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At least 703 records · Page 39Linked to original sources
PubMed2006

Modeling evaluation of the fluid-dynamic microenvironment in tissue-engineered constructs: a micro-CT based model.

Natural cartilage remodels both in vivo and in vitro in response to mechanical stresses, hence mechanical stimulation is believed to be a potential tool to modulate extra-cellular matrix synthesis in tissue-engineered cartilage. Fluid-induced shear is known to enhance chondrogenesis in engineered cartilage constructs. The quantification of the hydrodynamic environment is a condition required to study the biochemical response to shear of 3D engineered cell systems. We developed a computational model of culture medium flow through the microstructure of a porous scaffold, during direct- perfused culture. The 3D solid model of the scaffold micro-geometry was reconstructed from 250 micro-computed tomography (micro-CT) images. The results of the fluid dynamic simulations were analyzed at the central portions of the fluid domain, to avoid boundary effects. The average, median and mode shear stress values calculated at the scaffold walls were 3.48, 2.90, and 2.45 mPa respectively, at a flow rate of 0.5 cm(3)/min, perfused through a 15 mm diameter scaffold, at an inlet fluid velocity of 53 microm/s. These results were compared to results estimated using a simplified micro-scale model and to results estimated using an analytical macro-scale porous model. The predictions given by the CT-based model are being used in conjunction with an experimental bioreactor model, in order to quantify the effects of fluid-dynamic shear on the growth modulation of tissue-engineered cartilage constructs, to potentially enhance tissue growth in vitro.

Bioreactors↗
PubMed2006

Flow characterization of a wavy-walled bioreactor for cartilage tissue engineering.

Cartilage tissue engineering requires the use of bioreactors in order to enhance nutrient transport and to provide sufficient mechanical stimuli to promote extracellular matrix (ECM) synthesis by chondrocytes. The amount and quality of ECM components is a large determinant of the biochemical and mechanical properties of engineered cartilage constructs. Mechanical forces created by the hydrodynamic environment within the bioreactors are known to influence ECM synthesis. The present study characterizes the hydrodynamic environment within a novel wavy-walled bioreactor (WWB) used for the development of tissue-engineered cartilage. The geometry of this bioreactor provides a unique hydrodynamic environment for mammalian cell and tissue culture, and investigation of hydrodynamic effects on tissue growth and function. The flow field within the WWB was characterized using two-dimensional particle-image velocimetry (PIV). The flow in the WWB differed significantly from that in the traditional spinner flask both qualitatively and quantitatively, and was influenced by the positioning of constructs within the bioreactor. Measurements of velocity fields were used to estimate the mean-shear stress, Reynolds stress, and turbulent kinetic energy components in the vicinity of the constructs within the WWB. The mean-shear stress experienced by the tissue-engineered constructs in the WWB calculated using PIV measurements was in the range of 0-0.6 dynes/cm2. Quantification of the shear stress experienced by cartilage constructs, in this case through PIV, is essential for the development of tissue-growth models relating hydrodynamic parameters to tissue properties.

Animals↗
PubMed2006

Engineering Escherichia coli to improve culture performance and reduce formation of by-products during recombinant protein production under transient intermittent anaerobic conditions.

