Wavelength-dependent collagen fragmentation during mid-IR laser ablation

Mid-infrared free-electron lasers have proven adept in surgical applications. When tuned to wavelengths between 6 and 7 microm, such lasers remove defined volumes of soft tissue with very little collateral damage. Previous attempts to explain the wavelength-dependence of collateral damage have invoked a wavelength-dependent loss of protein structural integrity. However, the molecular nature of this structural failure has been heretofore ill-defined. In this report, we evaluate several candidates for the relevant transition by analyzing the nonvolatile debris ejected during ablation. Porcine corneas were ablated with a free-electron laser tuned to 2.77 or 6.45 microm-wavelengths with matched absorption coefficients for hydrated corneas that respectively target either tissue water or protein. The debris ejected during these ablations was characterized via gel electrophoresis, as well as Fourier transform infrared spectroscopy, micro-Raman and 13C-NMR spectroscopy. We find that high-fluence (240 J/cm2) ablation at 6.45 microm, but not at 2.77 microm, leads to protein fragmentation accompanied by the accumulation of nitrile and alkyne species. The candidate transition most consistent with these observations is scission of the collagen protein backbone at N-alkylamide bonds. Identifying this transition is a key step toward understanding the observed wavelength-dependence of collateral damage in mid-infrared laser ablation.     

Quaternary conformational changes in human hemoglobin studied by laser photolysis of carboxyhemoglobin.

These experiments indicate that absorbance changes observed at the 425 nm isosbestic point of the Hb and HbCO following laser photolysis of HbCO provide a direct measure of the rates of quaternary conformational changes between rapidly reacting Hb (the immediate product of full photolysis) and slowly reacting normal deoxyhemoglobin. Hb, first observed by Gibson (Gibson, Q.H. (1959) Biochem. J. 71, 293-303), Has been interpreted as deoxyhemoglobin remaining in the liganded quaternary conformation following rapid removal of ligand by a light pulse. In borate buffers between pH 8.4 and 9.6 particularly simple pH-independent results were obtained which allowed the use of a Monod. Wyman, and Changeux model (Monod, J., Wyman, J., and Changeux, J (1965) J. Mol. Biol. 12, 88-118) to fit the data. In this case Hb is taken to be R state deoxyhemoglobin. Partial photolysis experiments at 425 nm show that the rate of the R - T conformational change at 20 degrees decreases by about a factor of 2 for each additional bound ligand. The rate of the ligand-free conformational change is found to be 920 +/- 60s(-1), 6400 +/- 600s(-1), and 15,700 +/- 700(-1) respectively at 3 degrees, 20 degrees, and 30 degrees. The previously uninterpreted effects of flash length and partial photolysis on the CO recombination kinetics can be explained in terms of the present model. Kinetic results obtained below pH 8 are found to be inconsistent with a two-state model. It appears that binding of inositol hexaphosphate produces a new rapidly reacting quaternary conformation of HbCO.     

Cytologic changes in small hepatocellular carcinomas after radiofrequency ablation

OBJECTIVE: To illustrate the cytologic changes in hepatocellular carcinoma (HCC) after radiofrequency ablation (RFA). STUDY DESIGN: The study included 20 patients with 23 HCC who had undergone RFA under ultrasound guidance. Baseline cytomorphology of HCC was evaluated by fine needle aspiration (FNA) in all cases. Triphasic helical computed tomography (CT) and FNA cytology were done to evaluate the efficacy of the treatment within two weeks after RFA. The cytologic specimens were stained with Riu's method (Romanowsky stain). RESULTS: A range of cytologic findings after RFA was found, including granular and amorphous debris with artefactual aggregation, degenerated cells or necrotic material, and dyshesive, degenerated cells in a necrotic background. The cytologic patterns included necrotic cells and debris in 14 tumors and fine, granular necrosis in 9. Helical CT showed no enhancement in any of the tumors after RFA. CONCLUSION: The cell patterns indicated complete necrosis in HCC after RFA.     

