Covalent Crosslinking of Colloidal Cellulose Nanocrystals for Multifunctional Nanostructured Hydrogels with Tunable Physicochemical Properties.

Cellulose nanocrystals (CNCs) have shown promise for the development of multifunctional materials for many research communities, ranging from bioresource engineering and biomedical engineering to materials science and engineering. However, accessible hydroxyl (OH) groups on the surface of colloidal CNCs at the (11̅0)ß/(100)α and (110)ß/(010)α facets and the intermolecular hydrogen bonding (H-bonds) between these OH groups account for the instability of self-assembled CNC structures in moist environments, limiting their practical uses to dry media. In this work, accessible OH groups of CNCs were crosslinked using two crosslinkers, that is, glutaraldehyde (GA) and epichlorohydrin (EC), to form nanoparticle-based hydrogels with tunable physicochemical properties. The intensity of the intermolecular H-bonds was controlled by the type and concentration of crosslinkers as well as the CNC concentration. Rheological analyses through the loss tangent were used to determine the degree of crosslinking with maximal values beyond 90%. Fourier-transform infrared spectroscopy demonstrated that H-bond intensity was inversely proportional to the degree of crosslinking for both GA and EC, indicating a dissimilar crosslinking mechanism for GA and EC in acidic and alkaline pH conditions, respectively. Atomic force microscopy and wettability analyses showed a significant increase in the surface roughness from 3.2 ± 0.41 nm (pure CNC) to 31.5 ± 1.08 nm (CNCs crosslinked by GA) and 23.8 ± 0.14 nm (CNCs crosslinked by EC) and water contact angle from 13° (pure CNC) to 108° (CNCs crosslinked by GA) and 104° (CNCs crosslinked by EC). Moreover, optimum water absorption values were found at 157.67 ± 2.01 g and 173.59 ± 1.26 g of water for 1 g of freeze-dried hydrogels for 10% GA and 1% EC, respectively. The results aligned with reaction conditions that led to maximal degrees of crosslinking and indicated the transformation of surface chemistry from a hydrophilic to a hydrophobic network as well as tunable topology and aqueous stability of self-assembled structures made from crosslinked CNCs. This technology demonstrated the potential of crosslinked CNCs with tunable physicochemical properties for use as advanced building blocks to produce 2D and 3D structures for their related functions.

Structures of three polycystic kidney disease-like domains from Clostridium histolyticum collagenases ColG and ColH.

Clostridium histolyticum collagenases ColG and ColH are segmental enzymes that are thought to be activated by Ca(2+)-triggered domain reorientation to cause extensive tissue destruction. The collagenases consist of a collagenase module (s1), a variable number of polycystic kidney disease-like (PKD-like) domains (s2a and s2b in ColH and s2 in ColG) and a variable number of collagen-binding domains (s3 in ColH and s3a and s3b in ColG). The X-ray crystal structures of Ca(2+)-bound holo s2b (1.4 Šresolution, R = 15.0%, Rfree = 19.1%) and holo s2a (1.9 Šresolution, R = 16.3%, Rfree = 20.7%), as well as of Ca(2+)-free apo s2a (1.8 Šresolution, R = 20.7%, Rfree = 27.2%) and two new forms of N-terminally truncated apo s2 (1.4 Šresolution, R = 16.9%, Rfree = 21.2%; 1.6 Šresolution, R = 16.2%, Rfree = 19.2%), are reported. The structurally similar PKD-like domains resemble the V-set Ig fold. In addition to a conserved ß-bulge, the PKD-like domains feature a second bulge that also changes the allegiance of the subsequent ß-strand. This ß-bulge and the genesis of a Ca(2+) pocket in the archaeal PKD-like domain suggest a close kinship between bacterial and archaeal PKD-like domains. Different surface properties and indications of different dynamics suggest unique roles for the PKD-like domains in ColG and in ColH. Surface aromatic residues found on ColH s2a-s2b, but not on ColG s2, may provide the weak interaction in the biphasic collagen-binding mode previously found in s2b-s3. B-factor analyses suggest that in the presence of Ca(2+) the midsection of s2 becomes more flexible but the midsections of s2a and s2b stay rigid. The different surface properties and dynamics of the domains suggest that the PKD-like domains of M9B bacterial collagenase can be grouped into either a ColG subset or a ColH subset. The conserved properties of PKD-like domains in ColG and in ColH include Ca(2+) binding. Conserved residues not only interact with Ca(2+), but also position the Ca(2+)-interacting water molecule. Ca(2+) aligns the N-terminal linker approximately parallel to the major axis of the domain. Ca(2+) binding also increases stability against heat and guanidine hydrochloride, and may improve the longevity in the extracellular matrix. The results of this study will further assist in developing collagen-targeting vehicles for various signal molecules.

