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Volumn 276, Issue 5315, 1997, Pages 1109-1112

Reversible unfolding of individual titin immunoglobulin domains by AFM

Author keywords

[No Author keywords available]

Indexed keywords

IMMUNOGLOBULIN;

EID: 0031011695     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.276.5315.1109     Document Type: Article
Times cited : (2708)

References (40)
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    • We found that we could increase the stability and probability of attachment by applying contact forces of several nanonewtons over several seconds.
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    • 2 tag for immobilization on solid surfaces. The identity of the cloned fragments was verified by DNA sequencing with the use of a standard automated sequencer. Expression of the fragments was induced in BL21(DE)3 by 0.1 mM isopropyl-β-D-thiogalactopyranoside at 37°C for 3 hours. The proteins were expressed solubly and were purified from bacterial lysates as described (25). Protein was stored frozen in aliquots in 20 mM sodium phosphate (pH 7) and 5 mM dithiothreitol (DTT). Circular dichroism spectroscopy in the far ultraviolet was recorded in storage buffer on a Jasco J-710 spectropolarimeter fitted with a thermostatted cell holder. For further details, see (1). The spectra were typical for the beta-barrel structure of titin Ig domains and confirmed the fold of the constructs. Native cardiac titin was purified from bovine heart tissue following the protocol of (26), except that the final ammonium sulfate precipitation was omitted. Electrophoresis of the preparation on 3% SDS-polyacrylamide gel electrophoresis showed essentially undegraded titin. The protein was stored at 0.5 mg/ml in 200 mM sodium phosphate (pH 7), 50% glycerol, 5 mM EGTA, 5 mM DTT, and leupeptin (2 μg/ml) at -20°C.
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    • The characteristic pattern of the force extension curves observed upon stretching of titin fragments is sensitive to denaturing and cross-linking agents. Incubation of Ig8 in a solution containing 6.6 M urea produced force extension curves that were either featureless or peaked at long extensions with a variable spacing. Treating Ig4 titin fragments with glutaraldehyde (0.1 to 5%) also eliminated the sawtooth pattern in the force extension curves. Instead, we could only observe a featureless and short-ranged force extension curve. These observations suggest that we could either destroy the tertiary structure of the protein by denaturation or that we could render it rigid by cross-linking.
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    • It cannot be ruled out that fibronectin III domains, which have a structure similar to that of Ig domains, also contribute to the sawtooth pattern of native titin.
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    • The first unfolding peak is typically camouflaged due to multiple adsorptions. The absolute position of the first peak varies because of a random pickup of the protein with respect to the anchoring cysteines, we believe, but also because of rearrangements of the adsorbed segments of the protein on the tip.
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    • Note that the unfolding force is the maximum force of the peaks above the baseline of the free cantilever on the approach part of the cycle and not the difference between the peaks and the troughs.
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    • Geometric effects can also diminish the observed rupture force of the first peaks. When the line defined by the cysteine tag to the gold surface and the adhesion point on the AFM tip is not in parallel with the direction of pulling, the measured rupture force of the first peak can be up to 15% below the actual force. However, this effect will be unmeasurable from the third peak on.
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    • Supported by the Deutsche Forschungsgemeinschaft. We thank J. I. Brauman, R. M. Simmons, H. P. Erickson, W. A. Linke, and A. Pastore for helpful discussions and A. Pastore for technical support. J.M.F. was supported by an Alexander V. Humboldt award.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.