Proteoglycan and collagen morphology in superficially scarred rabbit cornea

Summary We have examined the changes in collagen and proteoglycan morphology in superficial lamellar keratectomy wounds produced in rabbit corneas. The ultrastructural location within the tisse of keratan sulphate and chondroitin sulphate proteoglycans was demonstrated using the cationic dye Cuprolinic Blue under critical electrolyte conditions. Large proteoglycan filaments (up to 500 nm long) appeared in the early stages of wound healing; these were most common after two weeks' wound healing, after which they decreased both in number and size. At these early stages of scar formation, spaces containing proteoglycans were present amongst bundles of collagen fibrils. As proteoglycans play an important role in controlling corneal hydration, the presence of the large proteoglycan-filled spaces would result in an abnormally high water content which is found in early early scar tissue.     

Substrate-induced conformational changes in glycosyltransferases

Oligosaccharide chains of glycoproteins, glycolipids and glycosaminoglycans are synthesized by glycosyltransferases by the transfer of specific glycosyl moieties from activated sugar-nucleotide donors to specific acceptors. Structural studies on several of these enzymes have shown that one or two flexible loops at the substrate-binding site of the enzymes undergo a marked conformational change from an open to a closed conformation on binding the donor substrate. This conformational change, in which the loop acts as a lid covering the bound donor substrate, creates an acceptor-binding site. After the glycosyl unit is transferred from the donor to the acceptor, the saccharide product is ejected and the loop reverts to its native conformation, thereby releasing the remaining nucleotide moiety. The specificity of the sugar donor is determined by a few residues in the sugar-nucleotide-binding pocket of the enzyme that are conserved among the family members from different species.     

Tracing conformational changes in proteins

Background

Many proteins undergo extensive conformational changes as part of their functionality. Tracing these changes is important for understanding the way these proteins function. Traditional biophysics-based conformational search methods require a large number of calculations and are hard to apply to large-scale conformational motions.

Results

In this work we investigate the application of a robotics-inspired method, using backbone and limited side chain representation and a coarse grained energy function to trace large-scale conformational motions. We tested the algorithm on four well known medium to large proteins and we show that even with relatively little information we are able to trace low-energy conformational pathways efficiently. The conformational pathways produced by our methods can be further filtered and refined to produce more useful information on the way proteins function under physiological conditions.

Conclusions

The proposed method effectively captures large-scale conformational changes and produces pathways that are consistent with experimental data and other computational studies. The method represents an important first step towards a larger scale modeling of more complex biological systems.

     

Kinetics of a collagen-like polypeptide fragmentation after mid-IR free-electron laser ablation

Tissue ablation with mid-infrared irradiation tuned to collagen vibrational modes results in minimal collateral damage. The hypothesis for this effect includes selective scission of protein molecules and excitation of surrounding water molecules, with the scission process currently favored. In this article, we describe the postablation infrared spectral decay kinetics in a model collagen-like peptide (Pro-Pro-Gly)₁₀. We find that the decay is exponential with different decay times for other, simpler dipeptides. Furthermore, we find that collagen-like polypeptides, such as (Pro-Pro-Gly)₁₀, show multiple decay times, indicating multiple scission locations and cross-linking to form longer chain molecules. In combination with data from high-resolution mass spectrometry, we interpret these products to result from the generation of reactive intermediates, such as free radicals, cyanate ions, and isocyanic acid, which can form cross-links and protein adducts. Our results lead to a more complete explanation of the reduced collateral damage resulting from infrared laser irradiation through a mechanism involving cross-linking in which collagen-like molecules form a network of cross-linked fibers.     

Apoptosis of male germ-line stem cells after laser ablation of their niche

Male germ-line stem cells (GSCs) and their niche-the apical cells or hub cells-display a unique feature at the apices of insect testicular follicles. In the locust, Locusta migratoria, the niche consists of only one large apical cell surrounded by about 60 GSCs. The apical cell can be readily identified in the intact follicle. Using laser ablation it is feasible to destroy the apical cell exclusively without injuring neighboring GSCs or any other cells. The most immediate effect on GSCs is the loss of their structural polarity. Beginning about 3 h after laser treatment chromatin starts to clump and condense in individual GSCs, and some show the first signs of cellular breakdown. These symptoms intensify during the 96-h observation period after laser ablation of the apical cell. TUNEL staining and electron microscopic observations confirm an apoptotic cell death of the GSCs. Laser ablation of individual GSCs had no effect on neighboring GSCs or the apical cell. Destroyed apical cells were not replaced during the observation period. Mitotic divisions of GSCs ceased after about 24 h after apical cell ablation. It is speculated that it might be a general principle in stem cell-niche relationships that stem cells undergo apoptosis when the niche is dysfunctional. This could be a control mechanism to prevent tumor growth of orphaned GSCs.     

