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

Cynthia R. Sides, Rohana Liyanage, Jackson O. Lay, Sagaya Theresa Leena Philominathan, Osamu Matsushita, Joshua Sakon

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)505-519
Number of pages15
JournalJournal of the American Society for Mass Spectrometry
Volume23
Issue number3
DOIs
Publication statusPublished - Mar 2012
Externally publishedYes

Fingerprint

Proteolysis
Collagenases
Ionization
Mass spectrometry
Desorption
Mass Spectrometry
Lasers
Collagen
Protein Stability
Cobalt
X-Rays
Calcium
Crystal structure
Proteins
X rays
Phase stability
X ray scattering
Drug delivery
Metals
Modulation

Keywords

  • α Helix
  • β sheets
  • 3D structure
  • B-factors
  • Collagen
  • Collagen binding domain
  • Limited proteolysis
  • MALDI-TOF MS
  • Mapping protein surface
  • Secondary structure
  • Solution dynamics
  • Stability
  • X-ray structure

ASJC Scopus subject areas

  • Structural Biology
  • Spectroscopy

Cite this

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.; Philominathan, Sagaya Theresa Leena; Matsushita, Osamu; Sakon, Joshua.

In: Journal of the American Society for Mass Spectrometry, Vol. 23, No. 3, 03.2012, p. 505-519.

Research output: Contribution to journalArticle

Sides, Cynthia R. ; Liyanage, Rohana ; Lay, Jackson O. ; Philominathan, Sagaya Theresa Leena ; Matsushita, Osamu ; Sakon, Joshua. / Probing the 3-D structure, dynamics, and stability of bacterial collagenase collagen binding domain (apo- versus holo-) by limited proteolysis MALDI-TOF MS. In: Journal of the American Society for Mass Spectrometry. 2012 ; Vol. 23, No. 3. pp. 505-519.
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