Three E. coli strains, named VAL22, VAL23, and VAL24, were engineered at the level of mixed-acid fermentation pathways to improve culture performance under transient anaerobic conditions. VAL22 is a single mutant with an inactivated poxB gene that codes for pyruvate oxidase which converts pyruvate to acetate. VAL23 is a double mutant unable to produce lactate and formate due to deletions of the ldhA and pflB genes that code for lactate dehydrogenase and pyruvate-formate lyase, respectively. VAL24 is a triple mutant with ldhA and pflB deleted and poxB inactivated. Engineered strains were cultured under oscillating dissolved oxygen tension (DOT) in a scale-down system, to simulate gradients occurring in large-scale bioreactors. Kinetic and stoichiometric parameters of constant (10%) and oscillating DOT cultures of the engineered strains were compared with those of the parental strain, W3110. All strains expressed recombinant green fluorescent protein (GFP) as a protein model. Mutant strains showed improved specific growth rate, reduced by-product formation, and reduced specific glucose uptake rate compared to the parental strain, when cultured under oscillating DOT. In particular, lactate and formate production was abolished and acetate accumulation was reduced by 9-12%s. VAL24 showed the best performance, as specific growth and GFP production rates, and maximum GFP concentration were not affected by DOT gradients and were at least twofold higher than those of W3110 under constant DOT. Under oscillating DOT, VAL24 wasted about 40% less carbon into fermentation by-products than W3110. It was demonstrated that, although E. coli responds rapidly to DOT fluctuations by deviating to fermentative metabolism, such pathways can be eliminated as they are not necessary for bacterial survival during the short circulation times typical of large-scale cultures. The approach shown here opens new possibilities for designing metabolically engineered strains, with reduced sensitivity to DOT gradients and improved performance under typical conditions of large-scale cultures.

Acetyltransferases↗
PubMed2005

Liver tissue engineering at extrahepatic sites in mice as a potential new therapy for genetic liver diseases.

Liver tissue engineering using hepatocyte transplantation has been proposed as an alternative to whole-organ transplantation or liver-directed gene therapy to correct various types of hepatic insufficiency. Hepatocytes are not sustained when transplanted under the kidney capsule of syngeneic mice. However, when we transplanted hepatocytes with the extracellular matrix components extracted from Engelbreth-Holm-Swarm cells, hepatocytes survived for at least 140 days and formed small liver tissues. Liver engineering in hemophilia A mice reconstituted 5% to 10% of normal clotting activity, enough to reduce the bleeding time and have a therapeutic benefit. Conversely, the subcutaneous space did not support the persistent survival of hepatocytes with Engelbreth-Holm-Swarm gel matrix. We hypothesized that establishing a local vascular network at the transplantation site would reduce graft loss. To test this idea, we provided a potent angiogenic agent before hepatocyte transplantation into the subcutaneous space. With this procedure, persistent survival was achieved for the length of the experiment (120 days). To establish that these engineered liver tissues also retained their native regeneration potential in vivo, we induced two different modes of proliferative stimulus to the naive liver and confirmed that hepatocytes within the extrahepatic tissues regenerated with activity similar to that of naive liver. In conclusion, our studies indicate that liver tissues can be engineered and maintained at extrahepatic sites, retain their capacity for regeneration in vivo, and used to successfully treat genetic disorders.

Angiogenic Proteins↗
PubMed2002

Electrospun nanofibrous structure: a novel scaffold for tissue engineering.

The architecture of an engineered tissue substitute plays an important role in modulating tissue growth. A novel poly(D,L-lactide-co-glycolide) (PLGA) structure with a unique architecture produced by an electrospinning process has been developed for tissue-engineering applications. Electrospinning is a process whereby ultra-fine fibers are formed in a high-voltage electrostatic field. The electrospun structure, composed of PLGA fibers ranging from 500 to 800 nm in diameter, features a morphologic similarity to the extracellular matrix (ECM) of natural tissue, which is characterized by a wide range of pore diameter distribution, high porosity, and effective mechanical properties. Such a structure meets the essential design criteria of an ideal engineered scaffold. The favorable cell-matrix interaction within the cellular construct supports the active biocompatibility of the structure. The electrospun nanofibrous structure is capable of supporting cell attachment and proliferation. Cells seeded on this structure tend to maintain phenotypic shape and guided growth according to nanofiber orientation. This novel biodegradable scaffold has potential applications for tissue engineering based upon its unique architecture, which acts to support and guide cell growth.

Animals↗
PubMed2003

Engineering integrin-specific surfaces with a triple-helical collagen-mimetic peptide.