Cytomorphology and histomorphology after thermal endometrial ablation. Early changes

OBJECTIVE: Endometrial thermal balloon ablation is a recent technique for treatment of dysfunctional uterine bleeding. Although the histologic features of thermal injury to the endometrium have been documented, the cytologic features are not described in the literature. In this study, the cytomorphology of recent thermal injury was studied and correlated with histomorphology. STUDY DESIGN: The study consisted of 29 cases divided into 3 groups: (1) control group, hysterectomies without ablation; (2) specimen (in vitro) group, where ablation was carried out on the uteri in the immediate postoperative period; and (3) in vivo group, with intraoperative endometrial ablation. The cytotoxic effect of thermal injury was assessed by staining the smears for oxidative enzymes. RESULTS: The cytomorphologic features of in vivo thermal ablation were characteristic and composed of fragmented glands, numerous single cells with fuzzy cytoplasm and giant cells. Staining for dehydrogenases was absent from > 90% of endometrial cells in the in vivo cases as against 100% positivity in the control group. CONCLUSION: The cytomorphologic features described for the first time in this study can be applied to endometrial aspirate or brush smears to evaluate the efficacy of the technique and follow-up.     

pH-induced conformational changes of DNA

The electrophoretic mobility of calf thymus DNA has been measured in aqueous buffered solutions as a function of pH. In the pH range 3.7–3.0, two electrophoretic species appear. The faster one migrates with the mobility of native DNA, the slower one migrates with a mobility close to that of thermally denatured DNA. The ratio of the two species varies with pH. Decreasing the pH increase the relative amount of the slower-moving component. These results may be interpreted by assuming that the DNA used in these experiments has a broad heterogeneity of base composition and that the conformational stability with respect to pH increases with increasing G + C content.     

Cation-induced conformational changes in tRNA molecules

The uv absorption spectra and melting profiles of an initially ion-free solution of E. coli unfractionated tRNA are significantly modified by the addition of either Na⁺, Mg²⁺, or Mn²⁺ or of other first-series transition-metal ions such as Ni²⁺, Co²⁺, and Zn²⁺. The main effect of the addition of all monovalent or divalent cations examined is an increase of the ordered and stacking stabilized tRNA structure, as revealed by a drop in the absorption near 260 nm, as well as in the 4-TU absorption region. Sharp differences have, however, been detected in the 290–305-nm range in the presence of the various ions studied. When transition-metal ions were added to a tRNA solution, an absorption peak appeared at 294 nm. This effect is interpreted as a perturbation of the electronic structure of the bases due to direct binding of metal ions to the bases. An analysis of the variation in the spectrum as a function of metal concentration and of the thermal melting reversibility in the presence of various metal ions supports the conclusion that while all ions investigated are involved in binding to the phosphate groups of tRNA, transition-metal ions are also able to bind directly to the bases.     

Voltage-dependent conformational changes in connexin channels

Channels formed by connexins display two distinct types of voltage-dependent gating, termed V(j)- or fast-gating and loop- or slow-gating. Recent studies, using metal bridge formation and chemical cross-linking have identified a region within the channel pore that contributes to the formation of the loop-gate permeability barrier. The conformational changes are remarkably large, reducing the channel pore diameter from 15 to 20? to less than 4?. Surprisingly, the largest conformational change occurs in the most stable region of the channel pore, the 3(10) or parahelix formed by amino acids in the 42-51 segment. The data provide a set of positional constraints that can be used to model the structure of the loop-gate closed state. Less is known about the conformation of the V(j)-gate closed state. There appear to be two different mechanisms; one in which conformational changes in channel structure are linked to a voltage sensor contained in the N-terminus of Cx26 and Cx32 and a second in which the C-terminus of Cx43 and Cx40 may act either as a gating particle to block the channel pore or alternatively to stabilize the closed state. The later mechanism utilizes the same domains as implicated in effecting pH gating of Cx43 channels. It is unclear if the two V(j)-gating mechanisms are related or if they represent different gating mechanisms that operate separately in different subsets of connexin channels. A model of the V(j)-closed state of Cx26 hemichannel that is based on the X-ray structure of Cx26 and electron crystallographic structures of a Cx26 mutation suggests that the permeability barrier for V(j)-gating is formed exclusively by the N-terminus, but recent information suggests that this conformation may not represent a voltage-closed state. Closed state models are considered from a thermodynamic perspective based on information from the 3.5? Cx26 crystal structure and molecular dynamics (MD) simulations. The applications of computational and experimental methods to define the path of allosteric molecular transitions that link the open and closed states are discussed. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Osmolyte-induced changes in protein conformational equilibria