Treatment and prevention of chemotherapy-induced alopecia with PTH-CBD, a collagen-targeted parathyroid hormone analog, in a non-depilated mouse model.

Alopecia is a psychologically devastating complication of chemotherapy for which there is currently no effective therapy. PTH-CBD is a collagen-targeted parathyroid hormone analog that has shown promise as a therapy for alopecia disorders. This study compared the efficacy of prophylactic versus therapeutic administration of PTH-CBD in chemotherapy-induced alopecia using a mouse model that mimics the cyclic chemotherapy dosing used clinically. C57BL/6J mice were treated with a single subcutaneous injection of PTH-CBD (320 mcg/kg) or vehicle control before or after hair loss developing from three courses of cyclophosphamide chemotherapy (50-150 mg/kg/week). Mice receiving chemotherapy alone developed hair loss and depigmentation over 6-12 months. Mice pretreated with PTH-CBD did not develop these changes and maintained a normal-appearing coat. Mice treated with PTH-CBD after development of hair loss showed a partial recovery. Observations of hair loss were confirmed quantitatively by gray scale analysis. Histological examination showed that in mice receiving chemotherapy alone, there were small, dystrophic hair follicles mostly in the catagen phase. Mice receiving PTH-CBD before chemotherapy showed a mix of normal-appearing telogen and anagen hair follicles with no evidence of dystrophy. Mice receiving PTH-CBD therapy after chemotherapy showed intermediate histological features. PTH-CBD was effective in both the prevention and the treatment of chemotherapy-induced alopecia in mice, but pretreatment appears to result in a better cosmetic outcome. PTH-CBD shows promise as an agent in the prevention of this complication of chemotherapy and improving the quality of life for cancer patients.

Parathyroid hormone linked to a collagen binding domain promotes hair growth in a mouse model of chemotherapy-induced alopecia in a dose-dependent manner.

Chemotherapy-induced alopecia is a major source of psychological stress in patients undergoing cancer chemotherapy, and it can influence treatment decisions. Although there is currently no therapy for alopecia, a fusion protein of parathyroid hormone and collagen binding domain (PTH-CBD) has shown promise in animal models. The aim of this study was to determine whether there are dose-dependent effects of PTH-CBD on chemotherapy-induced alopecia in a mouse model. C57BL/6J mice were waxed to synchronize hair follicles; treated on day 7 with vehicle or PTH-CBD (100, 320, and 1000 mcg/kg subcutaneous injection); and treated on day 9 with vehicle or cyclophosphamide (150 mg/kg intraperitoneally). Mice were photographed every 3-4 days and killed on day 63 for histological analysis. Photographs were quantified by gray scale analysis to assess hair content. Mice not receiving chemotherapy showed regrowth of hair 2 weeks after waxing and normal histology after 2 months. Mice receiving chemotherapy alone showed marked hair loss after chemotherapy, which was sustained for 10 days and was followed by rapid regrowth of a normal coat. Histological analysis revealed rapid cycling dystrophic anagen/catagen follicles. Animals receiving chemotherapy and PTH-CBD showed decreased hair loss and more rapid regrowth of hair than that seen with chemotherapy alone (increased hair growth by gray scale analysis, P<0.05), and the effects were dose dependent. Histologically, hair follicles in animals receiving the highest dose of PTH-CBD were in a quiescent phase, similar to that in mice that did not receive chemotherapy. Single-dose subcutaneous administration of PTH-CBD showed dose-dependent effects in minimizing hair loss and speeding up recovery from chemotherapy-induced alopecia.

Structural comparison of ColH and ColG collagen-binding domains from Clostridium histolyticum.

