Probing the 3-D structure, dynamics, and stability of bacterial collagenase collagen binding domain (apo- versus holo-) by limited proteolysis MALDI-TOF MS

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[1] Sides, Cynthia R.

[2] Liyanage, Rohana

[3] Lay Jr., Jackson O.

[4] 1,Philominathan, Sagaya Theresa Leena

[5] Matsushita, Osamu

[6] Sakon, Joshua

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Liyanage, R. (rliyana@uark.edu) | 1600年 / Springer New York LLC卷 / 23期

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Pairing limited proteolysis and matrix-assisted laser desorption/ ionization-time of flight mass spectrometry (MALDI-TOF MS) to probe clostridial collagenase collagen binding domain (CBD) reveals the solution dynamics and stability of the protein; as these factors are crucial to CBD effectiveness as a drug-delivery vehicle. MS analysis of proteolytic digests indicates initial cleavage sites; thereby specifying the less stable and highly accessible regions of CBD. Modulation of protein structure and stability upon metal binding is shown through MS analysis of calcium-bound and cobalt-bound CBD proteolytic digests. Previously determined X-ray crystal structures illustrate that calcium binding induces secondary structure transformation in the highly mobile N-terminal arm and increases protein stability. MS-based detection of exposed residues confirms protein flexibility; accentuates N-terminal dynamics; and demonstrates increased global protein stability exported by calcium binding. Additionally; apo- and calcium-bound CBD proteolysis sites correlate well with crystallographic B-factors; accessibility; and enzyme specificity. MS-observed cleavage sites with no clear correlations are explained either by crystal contacts of the X-ray crystal structures or by observed differences between Molecules A and B in the X-ray crystal structures. The study newly reveals the absence of the βA strand and thus the very dynamic N-terminal linker; as corroborated by the solution X-ray scattering results. Cobalt binding has a regional effect on the solution phase stability of CBD; as limited proteolysis data implies the capture of an intermediate-CBD solution structure when cobalt is bound. © 2011 American Society for Mass Spectrometry;

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Journal article (JA)

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[1] Probing the 3-D Structure, Dynamics, and Stability of Bacterial Collagenase Collagen Binding Domain (apo- versus holo-) by Limited Proteolysis MALDI-TOF MS
Sides, Cynthia R.
Liyanage, Rohana
Lay, Jackson O., Jr.
Philominathan, Sagaya Theresa Leena
Matsushita, Osamu
Sakon, Joshua
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY, 2012, 23 (03) : 505 - 519

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