Integrin-mediated cell adhesion to extracellular matrix proteins anchors cells and triggers signals that direct cell function. The integrin alpha(2)beta(1) recognizes the glycine-phenylalanine-hydroxyproline-glycine-glutamate-arginine (GFOGER) motif in residues 502-507 of the alpha(1)(I) chain of type I collagen. Integrin recognition is entirely dependent on the triple-helical conformation of the ligand similar to that of native collagen. This study focuses on engineering alpha(2)beta(1)-specific bioadhesive surfaces by immobilizing a triple-helical collagen-mimetic peptide incorporating the GFOGER binding sequence onto model nonadhesive substrates. Circular dichroism spectroscopy verified that this peptide adopts a stable triple-helical conformation in solution. Passively adsorbed GFOGER-peptide exhibited dose-dependent HT1080 cell adhesion and spreading comparable to that observed on type I collagen. Subsequent antibody blocking conditions verified the involvement of integrin alpha(2)beta(1) in these adhesion events. Focal adhesion formation was observed by immunofluorescent staining for alpha(2)beta(1) and vinculin on MC3T3-E1 cells. Model functionalized surfaces then were engineered using three complementary peptide-tethering schemes. These peptide-functionalized substrates supported alpha(2)beta(1)-mediated cell adhesion and focal adhesion assembly. Our results suggest that this peptide is active in an immobilized conformation and may be applied as a surface modification agent to promote alpha(2)beta(1)-specific cell adhesion. Engineering surfaces that specifically target certain integrin-ligand interactions and signaling cascades provides a biomolecular strategy for optimizing cellular responses in biomaterials and tissue engineering applications.

3T3 Cells↗
PubMed2005

Combined chondrocyte-copolymer implantation with slow release of basic fibroblast growth factor for tissue engineering an auricular cartilage construct.

Basic fibroblast growth factor (b-FGF) may have a role in tissue-engineered chondrogenesis. However, when applied in solution, b-FGF rapidly diffuses from the implant site. In another approach for tissue engineering, poly-lactide-based copolymers have shown promise as scaffolds for chondrocytes used to tissue engineer auricular cartilage in the shape of an ear. This study evaluated the effectiveness of b-FGF impregnated in gelatin microspheres to achieve slow growth factor release for augmenting the in vivo chondrogenic response. Whereas 125I-labeled b-FGF injected in solution showed rapid in vivo clearance from the injection site (only 3% residual after 24 h), when incorporated into gelatin microspheres, 44% and 18% of the b-FGF remained at 3 and 14 days, respectively. Canine chondrocytes were isolated and grown in vitro onto ear-shaped poly-lactide/caprolactone copolymers for 1 week, then implanted into the dorsal subcutaneous tissue of nude mice; implants contained b-FGF either in free solution or in gelatin microspheres. A third group underwent preinjection of b-FGF in gelatin microspheres 4 days before chondrocyte-copolymer implantation. The implants with b-FGF-incorporated microspheres showed the greatest chondrogenic characteristics at 5 and 10 weeks postoperatively: good shape and biomechanical trait retention, strong (histologic) metachromasia, rich vascularization of surrounding tissues, and increased gene expression for type II collagen (cartilage marker) and factor VIII-related antigen (vascular marker). In the case of implant site preadministration with b-FGF-impregnated microspheres, the implant architecture was not maintained as well, and reduced vascularization and metachromasia was also apparent. In conclusion, these findings indicate that a sustained release of b-FGF augments neovascularization and chondrogenesis in a tissue-engineered cartilage construct.

Animals↗
PubMed2005

Performance of polyoxymethylene plastic (POM) as a component of a tissue engineering bioreactor.