Clostridium histolyticum secretes collagenases, ColG and ColH, that cause extensive tissue destruction in myonecrosis. The C-terminal collagen-binding domain (CBD) of collagenase is required for insoluble collagen fibril binding and subsequent collagenolysis. The high-resolution crystal structures of ColG-CBD (s3b) and ColH-CBD (s3) are reported in this paper. The new X-ray structure of s3 was solved at 2.0-Å resolution (R = 17.4%; R(free) = 23.3%), while the resolution of the previously determined s3b was extended to 1.4 Å (R = 17.9%; R(free) = 21.0%). Despite sharing only 30% sequence identity, the molecules resemble one another closely (root mean square deviation [RMSD] C(α) = 1.5 Å). All but one residue, whose side chain chelates with Ca(2+), are conserved. The dual Ca(2+) binding site in s3 is completed by an unconserved aspartate. Differential scanning calorimetric measurements showed that s3 gains thermal stability, comparable to s3b, by binding to Ca(2+) (holo T(m) = 94.1°C; apo T(m) = 70.2°C). holo s3 is also stabilized against chemical denaturants urea and guanidine HCl. The three most critical residues for collagen interaction in s3b are conserved in s3. The general shape of the binding pocket is retained by altered loop structures and side chain positions. Small-angle X-ray scattering data revealed that s3 also binds asymmetrically to minicollagen. Besides the calcium-binding sites and the collagen-binding pocket, architecturally important hydrophobic residues and the hydrogen-bonding network around the cis-peptide bond are well conserved within the metallopeptidase subfamily M9B. CBDs were previously shown to bind to the extracellular matrix of various tissues. Compactness and extreme stability in physiological Ca(2+) concentration possibly make both CBDs suitable for targeted growth factor delivery.

Therapy for alopecia areata in mice using parathyroid hormone agonists and antagonists, linked to a collagen-binding domain.

Alopecia areata is a common form of hair loss in which autoimmune-mediated destruction of hair follicles causes patchy hair loss, for which there is no adequate therapy. Parathyroid hormone (PTH) induces the hair cycle and promotes hair growth. PTH-CBD is a fusion protein of PTH and a bacterial collagen-binding domain (CBD), leading to targeted delivery to and retention in the skin collagen. We tested the effects of a single dose of PTH-CBD (low or high dose) on an animal model for alopecia areata, the C3H/HeJ engrafted mouse. In all the treated animals, there was a rapid (1-4 days) increase in hair growth, with sustained effects observed over a 2-month period (7/10 total treated mice<40% hair loss based on gray scale analysis, vs. 2/5 in vehicle control animals). Histological examination revealed massive stimulation of anagen VI hair follicles in treated animals despite an ongoing immune response. PTH-CBD thus shows promise as a therapy for alopecia areata, likely in conjunction with a mild immune suppressant, such as hydrocortisone cream.

Treatment for chemotherapy-induced alopecia in mice using parathyroid hormone agonists and antagonists linked to a collagen binding domain.

Parathyroid hormone (PTH) agonists and antagonists have been shown to improve hair growth after chemotherapy; however, rapid clearance and systemic side-effects complicate their usage. To facilitate delivery and retention to skin, we fused PTH agonists and antagonists to the collagen binding domain (CBD) of Clostridium histolyticum collagenase. in-vitro studies showed that the agonist fusion protein, PTH-CBD, bound collagen and activated the PTH/parathyroid hormone-related peptide receptor in SaOS-2 cells. The antagonist fusion proteins, PTH(7-33)-CBD and PTH([-1]-33)-CBD, also bound collagen and antagonized PTH(1-34) effect in SaOS-2 cells; however, PTH(7-33)-CBD had lower intrinsic activity. Distribution studies confirmed uptake of PTH-CBD to the skin at 1 and 12 hr after subcutaneous injection. We assessed in vivo efficacy of PTH-CBD and PTH(7-33)-CBD in C57BL/6J mice. Animals were depilated to synchronize the hair follicles; treated on Day 7 with agonist, antagonist, or vehicle; treated on Day 9 with cyclophosphamide (150 mg/kg i.p.) or vehicle; and sacrificed on Day 39. Normal mice (no chemo and no treatment) showed rapid regrowth of hair and normal histology. Chemo+Vehicle mice showed reduced hair regrowth and decreased pigmentation; histology revealed reduced number and dystrophic anagen/catagen follicles. Chemo+Antagonist mice were grossly and histologically indistinguishable from Chemo+Vehicle mice. Chemo+Agonist mice showed more rapid regrowth and repigmentation of hair; histologically, there was a normal number of hair follicles, most of which were in the anagen phase. Overall, the agonist PTH-CBD had prominent effects in reducing chemotherapy-induced damage of hair follicles and may show promise as a therapy for chemotherapy-induced alopecia.

A single injection of the anabolic bone agent, parathyroid hormone-collagen binding domain (PTH-CBD), results in sustained increases in bone mineral density for up to 12 months in normal female mice.

