The pi-helix formation between Asp369 and Thr375 as a key factor in E1-E2 conformational change of Na+/K+-ATPase.

Molecular modeling of the H4-H5-loop of the alpha2 isoform of Na+/K+-ATPase in the E1 and E2 conformations revealed that twisting of the nucleotide (N) domain toward the phosphorylation (P) domain is connected with the formation of a short pi-helix between Asp369 and Thr375. This conformational change close to the hinge region between the N-domain and the P-domain could be an important event leading to a bending of the N-domain by 64.7 degrees and to a shortening of the distance between the ATP binding site and the phosphorylation site (Asp369) by 1.22 nm from 3.22 nm to 2.00 nm. It is hypothesized that this shortening mechanism is involved in the Na+-dependent formation of the Asp369 phospho-intermediate as part of the overall Na+/K+-ATPase activity.

Comparative modeling of the H4-H5-loop of the alpha2-isoform of Na+/K+-ATPase alpha-subunit in the E1 conformation.

Restraint-based comparative modeling was used for calculation and visualization of the H4-H5-loop of Na+/K+-ATPase from mouse brain (Mus musculus, adult male brain, alpha2-isoform) between the amino acid residues Cys 336 and Arg 758 in the E1 conformation The structure consists of two well separated parts. The N-domain is formed by a seven-stranded antiparallel beta-sheet with two additional beta-strands and five alpha-helices sandwiching it, the P-domain is composed of a typical Rossman fold. The ATP-binding site was found on the N-domain to be identical in both alpha2- and alpha1-isoforms. The phosphorylation Asp 369 residue was found in the central part of the P-domain, located at the C-terminal end of the central beta-sheet. The distance between the alpha-carbon of Phe 475 at the ATP-binding site and the alpha-carbon of Asp 369 at the phosphorylation site is 3.22 nm. A hydrogen bond between the oxygen atom of Asp 369 and the nitrogen atom of Lys 690 was clearly detected and assumed to play a key role in maintaining the proper structure of the phosphorylaton site in E1 conformation.

Biochemical and biophysical aspects of collagen nanostructure in the extracellular matrix.

ECM is composed of different collagenous and non-collagenous proteins. Collagen nanofibers play a dominant role in maintaining the biological and structural integrity of various tissues and organs, including bone, skin, tendon, blood vessels, and cartilage. Artificial collagen nanofibers are increasingly significant in numerous tissue engineering applications and seem to be ideal scaffolds for cell growth and proliferation. The modern tissue engineering task is to develop three-dimensional scaffolds of appropriate biological and biomechanical properties, at the same time mimicking the natural extracellular matrix and promoting tissue regeneration. Furthermore, it should be biodegradable, bioresorbable and non-inflammatory, should provide sufficient nutrient supply and have appropriate viscoelasticity and strength. Attributed to collagen features mentioned above, collagen fibers represent an obvious appropriate material for tissue engineering scaffolds. The aim of this minireview is, besides encapsulation of the basic biochemical and biophysical properties of collagen, to summarize the most promising modern methods and technologies for production of collagen nanofibers and scaffolds for artificial tissue development.

Composite hyaluronate-type I collagen-fibrin scaffold in the therapy of osteochondral defects in miniature pigs.

The potential of novel scaffold containing sodium hyaluronate, type I collagen, and fibrin was investigated in the regeneration of osteochondral defects in miniature pigs. Both autologous chondrocyte-seeded scaffolds and non-seeded scaffolds were implanted into two defects located in the non-weight-bearing zone of the femoral trochlea (defect A was located more distally and medially, defect B was located more proximally and laterally). Control defects were left untreated. Twelve weeks after the operation, the knees were evaluated in vivo using MRI. Six months after the implantation, the defects were analyzed using MRI, histological, and immunohistochemical analysis. In the A defects of chondrocyte-seeded scaffold group, hyaline cartilage and fibrocartilage was formed, containing type II collagen, acidic and neutral glycosaminoglycans while the non-seeded scaffold group was predominantly filled with fibrocartilage. Defects in the control group were predominantly filled with fibrous tissue. Histomorphometric analysis of photomicrographs revealed a significantly higher amount of hyaline cartilage in the cell-seeded scaffold group in A defects than in other groups. Both scaffold groups in A defects showed significantly less fibrous tissue than cell-seeded defects B and the control group. Both histological and MRI analysis proved that the novel composite scaffold has a potential to regenerate osteochondral defects within six months.

Fluorescent advanced glycation end products in the detection of factual stages of cartilage degeneration.

Patients treated for knee disorders were included in this study. They were examined clinically (Lequesne and Tegner scores) and by standard X-ray investigation. Patients underwent a surgical procedure, either arthroscopy or knee replacement. At the initial phase of surgery, a sample of cartilage was taken for laboratory examination. Progression of the disorder and the clinical examination was correlated with the actual state of the cartilage using a novel fluorescence approach. The intrinsic fluorescence of cartilages was shown as a suitable and sensitive method for detection of the actual state of cartilages because the correlation with X-ray examination and clinical status was found. Intrinsic fluorescence properties of cartilages from patients with chondropathy and osteoarthritis were described and found to be age-dependent. We also observed a higher concentration of advanced glycation end products due to inflammatory and/or degenerative processes in the cartilage. In addition, acute pathological changes due to diseases such as meniscal lesions or anterior cruciate ligament rupture caused a significant increase of formation of advanced glycation end products even in the group of young patients. In fact, such an observation could be crucial and important for the detection of knee conditions suspected of early meniscal and/or ACL lesions especially among young patients.

Protein structure and dynamics determined by protein modeling combined with spectroscopic techniques.

Beside of the protein crystals, another attractive option in protein structure analysis has recently appeared: computer modeling of the protein structure based on homology and similarity with proteins of already known structures. We used the combination of computer modeling with spectroscopic techniques, such as steady-state or time-resolved fluorescence spectroscopy or Raman spectroscopy, and with molecular biology techniques. This method could achieve reliable results comparable with resolution obtained from crystal structures. Molecular modeling of the ATP site within the H4-H5-loop revealed eight amino acids residues, namely besides the previously reported amino acids Asp443, Lys480, Lys501, Gly502 and Arg544, also Glu446, Phe475 and Gln482, which form the complete ATP recognition site. Moreover, we proved that a hydrogen bond between Arg423 and Glu472 supported the connection of two opposite halves of the ATP-binding pocket. Similarly, the conserved residue Pro489 is important for the proper interaction of the third and fourth-strands, which both contain residues that take part in the ATP-binding (Ref. 34).