Polyoxymethylene (POM, acetal homopolymer, polyacetal), commercialized as Delrin by DuPont, is an engineering resin with mechanical properties that make it useful for the prototyping and manufacture of laboratory apparatus. These properties include excellent, "metal-like," machining characteristics and dimensional stability, as well as thermal stability, which allows steam sterilization. Historically, POM has been used widely, including as a surgical implant material. For these reasons, we have used this plastic as a media-wetted component in a tissue-engineering bioreactor, with good results. However, a study by LaIuppa et al.5 suggested that POM is unsuitable for use in a cell culture environment (LaIuppa et al. J Biomed Mater Res 1997;36:347-359). POM is based on the polymerization of formaldehyde, and, in addition, contains stabilizers and/or fillers. All of these could potentially be released into the medium, e.g., as formaldehyde or other thermal breakdown products, especially upon repeated autoclaving. The cited report thus appeared plausible, although contrary to our observations. In this study, we specifically assessed whether media conditioned by long-term exposure to machined white POM had a negative effect on the proliferation and chondrogenic differentiation of human mesenchymal stem cells (MSCs). We selected this cell system, as cartilage tissue engineering is the primary application of our bioreactor system. The POM samples were steam-autoclaved 1 to 20 times, to assess the possibility of any toxic thermal breakdown product release into the media. We found that MSCs did not attach directly to machined POM. Because cells that escape from the tissue construct cannot colonize the reactor and compete for nutrients, this is a desirable characteristic of a material used in a tissue-engineering bioreactor. Furthermore, the use of POM-conditioned media had no detectable impact on the proliferation rate of MSCs measured over a one-week period; nor was any effect on chondrogenic differentiation observed at up to 3 weeks in culture. In summary, the use of POM as a culture medium-wetted component appears to be innocuous, at least for human MSCs. The contrast of these findings to those of LaIuppa et al.5 may reflect a cell-type specific sensitivity, or may be due to different handling of the material.

Acetals↗
PubMed2006

Enhancement of in vivo endothelialization of tissue-engineered vascular grafts by granulocyte colony-stimulating factor.

Successful reconstruction of large-diameter blood vessel in humans has been demonstrated using the tissue engineering technique, but improvement in patency of small-diameter bioartificial vascular graft remains a great challenge. This study reports that granulocyte colony-stimulating factor (G-CSF) can enhance in vivo endothelialization of tissue-engineered vascular grafts, which could be used to improve patency of small-diameter vascular graft. Vascular grafts were tissue engineered with decellularized canine abdominal aortas and canine autologous bone marrow-derived cells. Prior to cell seeding onto decellularized graft matrices, bone marrow-derived cells were induced to differentiate into endothelial cells and smooth muscle cells. The cell-seeded vascular grafts were implanted into the abdominal aortas of bone marrow donor dogs. Before and after graft implantation, G-CSF was administered subcutaneously to the dogs (n = 3). The grafts implanted into the dogs not receiving G-CSF were used as controls (n = 3). Eight weeks after implantation, grafts in both groups showed regeneration of vascular tissues including endothelium and smooth muscle. Importantly, endothelium formation was more extensive in the G-CSF-treated grafts than in the control grafts, as assessed with reverse transcription polymerase chain reaction, western blot, and immunohistochemistry. In addition, intimal hyperplasia was significantly reduced in the G-CSF-treated grafts compared to the control grafts. This study suggests that G-CSF administration could be applied to improve patency of small-diameter tissue-engineered vascular grafts.

Animals↗
PubMed2006

Biomaterials and scaffolds for ligament tissue engineering.

Tissue engineering has achieved much progress in an attempt to improve and recover impaired functions of tissues and organs. Although many studies have been done, progress for tissue-engineered anterior cruciate ligaments (ACLs) has been slow due to their complex structures and mechanical properties. In this review, the ACL anatomical structure, progresses achieved, material selection, structure design, and future direction have been discussed, while the challenges and requirements from materials and scaffolds are highlighted. There is a considerably huge amount work that needs to be carried out; as such, future direction in ligament tissue engineering is proposed in hope that this review will give information on future ligament tissue engineering.