Parathyroid hormone (PTH) is the most effective osteoporosis treatment, but it is only effective if administered by daily injections. We fused PTH(1-33) to a collagen binding domain (PTH-CBD) to extend its activity, and have shown an anabolic bone effect with monthly dosing. We tested the duration of action of this compound with different routes of administration. Normal young C57BL/6J mice received a single intraperitoneal injection of PTH-CBD (320 µg/kg). PTH-CBD treated mice showed a 22.2 % increase in bone mineral density (BMD) at 6 months and 12.8 % increase at 12 months. When administered by subcutaneous injection, PTH-CBD again caused increases in BMD, 15.2 % at 6 months and 14.3 % at 12 months. Radiolabeled PTH-CBD was concentrated in bone and skin after either route of administration. We further investigated skin effects of PTH-CBD, and histological analysis revealed an apparent increase in anagen VI hair follicles. A single dose of PTH-CBD caused sustained increases in BMD by >10 % for 1 year in normal mice, regardless of the route of administration, thus showing promise as a potential osteoporosis therapy.

Ca2+ -induced orientation of tandem collagen binding domains from clostridial collagenase ColG permits two opposing functions of collagen fibril formation and retardation.

To penetrate host tissues, histotoxic clostridia secrete virulence factors including enzymes to hydrolyze extracellular matrix. Clostridium histolyticum, recently renamed as Hathewaya histolytica, produces two classes of collagenase (ColG and ColH). The high-speed AFM study showed that ColG collagenase moves unidirectionally to plane collagen fibril and rebundles fibril when stalled . The structural explanation of the roles for the tandem collagen-binding segment (CBDs) is illuminated by its calcium-bound crystal structure at 1.9 Å resolution (Rwork = 15.0%; Rfree = 19.6%). Activation may involve calcium-dependent domain rearrangement supported by both small-angle X-ray scattering and size exclusion chromatography. At pCa ≥ 5 (pCa = -log[Ca2+ ]), the tandem CBD adopts an extended conformation that may facilitate secretion from the bacterium. At pCa ≤ 4, the compact structure seen in the crystal structure is adopted. This arrangement positions the two binding surfaces ~ 55 Å apart, and possibly ushers ColG along tropocollagen molecules that allow for unidirectional movement. A sequential binding mode where tighter binding CBD2 binds first could aid in processivity as well. Switch from processive collagenolysis to fibril rearrangement could be concentration dependent. Collagen fibril formation is retarded at 1 : 1 molar ratio of tandem CBD to collagen. Tandem CBD may help isolate a tropocollagen molecule from a fibril at this ratio. At 0.1 : 1 to 0.5 : 1 molar ratios fibril self-assembly was accelerated. Gain of function as a result of gene duplication of CBD for the M9B enzymes is speculated. The binding and activation modes described here will aid in drug delivery design. ACCESSION CODES: The full atomic coordinates of the tandem CBD and its corresponding structure factor amplitudes have been deposited in the Protein Data Bank (PDB accession code 5IKU). Small-angle X-ray scattering data and corresponding ab initio models have been submitted to the Small Angle Scattering Biological Data Bank (SASBDB). Accession codes CL2, collagenase module 2, CN2, CP2 are assigned to envelopes for tandem CBD at -log[Ca2+ ] (pCa) 3, 4, 5, and 6, respectively. Accession code DC64 was assigned to the complex of polycystic kidney disease-CBD1-CBD2 with mini-collagen.

Alternate carbohydrate and nontraditional inducer leads to increased productivity of a collagen binding domain fusion protein via fed-batch fermentation.

The production of collagen binding domain fusion proteins is of significant importance because of their potential as therapeutic biomaterials. It was previously reported that the expression of collagen-binding domain fusion proteins in Escherichia coli was higher when expressed using lactose as an inducer and chemically defined growth media on a shake flask scale. In an effort to further investigate factors that affect expression levels on a fed-batch scale, alternative induction techniques were tested in conjunction with fed-batch fermentation. In this paper, we discuss ten fed-batch fermentation experiments utilizing either glucose or glycerol feed and using lactose or isopropyl-ß-d-thiogalactopyranoside (IPTG) as an induction source. It was found that glycerol-fed fermentations induced with lactose allowed for greater expression of target protein, though lesser cell densities were achieved.

Fluorescent ampicillin analogues as multifunctional disguising agents against opsonization.

