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

Journal of the American Society for Mass Spectrometry (2011-12-31)
Cynthia R Sides, Rohana Liyanage, Jackson O Lay, Sagaya Theresa Leena Philominathan, Osamu Matsushita, Joshua Sakon
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Collagenase from Clostridium histolyticum, suitable for release of physiologically active rat hepatocytes, Type IV, 0.5-5.0 FALGPA units/mg solid, ≥125 CDU/mg solid
Sigma-Aldrich
Collagenase from Clostridium histolyticum, for general use, Type I, 0.25-1.0 FALGPA units/mg solid, ≥125 CDU/mg solid
Sigma-Aldrich
Collagenase from Clostridium histolyticum, suitable for release of physiologically active rat epididymal adipocytes, Type II, 0.5-5.0 FALGPA units/mg solid, ≥125 CDU/mg solid
Sigma-Aldrich
Collagenase from Clostridium histolyticum, release of rat epididymal adipocytes and hepatocytes tested (for methodology see Type II and Type IV), Type VIII, 0.5-5.0 FALGPA units/mg solid, ≥125 CDU/mg solid
Sigma-Aldrich
Collagenase from Clostridium histolyticum, Type IA, 0.5-5.0 FALGPA units/mg solid, ≥125 CDU/mg solid, For general use
Sigma-Aldrich
Collagenase from Clostridium histolyticum, release of physiologically active rat pancreatic islets tested, Type XI, 2-5 FALGPA units/mg solid, ≥800 CDU/mg solid
Sigma-Aldrich
Collagenase from Clostridium histolyticum, powder, Suitable for the digestion and isolation of physiologically active pancreatic islet cells, suitable for cell culture
Sigma-Aldrich
Collagenase from Clostridium histolyticum, lyophilized powder, ≥125 CDU/mg solid (CDU = collagen digestion units), 0.5-5.0 FALGPA units/mg solid
Sigma-Aldrich
Collagenase from Clostridium histolyticum, release of physiologically active rat pancreatic islets tested, Type V, ≥1 FALGPA units/mg solid, >125 CDU/mg solid
Sigma-Aldrich
Endoproteinase Glu-C from Staphylococcus aureus V8, Type XVII-B, lyophilized powder, 500-1,000 units/mg solid
Sigma-Aldrich
Collagenase from Clostridium histolyticum, 0.2 μm filtered, release of physiologically active rat hepatocytes tested, Type IV-S, 0.5-5.0 FALGPA units/mg solid, ≥125 CDU/mg solid
Sigma-Aldrich
Endoproteinase Glu-C from Staphylococcus aureus V8, suitable for protein sequencing, lyophilized powder
Sigma-Aldrich
Collagenase from Clostridium histolyticum, high purity, purified by chromatography, Type VII, ≥4 FALGPA units/mg solid, lyophilized powder, ≥700 CDU/mg solid (CDU = collagen digestion units)
Sigma-Aldrich
Collagenase from Clostridium histolyticum, lyophilized powder (from 0.2μm filtered solution), 0.5-5.0 FALGPA units/mg solid, suitable for cell culture
Sigma-Aldrich
Collagenase from Clostridium histolyticum, lyophilized powder (from sterile-filtered solution), Suitable for digestion and isolation of physiologically active pancreatic islet cells, suitable for cell culture
Sigma-Aldrich
Collagenase from Clostridium histolyticum, 0.2 μm filtered, release of physiologically active rat epididymal adipocytes tested, Type II-S, 0.5-5.0 FALGPA units/mg solid
Sigma-Aldrich
Collagenase from Clostridium histolyticum, 0.2 μm filtered, for general use, Type I-S, 0.2-1.0 FALGPA units/mg solid, ≥125 CDU/mg solid
Sigma-Aldrich
Collagenase from Clostridium histolyticum, sterile; 0.2 μm filtered, Type IA-S, 0.5-5.0 FALGPA units/mg solid, ≥125 CDU/mg solid
Sigma-Aldrich
Collagenase from Clostridium histolyticum, 0.2 μm filtered, high purity, purified by chromatography, Type VII-S, ≥4 FALGPA units/mg solid, ≥700 CDU/mg solid (CDU = collagen digestion units)
Sigma-Aldrich
Collagenase from Clostridium histolyticum, Sigma Blend Type F, ≥2.0 FALGPA units/mg solid
Sigma-Aldrich
Collagenase from Clostridium histolyticum, Sigma Blend Type H, ≥1.0 FALGPA units/mg solid
Sigma-Aldrich
Collagenase from Clostridium histolyticum, powder, suitable for cell culture, ≥4 FALGPA units/mg solid, high purity, ≥700 CDU/mg solid (CDU = collagen digestion units)
Sigma-Aldrich
Collagenase from Clostridium histolyticum, 0.2 μm filtered, suitable for release of physiologically active rat pancreatic islets, Type V-S, ≥1 FALGPA units/mg solid, ≥125 CDU/mg solid
Sigma-Aldrich
Collagenase from Clostridium histolyticum, purified by chromatography, ≥0.25 FALGPA units/mg solid, ≥250 CDU/mg solid (CDU = collagen digestion units), lyophilized powder
Sigma-Aldrich
Collagenase from Clostridium histolyticum, Sigma Blend Type L, ≤1.0 FALGPA units/mg solid

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