Determination of Fluorescent Probe Orientations on Biomolecules by Conformational Searching: Algorithm Testing and Applications to the Atomic Model of Myosin

Root, Douglas D.; Shangguan, Xin; Xu, Jin; McAllister, Michael A.

doi:10.1006/jsbi.1999.4127

Cited by 20 publications

(19 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…(2), k 2 is an orientation factor having a value of 2/3 in these experiments, Q D is the quantum yield of the donor, given by t d /t Tb where t Tb is the lifetime of free Tb(III) when directly excited with the laser (4.75 ms) (Root et al, 1999). The refractive index h is 1.4 for biological samples in water and, in equation 3, F D (l) is the corrected fluorescence intensity of the donor, and e is the extinction coefficient of the acceptor.…”

Section: Lret Studiesmentioning

confidence: 99%

Strategy for selecting and characterizing linker peptides for CBM9‐tagged fusion proteins expressed in Escherichia coli

Kavoosi

Creagh

Kilburn

et al. 2007

Biotech & Bioengineering

View full text Add to dashboard Cite

The influence of linker design on fusion protein production and performance was evaluated when a family 9 carbohydrate-binding module (CBM9) serves as the affinity tag for recombinant proteins expressed in Escherichia coli. Two bioinformatic strategies for linker design were applied: the first identifies naturally occurring linkers within the proteome of the host organism, the second involves screening peptidases and their known specificities using the bioinformatics software MEROPS to design an artificial linker resistant to proteolysis within the host. Linkers designed using these strategies were compared against traditional poly-glycine linkers. Although widely used, glycine-rich linkers were found by tandem MS data to be susceptible to hydrolysis by E. coli peptidases. The natural (PT)(x)P and MEROPS-designed S(3)N(10) linkers were significantly more stable, indicating both strategies provide a useful approach to linker design. Factor X(a) processing of the fusion proteins depended strongly on linker chemistry, with poly(G) and S(3)N(10) linkers showing the fastest cleavage rates. Luminescence resonance energy transfer studies, used to measure average distance of separation between GFP and Tb(III) bound to a strong calcium-binding site of CBM9, revealed that, for a given linker chemistry, the separation distance increases with increasing linker length. This increase was particularly large for poly(G) linkers, suggesting that this linker chemistry adopts a hydrated, extended configuration that makes it particularly susceptible to proteolysis. Differential scanning calorimetry studies on the PT linker series showed that fusion of CBM9 to GFP did not alter the T(m) of GFP but did result in a destabilization, as seen by both a decrease in T(m) and DeltaH(cal), of CBM9. The degree of destabilization increased with decreasing length of the (PT)(x)P linker such that DeltaT(m) = -8.4 degrees C for the single P linker.

show abstract

Section: Lret Studiesmentioning

confidence: 99%

Strategy for selecting and characterizing linker peptides for CBM9‐tagged fusion proteins expressed in Escherichia coli

Kavoosi

Creagh

Kilburn

et al. 2007

Biotech & Bioengineering

View full text Add to dashboard Cite

show abstract

“…7,9,[13][14][15] Oligonucleotides were provided by Bio-Synthesis, Inc. (Lewisville, TX). Lanthanide chelates were attached to the 5Ј or 3Ј ends of oligonucleotides via C3 amine linkages.…”

Section: Probe Synthesismentioning

confidence: 99%

“…The force field was then changed to OPLS-AA and the lanthanide chelate and fluorescein were modeled onto the structure using conformational searching as previously described. 14 The structure was then entered into more rounds of MC/SD simulations and multiple minimizations until no lower energy structures were obtained. Parameters for the lanthanide ion were based on previously published values 21 and were refined to reproduce experimental hydration free energies, bond lengths, and binding energies to aminocarboxylate chelates in the generalized Born/surface area continuum solvent model used for these simulations.…”