Cancer nanomedicines are opening new paradigms in cancer management and recent research points to how they can vastly improve imaging and therapy through multimodality and multifunctionality. However, challenges to achieving optimal efficacy are manifold starting from processing materials and evaluating their intended effectiveness on biological tissue, to developing new strategies aimed at improving transport of these materials through the biological milieu to the target tissue. Here, we report a fluorescent derivative of a beta-lactam antibiotic, ampicillin (termed iAmp) and its multifunctional physicobiochemical characteristics and potential as a biocompatible shielding agent and an effective dispersant. Carbon nanotubes (CNTs) were chosen to demonstrate the efficacy of iAmp. CNTs are known for their versatility and have been used extensively for cancer theranostics as photothermal and photoacoustic agents, but have limited solubility in water and biocompatibility. Traditional dispersants are associated with imaging artifacts and are not fully biocompatible. The chemical structure of iAmp is consistent with a deamination product of ampicillin. Although the four-membered lactam ring is intact, it does not retain the antibiotic properties. The iAmp is an effective dispersant and simultaneously serves as a fluorescent label for single-walled CNTs (SWNTs) with minimal photobleaching. The iAmp also enables bioconjugation of SWNTs to bio-ligands such as antibodies through functional carboxyl groups. Viability tests show that iAmp-coated SWNTs have minimal toxicity. Bio-stability tests under physiological conditions reveal that iAmp coating not only remains stable in a biologically relevant environment with high protein and salt concentrations, but also renders SWNTs transparent against nonspecific protein adsorption, also known as protein corona. Mammalian tissue culture studies with macrophages and opsonins validate that iAmp coating affords immunological resistance to SWNTs. Furthermore, iAmp coating offers protection to SWNTs against their nonspecific adsorption across disparate cell types, which has precluded a targeted strategy, and enables selective molecular targeting. The iAmp can therefore be used as an efficient dispersant, a photostable fluorescent agent, and a biocompatible disguising agent, alleviating CNTs' drawbacks and rendering them suitable for nanotheranostic and drug delivery applications.

Catalytically enhanced endocellulase Cel5A from Acidothermus cellulolyticus.

Expression of a collagen-binding domain fusion protein: effect of amino acid supplementation, inducer type, and culture conditions.

Collagen binding domain fusion proteins are of significant importance because of their potential as therapeutic biomaterials. In this paper, we investigate the production of such therapeutic proteins via fermentation of Escherichia coli on both an undefined medium and a defined medium. Defined media with amino acid supplementation provided higher amounts of therapeutic protein than undefined media with no supplementation. Additionally, utilizing lactose instead of isopropyl-ß-d-thio-galactoside (IPTG) for induction and extending batch time yielded higher amounts of the model therapeutic.

Proteins with simplified hydrophobic cores compared to other packing mutants.

Efforts to design proteins with greatly reduced sequence diversity have often resulted in proteins with so-called molten globule properties. Substitutions were made at six neighboring sites in the major hydrophobic core of staphylococcal nuclease to create variants with all leucine, all isoleucine or all valine at these sites. The mutant proteins with simplified cores constructed here are quite unstable and have poorly packed cores, attested to by interaction energies. Eight related mutants with greater sequence diversity were also constructed. Comparison to these mutants and 159 other permutations of these 3 aliphatic side chains at these same 6 sites previously constructed shows that the simplified cores are not unusual in their stabilities or interaction energies. Further, crystal structures of the two mutants with the worst packing, as measured by interaction energies, showed no unusual disorder in the core. Therefore, reduction of sequence diversity is not necessarily incompatible with a single stable native structure. Other factors must also contribute to previous protein design failures.

Exploration of cellulose surface-binding properties of acidothermus cellulolyticus Cel5A by site-specific mutagenesis.

Understanding the interactions between cellulases and cellulosic substrates is critical to the development of an efficient artificial cellulase system for conversion of biomass to sugars. We directed specific mutations to the interactive surface of the Acidothermus cellulolyticus EI endoglucanase catalytic domain. The cellulose-binding domain is not translated in these mutants. Amino acid mutations were designed either to change the surface charge of the protein or to modify the potential for hydrogen bonding with cellulose. The relationship between cellulase-to-cellulose (Avicel PH101) binding and hydrolysis activity was determined for various groupings of mutations. While a significant increase in hydrolysis activity was not observed, certain clusters of residues did significantly alter substrate binding and some interesting correlations emerged. In the future, these observations may be used to aid the design of endoglucanases with improved performance on pretreated biomass.