Biocompatible Materials↗
PubMed2007

Engineering cartilage with human nasal chondrocytes and a silanized hydroxypropyl methylcellulose hydrogel.

Tissue engineering strategies, based on developing three-dimensional scaffolds capable of transferring autologous chondrogenic cells, holds promise for the restoration of damaged cartilage. In this study, the authors aimed at determining whether a recently developed silanized hydroxypropyl methylcellulose (Si-HPMC) hydrogel can be a suitable scaffold for human nasal chondrocytes (HNC)-based cartilage engineering. Methyltetrazolium salt assay and cell counting experiments first revealed that Si-HPMC enabled the proliferation of HNC. Cell tracker green staining further demonstrated that HNC were able to form nodular structures in this three-dimensional scaffold. HNC phenotype was then assessed by RT-PCR analysis of type II collagen and aggrecan expression as well as alcian blue staining of extracellular matrix. Our data indicated that Si-HPMC allowed the maintenance and the recovery of a chondrocytic phenotype. The ability of constructs HNC/Si-HPMC to form a cartilaginous tissue in vivo was finally investigated after 3 weeks of implantation in subcutaneous pockets of nude mice. Histological examination of the engineered constructs revealed the formation of a cartilage-like tissue with an extracellular matrix containing glycosaminoglycans and type II collagen. The whole of these results demonstrate that Si-HPMC hydrogel associated to HNC is a convenient approach for cartilage tissue engineering.

Aggrecans↗
PubMed2005

Evaluation of different scaffolds for BMP-2 genetic orthopedic tissue engineering.

To better understand the effects of scaffold materials for bone morphogenetic protein 2 (BMP-2) genetic tissue engineering in vivo, several gels, including alginate, collagen, agarose, hyaluronate, fibrin, or Pluronic, were mixed with adenovirus-mediated human BMP-2 gene (Adv-hBMP-2) transduced bone marrow stromal cells (BMSCs) and injected into the muscles of athymic mice to evaluate the resulting osteogenesis and chondrogenesis. These gel and gene-transduced BMSC mixtures were also loaded onto beta-TCP/HAP biphasic calcined bone (BCB) and observed under scanning electron microscopy (SEM). In addition, these composite scaffolds were implanted into the subcutaneous site of athymic mice to construct tissue-engineered bone. After injection, collagen, hyaluronate, or alginate gel mixed with gene-transduced BMSCs induced more bone formation than a cell suspension in alpha-MEM. The agarose-gene-transduced BMSC gel was found to contain much more hyaline cartilage. SEM showed the BMSCs could survive in alginate, agarose, and collagen gel in vitro for up to 8 d. After implantation of tissue-engineered bone, the alginate, collagen, and agarose gel could promote new bone formation within a BCB in vivo. Little or no bone formed after injection of fibrin or Pluronic gel mixed with BMSCs or implantation with BCB. These findings help to elucidate the effects of various scaffold materials for future research in orthopedic tissue engineering using BMP-2 transduced cells.

Adenoviridae↗
PubMed2003

Engineering of technetium-99m-binding artificial receptors for imaging gene expression.

