Bulletin of the American Physical Society
2009 APS March Meeting
Volume 54, Number 1
Monday–Friday, March 16–20, 2009; Pittsburgh, Pennsylvania
Session X39: Focus Session: Crystal Growth of and Moderated by Proteins |
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Sponsoring Units: DBP Chair: Raymond Friddle, Lawrence Livermore National Laboratory Room: 411 |
Thursday, March 19, 2009 2:30PM - 3:06PM |
X39.00001: Investigating protein controls on crystal growth: how competing timescales and electrostatic interactions lead to bi-stable and catalytic growth Invited Speaker: Structural relationships, chemical interactions, and mechanistic impacts of proteins at the surfaces of growing crystals are poorly understood, despite of their central role in directing formation of mineralized tissues. Here we describe results of in situ AFM investigations into the interactions of aspartic acid-rich peptides and proteins with single crystals of calcium oxalates and carbonates. Using specially designed cantilevers, we have obtained true single molecule resolution and directly imaged protein interactions with atomic steps. We show how the slow adsorption dynamics, strong electrostatic interactions and tendency towards clustering peculiar to macromolecules lead alternately to acceleration and inhibition, as well as switching of growth between two distinct states. We provide a mechanistic model for the observed behavior in terms of altered activation energies and competing timescales for macromolecule adsorption and solute attachment. [Preview Abstract] |
Thursday, March 19, 2009 3:06PM - 3:18PM |
X39.00002: Temperature-dependence of global radiation damage to protein crystals Matthew Warkentin, Robert Thorne We have measured the global radiation sensitivity of model protein crystals as a function of temperature in the range 300 K to 100 K. Our data show three regimes, each characterized by a different activation energy for damage. Between 300 and 225 K, the activation energy is considerably less than would be expected if it was limited by the diffusion of radicals or (diffusive) conformational changes in the molecular structure. This suggests that some other mechanism is the bottleneck for radiation damage in this temperature range. From 225 to 160 K, the activation energy is consistent with radiation damage being diffusion limited. Below 160 K, the activation energy is very small, and is consistent with activation energies for damage of small biomolecules. These results shed light on the underlying mechanisms of radiation damage in protein crystallography, and also inform temperature-dependent studies of protein crystals about radiation damage at temperatures other than 100 K. [Preview Abstract] |
Thursday, March 19, 2009 3:18PM - 3:30PM |
X39.00003: Directly probing the antifreeze glycoprotein kinetics at the ice/solution interface Salvador Zepeda, Etsuro Yokoyama, Yoshinor Furukawa Antifreeze proteins (AFP) and glycoproteins (AFGP) help fish, plants, insects and bacteria survive sub-freezing environments. It is well known that these proteins function via some surface interaction, but the exact mechanism has eluded scientists. Aside from mutagenesis experiments directed towards examining the functional importance of specific residues, conclusions about the mechanism have been drawn from indirect studies or more precisely from studies that describe the proteins effects on the ice interface. Our work is aimed at directly studying the protein kinetics at the ice/solution interface. Fluorescent microscopy is used to determine interaction planes, surface concentrations as well as adsorption characteristics and the segregation constants, while fourier transform infra-red attenuated total reßectance (FTIR-ATR) is used to determine the protein structure vs. temperature in the liquid and solid states as well as the ice interface characteristics. All data show that AFGP do not function by the characteristic Gibbs-Thomson mechanism. While the surface coverage is similar for the AFPIII, segregation (amount in ice/amount in solution) is non-zero. [Preview Abstract] |
Thursday, March 19, 2009 3:30PM - 3:42PM |
X39.00004: Ice-binding protein investigation using microfluidic devices Yeliz Celik, Natalya Pertaya, Christophere P. Garnham, Peter L. Davies, Ido Braslavsky Ice-binding proteins (IBPs) inhibit ice crystal growth and recrystallization. We have developed a novel microfluidic device capable of precise local temperature control in order to grow single ice crystals. This device allows us to expose an ice crystal to an adjustable IBP concentration. We have used this device in conjunction with fluorescence microscopy to examine the affinities hyperactive IBPs have to specific ice planes in comparison with those of a moderately active fish IBP. We also demonstrate that hyperactive IBPs bind irreversibly to ice surfaces. The direct visualization of IBPs on ice using the microfluidic devices reveal the kinetics of attachment of these proteins to ice surfaces, as well as their concentration effects and facet preferences. [Preview Abstract] |