Section: Molecular Modelingmentioning

confidence: 99%

See 1 more Smart Citation

Detection of single nucleotide variations by a hybridization proximity assay based on molecular beacons and luminescence resonance energy transfer

et al. 2004

Self Cite

View full text Add to dashboard Cite

A powerful combination of molecular beacon and luminescence resonance energy transfer technology reveals alterations in nucleic acid structure by as little as a single nucleotide in a novel hybridization proximity assay. The assay measures the length of a single-stranded target when a terbium chelate-labeled molecular beacon hybridizes to one side of the nucleic acid segment to be measured and an acceptor probe carrying a convention fluorophore hybridizes to the opposite end of the target. Using a test sequence shortened incrementally by deleting single nucleotides, this assay reports a nearly linear relationship between sequence length and the distance separating acceptor and donor probes. Consequently, this assay can be used to detect alternative splicing, allele types, rearrangements, insertion, and deletion events by measuring separation distances within a predefined region. Furthermore, the use of terbium chelates in molecular beacons can produce exceptionally high signal-to-background ratios compared to the use of conventional fluorophores. Principles of optimal probe design are investigated experimentally and by computational simulations of plausible molecular beacon folding. Some molecular beacon designs form dimers that reduce their maximal response to target sequences. A simple assay to detect such dimers is reported as a tool to help improve the design of molecular beacons. Optimally designed molecular beacons with terbium chelates and hybridization proximity assays are expected to expand their applications in the analysis and screening of genetic diseases.

show abstract

“…The myosin heavy chain was truncated to residue 792 to leave only the motor domain. Energy minimization and subsequent simulations of the entire modified chicken skeletal myosin S1 was performed using the AMBER* force field and a generalized Born / solvation area model as previously [79]. For both cleft opening and closing simulations, 11 pairs of distance constraints were applied around the 50 kDa cleft.…”

Section: Computational Proceduresmentioning

confidence: 99%

Fluorescence labeling and computational analysis of the strut of myosin’s 50 kDa cleft

Gawalapu¹,

Root²

2006

Archives of Biochemistry and Biophysics

Self Cite

View full text Add to dashboard Cite

Gawalapu, Ravi Kumar. Fluorescence labeling and computational analysis of the strut of myosin's 50 kDa cleft. Doctor of Philosophy (Biochemistry), August 2007, 148 pp., 3 tables, 60 illustrations, 120 references, 120 titles.In order to understand the structural changes in myosin S1, fluorescence polarization and computational dynamics simulations were used. Dynamics simulations on the S1 motor domain indicated that significant flexibility was present throughout the molecular model. The constrained opening versus closing of the 50 kDa cleft appeared to induce opposite directions of movement in the lever arm. A sequence called the "strut" which traverses the 50 kDa cleft and may play an important role in positioning the actomyosin binding interface during actin binding is thought to be intimately linked to distant structural changes in the myosin's nucleotide cleft and neck regions. To study the dynamics of the strut region, a method of fluorescent labeling of the strut was discovered using the dye CY3. CY3 served as a hydrophobic tag for purification by hydrophobic interaction chromatography which enabled the separation of labeled and unlabeled species of S1 including a fraction labeled specifically at the strut sequence. The high specificity of labeling was verified by proteolytic digestions, gel electrophoresis, and mass spectroscopy.Analysis of the labeled S1 by collisional quenching, fluorescence polarization, and actinactivated ATPase activity were consistent with predictions from structural models of the probe's location. Although the fluorescent intensity of the CY3 was insensitive to actin binding, its fluorescence polarization was notably affected. Intriguingly, the mobility of the probe increases upon S1 binding to actin suggesting that the CY3 becomes displaced from interactions with the surface of S1 and is consistent with a structural change in the strut due to cleft motions. Labeling the strut reduced the affinity of S1 for actin but did not prevent actin-activated ATPase activity which makes it a potentially useful probe of the actomyosin interface. The different conformations of myosin S1 indicated that the strut is not as flexible as several other key regions of myosin as determined by the application of force constraints to elastic portions of the myosin

show abstract

Determination of Fluorescent Probe Orientations on Biomolecules by Conformational Searching: Algorithm Testing and Applications to the Atomic Model of Myosin

Cited by 20 publications

References 42 publications

Strategy for selecting and characterizing linker peptides for CBM9‐tagged fusion proteins expressed in Escherichia coli

Strategy for selecting and characterizing linker peptides for CBM9‐tagged fusion proteins expressed in Escherichia coli

Detection of single nucleotide variations by a hybridization proximity assay based on molecular beacons and luminescence resonance energy transfer

Fluorescence labeling and computational analysis of the strut of myosin’s 50 kDa cleft

Contact Info

Product

Resources

About