scholarly journals Insight into helicase mechanism and function revealed through single-molecule approaches

2010 ◽  
Vol 43 (2) ◽  
pp. 185-217 ◽  
Author(s):  
Jaya G. Yodh ◽  
Michael Schlierf ◽  
Taekjip Ha
Keyword(s):  
Single Molecule ◽  
Conformational Dynamics ◽  
Magnetic Tweezers ◽  
Repetitive Behaviors ◽  
Helicase Activity ◽  
Step Size ◽  
Mechanistic Insight ◽  
Combination Of Methods ◽  
And Function ◽  
Insight Into

AbstractHelicases are a class of nucleic acid (NA) motors that catalyze NTP-dependent unwinding of NA duplexes into single strands, a reaction essential to all areas of NA metabolism. In the last decade, single-molecule (sm) technology has proven to be highly useful in revealing mechanistic insight into helicase activity that is not always detectable via ensemble assays. A combination of methods based on fluorescence, optical and magnetic tweezers, and flow-induced DNA stretching has enabled the study of helicase conformational dynamics, force generation, step size, pausing, reversal and repetitive behaviors during translocation and unwinding by helicases working alone and as part of multiprotein complexes. The contributions of these sm investigations to our understanding of helicase mechanism and function will be discussed.

scholarly journals S06A1 Mechanistic insight into RNA acetylation to maintain the decoding fidelity in bacteria(Structural Biology of ncRNA Processing and Function)

2007 ◽  
Vol 47 (supplement) ◽  
pp. S8
Author(s):  
Sarin Chimnaronk ◽  
Tateki Suzuki ◽  
Tetsuhiro Manita ◽  
Yoshiho Ikeuchi ◽  
Min Yao ◽  
...  
Keyword(s):  
Structural Biology ◽  
Mechanistic Insight ◽  
And Function ◽  
Insight Into ◽  
Decoding Fidelity

scholarly journals Transcription factors Foxo3a and Foxo1 couple the E3 ligase Cbl-b to the induction of Foxp3 expression in induced regulatory T cells

2010 ◽  
Vol 207 (7) ◽  
pp. 1381-1391 ◽  
Author(s):  
Yohsuke Harada ◽  
Yasuyo Harada ◽  
Chris Elly ◽  
Ge Ying ◽  
Ji-Hye Paik ◽  
...  
Keyword(s):  
T Cells ◽  
Ring Finger ◽  
Foxp3 Expression ◽  
Proximal Region ◽  
Binding Motif ◽  
Mechanistic Insight ◽  
T Cell Regulation ◽  
And Function ◽  
Deficient Mice ◽  
Insight Into

The transcription factor Foxp3 is essential for optimal regulatory T (T reg) cell development and function. Here, we show that CD4+ T cells from Cbl-b RING finger mutant knockin or Cbl-b–deficient mice show impaired TGF-β–induced Foxp3 expression. These T cells display augmented Foxo3a phosphorylation, but normal TGF-β signaling. Expression of Foxo3a rescues Foxp3 expression in Cbl-b–deficient T cells, and Foxo3a deficiency results in defective TGF-β–driven Foxp3 induction. A Foxo3a-binding motif is present in a proximal region of the Foxp3 promoter, and is required for Foxo3a association. Foxo1 exerts similar effects as Foxo3a on Foxp3 expression. This study reveals that Foxo factors promote transcription of the Foxp3 gene in induced T reg cells, and thus provides new mechanistic insight into Foxo-mediated T cell regulation.

scholarly journals Single Molecule FRET Studies of Protein Conformational Landscapes: 3 Prototypic Examples for the Relation Between Conformational Dynamics and Function

2011 ◽  
Vol 100 (3) ◽  
pp. 474a-475a
Author(s):  
Markus Richert ◽  
Dymitro Rodnin ◽  
Carola S. Hengstenberg ◽  
Thomas Peulen ◽  
Alessandro Valeri ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Dynamics ◽  
Single Molecule Fret ◽  
Dynamics And Function ◽  
And Function

Structural and mechanistic insight into alkane hydroxylation by Pseudomonas putida AlkB

2014 ◽  
Vol 460 (2) ◽  
pp. 283-293 ◽  
Author(s):  
Hernan Alonso ◽  
Oded Kleifeld ◽  
Adva Yeheskel ◽  
Poh C. Ong ◽  
Yu C. Liu ◽  
...  
Keyword(s):  
Ab Initio ◽  
Pseudomonas Putida ◽  
Structure And Function ◽  
Alkane Hydroxylation ◽  
Mechanistic Insight ◽  
Unknown Structure ◽  
And Function ◽  
Alkane Hydroxylases ◽  
Insight Into

Integral membrane non-haem di-iron alkane hydroxylases (AlkBs) are enzymes of unknown structure that allow bacteria to grow on alkanes. Catalysis-linked modifications with the inhibitor 1-octyne, mutagenesis studies and ab initio modelling provided novel insights into the structure and function of AlkB.

scholarly journals A nanobody-based molecular toolkit provides new mechanistic insight into clathrin-coat initiation

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Linton M Traub
Keyword(s):  
Decisive Role ◽  
Subcellular Location ◽  
Combined Approach ◽  
Mechanistic Insight ◽  
Early Endosomes ◽  
The Stability ◽  
And Function ◽  
Insight Into ◽  
Complementarity Determining Region