BACKGROUND: Optimization of gene therapy protocols requires accurate and non-invasive quantification of vector delivery and gene expression. To facilitate non-invasive imaging of gene expression, we have genetically engineered 'artificial receptors', i.e. membrane proteins that bind (99m)Tc-oxotechnetate ((99m)TcOT) via transchelation from a complex with glucoheptonate. The latter is a component of a widely used clinical imaging kit. METHODS: The engineered marker proteins were designed as type I and II membrane proteins and consisted of (1) an (99m)TcOT-binding domain, metallothionein (MT), and (2) a membrane-anchoring domain. Engineered constructs were used for transfection of COS-1 and 293 cells; the expression of mRNA was verified by RT-PCR. RESULTS: Immunofluorescent analysis, cell fractionation and immunoblotting revealed expression of marker proteins on plasma membrane. Transfection of cells resulted in strong positive staining of plasma membrane with anti-His-tag antibodies. Scintigraphic imaging in vitro confirmed the ability of transfected cells to bind (99m)TcOT. The fraction of bound radioactivity reached a peak (3.53%) when 0.93 MBq (99m)TcOT was added to transfected COS-1 cells. The experiment-to-control signal ratio was equal to 32 at the same added dose. CONCLUSIONS: (1) Both types of engineered 'artificial receptors' were expressed on the surface of eukaryotic cells; (2) marker proteins were functional in binding (99m)TcOT; and (3) type II membrane proteins were more efficient in binding (99m)TcOT than type I proteins. We anticipate that the developed approach could be useful for 'tagging' transfected cells with (99m)TcOT enabling imaging of tracking in vivo transduced cells or cell therapies.

Cell Membrane↗
PubMed2006

Multi-template approach to modeling engineered disulfide bonds.

The key issue for disulfide bond engineering is to select the most appropriate location in the protein. By surveying the structure of experimentally engineered disulfide bonds, we found about half of them that have geometry incompatible with any native disulfide bond geometry. To improve the current prediction methods that tend to apply either ideal geometrical or energetical criteria to single three-dimensional structures, we have combined a novel computational protocol with the usage of multiple protein structures to take into account protein backbone flexibility. The multiple structures can be selected from either independently determined crystal structures for identical proteins, models of nuclear magnetic resonance experiments, or crystal structures of homology-related proteins. We have validated our approach by comparing the predictions with known disulfide bonds. The accuracy of prediction for native disulfide bonds reaches 99.6%. In a more stringent test on the reported engineered disulfide bonds, we have obtained a success rate of 93%. Our protocol also determines the oxido-reduction state of a predicted disulfide bond and the corresponding mutational cost. From the energy ranking, the user can easily choose top predicted sites for mutagenesis experiments. Our method provides information about local stability of the engineered disulfide bond surroundings.

Bacterial Proteins↗
PubMed2001

Biochemical engineering of the side chain of sialic acids increases the biological stability of the highly sialylated cell adhesion molecule CEACAM1.

The biological half-life time of many glycoproteins is regulated via terminal sialic acids. In this study we determined the half-lives of two different cell adhesion molecules, CEACAM1 and the alpha1-integrin subunit, in PC12-cells before and after biochemical engineering the side chain of sialic acids by the use of N-propanoylmannosamine. Both are transmembrane glycoproteins. While the immunoglobulin superfamily member CEACAM1 mediates homophilic cell-cell adhesion the alpha1-integrin subunit is involved in cell-matrix interactions. We found that the half-life of the highly sialylated CEACAM1 is increased from 26 to 40 h by replacement of the N-acetylneuraminic acid by the novel, engineered N-propanoylneuraminic acids, whereas the half-life of the alpha1-integrin subunit remains unaffected under the same conditions. This demonstrates that biochemical engineering not only modulates the structure of cell surface sialic acids, but that biochemical engineering also influences biological stability of defined glycoproteins.

Animals↗
PubMed1999

A field release of genetically engineered gypsy moth (Lymantria dispar L.) nuclear polyhedrosis virus (LdNPV).

