicable to both reactions.

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