Pharmacokinetics in rats of a long-acting human parathyroid hormone-collagen binding domain peptide construct.

The pharmacokinetics of a hybrid peptide consisting of the N-terminal biologically active region of human parathyroid hormone (PTH) linked to a collagen-binding domain (CBD) were evaluated in female Sprague-Dawley rats. The peptide, PTH-CBD, consists of the first 33 amino acids of PTH linked as an extension of the amino acid chain to the CBD peptide derived from ColH collagenase of Clostridium histolyticum. Serum concentrations arising from single dose administration by the subcutaneous and intravenous routes were compared with those measured following route-specific mole equivalent doses of PTH(1-34). Population-based modeling demonstrated similar systemic absorption kinetics and bioavailability for both peptides. Exposure to PTH-CBD was sixfold higher because of a systemic clearance of approximately 20% relative to PTH(1-34); however, these kinetics were consistent with more than 95% of a dose being eliminated from serum within 24 h. Results obtained support continued investigation of PTH-CBD as a bone-targeted anabolic agent for the treatment of postmenopausal osteoporosis.

Bacterial collagen-binding domain targets undertwisted regions of collagen.

Clostridium histolyticum collagenase causes extensive degradation of collagen in connective tissue that results in gas gangrene. The C-terminal collagen-binding domain (CBD) of these enzymes is the minimal segment required to bind to a collagen fibril. CBD binds unidirectionally to the undertwisted C-terminus of triple helical collagen. Here, we examine whether CBD could also target undertwisted regions even in the middle of the triple helix. Collageneous peptides with an additional undertwisted region were synthesized by introducing a Gly → Ala substitution [(POG)(x) POA(POG)(y)]3, where x + y = 9 and x > 3). ¹H-¹5N heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR) titration studies with ¹5N-labeled CBD demonstrated that the minicollagen binds to a 10 Å wide 25 Å long cleft. Six collagenous peptides each labeled with a nitroxide radical were then titrated with ¹5N-labeled CBD. CBD binds to either the Gly → Ala substitution site or to the C-terminus of each minicollagen. Small-angle X-ray scattering measurements revealed that CBD prefers to bind the Gly → Ala site to the C-terminus. The HSQC NMR spectra of ¹5N-labeled minicollagen and minicollagen with undertwisted regions were unaffected by the titration of unlabeled CBD. The results imply that CBD binds to the undertwisted region of the minicollagen but does not actively unwind the triple helix.

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

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 ßA 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.

Aqueous-phase synthesis of monodisperse plasmonic gold nanocrystals using shortened single-walled carbon nanotubes.

Monodisperse gold nanocrystals with unique near-infrared optical properties were synthesized by simple mixing of highly shortened and well disperse single-walled carbon nanotubes and chloroauric acid in water at ambient conditions with a step-wise increase of gold ion concentration.

Ca2+-induced linker transformation leads to a compact and rigid collagen-binding domain of Clostridium histolyticum collagenase.

Clostridium histolyticum collagenase is responsible for extensive tissue destruction in gas gangrene, and its activity is enhanced by calcium ions. The collagen-binding domain is the minimal segment of the enzyme required for binding to insoluble collagen fibrils and for subsequent collagenolysis. The collagen-binding domain is joined to another binding module by a conserved 14-amino-acid linker. The linker undergoes secondary structural transformation from an alpha-helix to a beta-strand and forms a nonprolyl cis-peptide in the presence of calcium ions. In this study, various biophysical methods were utilized to better understand the structure and functional role of the novel calcium-activated linker. Two Ca(2+) ions bind cooperatively with macroscopic association constants of K(1) = 5.01 x 10(5) m(-1) and K(2) = 2.28 x 10(5) m(-1). The chelation of the second calcium ion is enthalpically unfavorable, which could be a result of isomerization of the nonprolyl cis-peptide. The holo protein is more stable than the apo protein against thermal denaturation (DeltaT(m) approximately 20 degrees C) and chemical denaturation (DeltaDeltaG(H2O) approximately 3 kcal x mol(-1) for urea or guanidine HCl denaturation and Delta20% v/v in 2,2,2-trifluoroethanol). The compact holo collagen-binding domain is more resistant to proteolytic digestion than the apo collagen-binding domain. The orientation of the linker appears to play a crucial role in the stability and dynamics of the collagen-binding domain.