Examining solute-induced changes in protein conformational equilibria is a long-standing method for probing the role of water in maintaining protein stability. Interpreting the molecular details governing the solute-induced effects, however, remains controversial. We present experimental and theoretical data for osmolyte-induced changes in the stabilities of the A and N states of yeast iso-1-ferricytochrome c. Using polyol osmolytes of increasing size, we observe that osmolytes alone induce A-state formation from acid-denatured cytochrome c and N state formation from the thermally denatured protein. The stabilities of the A and N states increase linearly with osmolyte concentration. Interestingly, osmolytes stabilize the A state to a greater degree than the N state. To interpret the data, we divide the free energy for the reaction into contributions from nonspecific steric repulsions (excluded volume effects) and from binding interactions. We use scaled particle theory (SPT) to estimate the free energy contributions from steric repulsions, and we estimate the contributions from water-protein and osmolyte-protein binding interactions by comparing the SPT calculations to experimental data. We conclude that excluded volume effects are the primary stabilizing force, with changes in water-protein and solute-protein binding interactions making favorable contributions to stability of the A state and unfavorable contributions to the stability of the N state. The validity of our interpretation is strengthened by analysis of data on osmolyte-induced protein stabilization from the literature, and by comparison with other analyses of solute-induced changes in conformational equilibria.     

Translocation of latex beads after laser ablation of the avian neural crest

Previous studies from this laboratory (M. E. Bronner-Fraser, 1982, Dev. Biol.91, 50–63) have demonstrated that latex beads translocate ventrally after injection into avian embryos during the phase of neural crest migration, to settle in the vicinity of neural-crest-derived structures. In order to examine the role of host neural crest cells in the ventral translocation of implanted beads, latex beads have been injected into regions of embryos from which the neural crest cells have been ablated using a laser microbeam. Prior to their migratory phase, neural crest cells reside in the dorsal portion of the neural tube. Laser irradiation of the dorsal neural tube was used to reproducibly achieve either partial or complete ablation of neural crest cells from the irradiated regions. The effectiveness of the ablation was assessed by the degree of reduction in dorsal root ganglia, a neural crest derivative. Because of the rapidity and precision of this technique, it was possible to selectively remove neural crest cells without significantly altering other embryonic structures. The results indicate that, after injection of latex beads into the somites of embryos whose neural crest cells were removed by laser irradiation, the beads translocate ventrally in the absence of the endogenous neural crest.     

Multiple conformational changes in enzyme catalysis

Understanding the molecular mechanisms of enzyme catalysis and allosteric regulation has been a primary goal of biochemistry for many years. The dynamics of these processes, approached through a variety of kinetic methods, are discussed. The results obtained for many different enzymes suggest that multiple intermediates and conformations are general characteristics of the catalytic process and allosteric regulation. Ribonuclease, dihydrofolate reductase, chymotrypsin, aspartate aminotransferase, and aspartate transcarbamoylase are considered as specific examples. Typical and maximum rates of conformational changes and catalysis are also discussed, based on results obtained from model systems. The nature and rates of interconversion of the intermediates, along with structural information, can be used as the bases for understanding the incredible catalytic efficiency of enzymes. Potential roles of conformational changes in the catalytic process are discussed in terms of static and environmental effects, and in terms of dynamic coupling within the enzyme-substrate complex.     

pH-dependent conformational changes in thrombocyte proteins

A study was made of the medium pH influence on structural states of platelets by optical methods. Within the pH range (6-8), two pH induced reversible changes of platelet state were observed. A conclusion is made that the structural rearrangements in platelets induced in the medium by changes in hydrogen ion concentration may involve some rearrangements in platelet proteins, and thus acting as a factor regulating platelet function.