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Volumn 128, Issue 31, 2006, Pages 10034-10042

Revealing two-state protein-protein interactions of calmodulin by single-molecule spectroscopy

Author keywords

[No Author keywords available]

Indexed keywords

AMINO ACIDS; CONFORMATIONS; METABOLISM; MOLECULAR SPECTROSCOPY; OLIGOMERS; POLARIZATION;

EID: 33746969931     PISSN: 00027863     EISSN: None     Source Type: Journal    
DOI: 10.1021/ja057005m     Document Type: Article
Times cited : (69)

References (62)
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    • (1999) Science , vol.283 , Issue.5408 , pp. 1670-1676
    • Moerner, W.E.1    Orrit, M.2
  • 3
    • 0033548686 scopus 로고    scopus 로고
    • Fluorescence spectroscopy of single biomolecules
    • Weiss, S. Fluorescence Spectroscopy of Single Biomolecules. Science 1999, 283 (5408), 1676-1683.
    • (1999) Science , vol.283 , Issue.5408 , pp. 1676-1683
    • Weiss, S.1
  • 4
    • 33746978267 scopus 로고    scopus 로고
    • special issue on single molecule spectroscopy
    • For reviews, see: Acc. Chem. Res. (special issue on single molecule spectroscopy) 2005, 38 (7).
    • (2005) Acc. Chem. Res. , vol.38 , Issue.7
  • 5
    • 33745962697 scopus 로고    scopus 로고
    • Nienhaus, G. U., Ed.; Humana Press: New Jersey.
    • For a review, see: Protein-Ligand Interactions; Nienhaus, G. U., Ed.; Humana Press: New Jersey. 2005.
    • (2005) Protein-Ligand Interactions
  • 44
    • 33746989783 scopus 로고    scopus 로고
    • note
    • A threshold criterion was applied to the photon counts of both channels before a calculation of FRET efficiency, to remove experimental points where the photon counts are due to the fluctuation of background and/or cross-talk from the other channel. The threshold criterion equals 2× the standard deviation from the background and the cross-talk, assuming that the uncertainty is due to photon-counting shot noise in the background and the cross-talk.
  • 47
    • 33747021990 scopus 로고    scopus 로고
    • note
    • -1, the final concentration of each component in the mixture of CaM and C28W in the single-molecule FRET experiments are expected to be approximately C28W 9.802 nM, CaM 0.202 nM, and CaM/ C28W 0.198 nM, respectively. Though, the expected concentration ratio between CaM and CaM/C28W is close to 1:1, in the FRET experiments, the CaM/C28W appeared at a lower concentration, due to reasons, such as (1) the C28W peptide is not 100% labeled and (2) the interaction between CaM and C28W is in dynamics, and there are some times that CaM/C28W becomes dissociated.
  • 48
    • 33746997995 scopus 로고    scopus 로고
    • note
    • First, the energy transfer acceptor Texas Red is linked to the peptide by a single bond and should be very flexible. Both molecular structure modeling and time-resolved anisotropy of the tethered Texas Red have shown a large rotational freedom of the Texas Red probe (see Supporting Information for detail). Its orientation should be averaged within the time bin and is unlikely to contribute to a large change of the FRET efficiency, reflected by 2.3 times in the energy transfer rate (FRET efficiency change from 0.43 to 0.65). Second, the energy transfer donor FlAsH is locked in the matrix of the N-terminal domain of CaM, and its orientation change has to be induced by the conformational change of CaM.
  • 53
    • 33746954650 scopus 로고    scopus 로고
    • note
    • Since the lysines at the central helix elements of CaM are more accessible to the bifunctional linker molecules used in the tethering process, most CaM molecules in the polarization measurements are expected to be linked to the glass surface through its central part. Therefore, once the C28W peptide interacts with CaM, especially in the intermediate state (if exist), the N-terminal domain of CaM changing from unbound to bound with C28W, the restriction on the conformation freedom of the N-terminal domain of CaM changes dramatically. This can be reflected by the distribution and dynamics of fluorescence polarization of the FlAsH dye molecules labeled there.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.