The gypsy moth (Lymantria dispar L.) nuclear polyhedrosis virus was genetically engineered for nonpersistence by removal of the gene coding for polyhedrin production and stabilized using a coocclusion process. A beta-galactosidase marker gene was inserted into the genetically engineered virus (LdGEV) so that infected larvae could be tested for its presence using a colorimetric assay. In 1993, LdGEV-infected gypsy moths were released in a forested plot in Massachusetts to test for spread and persistence. A similar forested plot 2 km away served as a control. For 3 years (1993-1995), gypsy moths were established in the two plots in Massachusetts to serve as test and control populations. Each week, larvae were collected from both plots. These field-collected larvae were reared individually, checked for mortality, and then tested for the presence of beta-galactosidase. Other gypsy moth larvae were confined on LdGEV-contaminated foliage for 1 week and then treated as the field-collected larvae. The LdGEV was sought in bark, litter, and soil samples collected from each plot. To verify the presence of the LdGEV, polymerase chain reaction, slot blot DNA hybridization, and restriction enzyme analysis were also used on larval samples. Field-collected larvae infected with the engineered virus were recovered in the release plot in 1993, but not in subsequent years; no field-collected larvae from the control plot contained the engineered virus. Larvae confined on LdGEV-contaminated foliage were killed by the virus. No LdGEV was recovered from bark, litter, or soil samples from either of the plots.

Animals↗
PubMed2000

Exploring local and non-local interactions for protein stability by structural motif engineering.

In order to probe the relative contribution of local and non-local interactions to the thermodynamic stability of proteins, we have devised an experimental approach based on a combination of motif engineering and sequence shuffling. Candidate chain segments in an immunoglobulin V(L) domain were identified whose conformation is proposed to be dominated by non-local interactions. Locally interacting structural motifs of a different conformation were then constructed as replacements, by introducing motif consensus sequences. We find that all nine replacements we constructed systematically reduce the folding cooperativity. By comparing this destabilising effect with the folding transitions of shuffled sequences for three of these motifs, we estimate the contribution of local, native interactions to the free energy of folding. Our results suggest that local and non-local interactions contribute to stability by an approximately equal amount, but that local interactions stabilise by increasing the resistance to denaturation while non-local interactions increase folding cooperativity. The systematic loss of stability by sequence shuffling in these host-guest experiments suggests that the designed interactions indeed are present in the native state, thus consensus sequence engineering may be a useful tool in structure design, but non-local interactions must be taken into account for global stability engineering. Statistical approaches are powerful tools for engineering protein structure and stability, but an analysis based on local sequence propensities alone does not adequately represent the balance of sequence and context in protein structures.

Amino Acid Motifs↗
PubMed2002

Engineered improvements in DNA-binding function of the MATa1 homeodomain reveal structural changes involved in combinatorial control.

We have engineered enhanced DNA-binding function into the a1 homeodomain by making changes in a loop distant from the DNA-binding surface. Comparison of the free and bound a1 structures suggested a mechanism linking van der Waals stacking changes in this loop to the ordering of a final turn in the DNA-binding helix of a1. Inspection of the protein sequence revealed striking differences in amino acid identity at positions 24 and 25 compared to related homeodomain proteins. These positions lie in the loop connecting helix-1 and helix-2, which is involved in heterodimerization with the alpha 2 protein. A series of single and double amino acid substitutions (a1-Q24R, a1-S25Y, a1-S25F and a1-Q24R/S25Y) were engineered, expressed and purified for biochemical and biophysical study. Calorimetric measurements and HSQC NMR spectra confirm that the engineered variants are folded and are equally or more stable than the wild-type a1 homeodomain. NMR analysis of a1-Q24R/S25Y demonstrates that the DNA recognition helix (helix-3) is extended by at least one turn as a result of the changes in the loop connecting helix-1 and helix-2. As shown by EMSA, the engineered variants bind DNA with enhanced affinity (16-fold) in the absence of the alpha 2 cofactor and the variant alpha 2/a1 heterodimers bind cognate DNA with specificity and affinity reflective of the enhanced a1 binding affinity. Importantly, in vivo assays demonstrate that the a1-Q24R/S25Y protein binds with fivefold greater affinity than wild-type a1 and is able to partially suppress defects in repression by alpha 2 mutants. As a result of these studies, we show how subtle differences in residues at a surface distant from the functional site code for a conformational switch that allows the a1 homeodomain to become active in DNA binding in association with its cofactor alpha 2.

Amino Acid Sequence↗