Besides AP-2 and clathrin triskelia, clathrin coat inception depends on a group of early-arriving proteins including Fcho1/2 and Eps15/R. Using genome-edited cells, we described the role of the unstructured Fcho linker in stable AP-2 membrane deposition. Here, expanding this strategy in combination with a new set of llama nanobodies against EPS15 shows an FCHO1/2–EPS15/R partnership plays a decisive role in coat initiation. A nanobody containing an Asn-Pro-Phe peptide within the complementarity-determining region 3 loop is a function-blocking pseudoligand for tandem EPS15/R EH domains. Yet, in living cells, EH domains gathered at clathrin-coated structures are poorly accessible, indicating residence by endogenous NPF-bearing partners. Forcibly sequestering cytosolic EPS15 in genome-edited cells with nanobodies tethered to early endosomes or mitochondria changes the subcellular location and availability of EPS15. This combined approach has strong effects on clathrin coat structure and function by dictating the stability of AP-2 assemblies at the plasma membrane.

Magnetic tweezers: a sensitive tool to study DNA and chromatin at the single-molecule level

2003 ◽  
Vol 81 (3) ◽  
pp. 151-159 ◽  
Author(s):  
Jordanka Zlatanova ◽  
Sanford H Leuba
Keyword(s):  
Single Molecule ◽  
Magnetic Tweezers ◽  
Biological Macromolecules ◽  
High Definition ◽  
Single Molecule Level ◽  
Applied Tension ◽  
Sensitive Tool ◽  
Different Types ◽  
Distinct Features ◽  
Insight Into

The advent of single-molecule biology has allowed unprecedented insight into the dynamic behavior of biological macromolecules and their complexes. Unexpected properties, masked by the asynchronous behavior of myriads of molecules in bulk experiments, can be revealed; equally importantly, individual members of a molecular population often exhibit distinct features in their properties. Finally, the single-molecule approaches allow us to study the behavior of biological macromolecules under applied tension or torsion; understanding the mechanical properties of these molecules helps us understand how they function in the cell. In this review, we summarize the application of magnetic tweezers (MT) to the study of DNA behavior at the single-molecule level. MT can be conveniently used to stretch DNA and introduce controlled levels of superhelicity into the molecule and to follow to a high definition the action of different types of topoisomerases. Its potential for chromatin studies is also enormous, and we will briefly present our first chromatin results.Key words: single-molecules, chromatin, topoisomerases, magnetic tweezers, force.

scholarly journals Modular, ultra-stable, and highly parallel protein force spectroscopy in magnetic tweezers using peptide linkers

2018 ◽  
Author(s):  
Achim Löf ◽  
Philipp U Walker ◽  
Steffen M Sedlak ◽  
Tobias Obser ◽  
Maria A Brehm ◽  
...  
Keyword(s):  
Single Molecule ◽  
Force Spectroscopy ◽  
Magnetic Tweezers ◽  
Protein Domain ◽  
High Yield ◽  
Primary Hemostasis ◽  
Wide Range ◽  
And Function ◽  
Von Willebrand

Single-molecule force spectroscopy has provided unprecedented insights into protein folding, force-regulation, and function. Here, we present a modular magnetic tweezers force spectroscopy approach that uses elastin-like polypeptide linkers to provide a high yield of protein tethers. Our approach extends protein force spectroscopy into the low force (<1 pN) regime and enables ultra-stable measurements on many molecules in parallel. We present (un-)folding data for the single protein domain ddFLN4 and for the large multi-domain dimeric protein von Willebrand factor (VWF) that is critically involved in primary hemostasis. The measurements reveal exponential force-dependencies of unfolding and refolding rates, directly resolve the stabilization of the VWF A2 domain by Ca2+, and discover transitions in the VWF C-domain stem at low forces that likely constitute the first steps of VWF activation in vivo. Our modular attachment approach will enable precise and multiplexed force spectroscopy measurements for a wide range of proteins in the physiologically relevant force regime.

scholarly journals Probing conformational dynamics of an enzymatic active site by an in situ single fluorogenic probe under piconewton force manipulation

2016 ◽  
Vol 113 (52) ◽  
pp. 15006-15011 ◽  
Author(s):  
Nibedita Pal ◽  
Meiling Wu ◽  
H. Peter Lu
Keyword(s):  
Single Molecule ◽  
Active Site ◽  
Complex Dynamics ◽  
Conformational Dynamics ◽  
Enzymatic Reaction ◽  
Product Formation ◽  
Magnetic Tweezers ◽  
Complex Nature ◽  
Enzyme Substrate ◽  
Multiple Product

Unraveling the conformational details of an enzyme during the essential steps of a catalytic reaction (i.e., enzyme–substrate interaction, enzyme–substrate active complex formation, nascent product formation, and product release) is challenging due to the transient nature of intermediate conformational states, conformational fluctuations, and the associated complex dynamics. Here we report our study on the conformational dynamics of horseradish peroxidase using single-molecule multiparameter photon time-stamping spectroscopy with mechanical force manipulation, a newly developed single-molecule fluorescence imaging magnetic tweezers nanoscopic approach. A nascent-formed fluorogenic product molecule serves as a probe, perfectly fitting in the enzymatic reaction active site for probing the enzymatic conformational dynamics. Interestingly, the product releasing dynamics shows the complex conformational behavior with multiple product releasing pathways. However, under magnetic force manipulation, the complex nature of the multiple product releasing pathways disappears and more simplistic conformations of the active site are populated.

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