Thursday, March 19, 2009 3:42PM - 3:54PM |
X39.00005: Biomineralization of a Self-Assembled Extracellular Matrix for Bone Engineering Yizhi Meng, Yi-Xian Qin, Nadine Pernodet, Xiaolan Ba, Miriam Rafailovich, Elaine DiMasi The mineralization of extracellular matrix (ECM) proteins is an important process in need of new experimental approaches. We present a study of two subclones of MC3T3-E1 osteoblast-like cells, one which mineralizes and one which does not. Using atomic force microscopy we measure the ECM protein fiber morphology and the elastic modulus, which changes as biomineralization proceeds. The non-mineralizing subclone undergoes less remodeling of the ECM over the same development period, compared to the mineralizing subclone. By using synchrotron grazing-incidence x-ray diffraction along with optical and electron microscopy, the development of hydroxyapatite crystals is followed. Cells are shown to mineralize only when an adequately structured ECM is present. Confocal light microscopy indicates that actin restructuring is correlated with mineralization. Correct and complete development of the ECM network, which can be interrupted either by using the non-mineralizing cell line or by culturing cells on an inhospitable substrate, is necessary for osteoblasts to mineralize. We discuss implications for bone biomineralization and for the development of implant materials. [Preview Abstract] |
Thursday, March 19, 2009 3:54PM - 4:06PM |
X39.00006: Peptide Probe for Crystalline Hydroxyapatite: In Situ Detection of Biomineralization Marcus Cicerone, Matthew Becker, Carl Simon, Kaushik Chatterjee While cells template mineralization in vitro and in vivo, specific detection strategies that impart chemical and structural information on this process have proven elusive. Recently we have developed an in situ based peptide probe via phage display methods that is specific to crystalline hydroxyapatite (HA). We are using this in fluorescence based assays to characterize mineralization. One application being explored is the screening of tissue engineering scaffolds for their ability to support osteogenesis. Specifically, osteoblasts are being cultured in hydrogel scaffolds possessing property gradients to provide a test bed for the HA peptide probe. Hydrogel properties that support osteogenesis and HA deposition will be identified using the probe to demonstrate its utility in optimizing design of tissue scaffolds. [Preview Abstract] |
Thursday, March 19, 2009 4:06PM - 4:18PM |
X39.00007: Superoxide dismutase activity of Cu-bound prion protein Miroslav Hodak, Wenchang Lu, Jerry Bernholc Misfolding of the prion protein, PrP, has been linked to a group of neurodegenerative diseases, including the mad cow disease in cattle and the Creutzfeldt-Jakob disease in humans. The normal function of PrP is still unknown, but it was found that the PrP can efficiently bind Cu(II) ions. Early experiments suggested that Cu-PrP complex possesses significant superoxide dismutase (SOD) activity, but later experiments failed to confirm it and at present this issue remains unresolved. Using a recently developed hybrid DFT/DFT method, which combines Kohn-Sham DFT for the solute and its first solvation shells with orbital-free DFT for the remainder of the solvent, we have investigated SOD activity of PrP. The PrP is capable of incorporating Cu(II) ions in several binding modes and our calculations find that each mode has a different SOD activity. The highest activity found is comparable to those of well-known SOD proteins, suggesting that the conflicting experimental results may be due to different bindings of Cu(II) in those experiments. [Preview Abstract] |
Thursday, March 19, 2009 4:18PM - 4:54PM |
X39.00008: Mechanisms of function by AF(G)Ps in ice crystal growth prevention, modification and recrystallization Invited Speaker: Antifreeze activity by proteins and glycoproteins is a form of biomineralization process. Contrasting to covalent or ionic crystals, these hydrogen-bonded molecular crystals (ice) have much weaker crystalline bonding energy profiles. Thus, when interacting with proteins that are equally easy to change in conformation by variations of the hydrophilic/hydrophobic environment, some unique findings are suggestive that local conditions play significant roles in the activity level of these proteins or glycoproteins as they affect ice crystal growth, modification and recrystallization. We review some of these experiments and provide ideas for their functioning mechanisms. [Preview Abstract] |
Thursday, March 19, 2009 4:54PM - 5:06PM |
X39.00009: Atomistic Investigation of Cu-Induced Misfolding in the Onset of Parkinson's Disease Francis Rose, Miroslav Hodak, Jerry Bernholc A nucleation mechanism for the misfolding of $\alpha$-synuclein, the protein implicated in Parkinson's Disease (PD), is investigated using computer simulations. Through a combination of ab initio and classical simulation techniques, the conformational evolution of copper-ion-initiated misfolding of $\alpha$-synuclein is determined. Based on these investigations and available experimental evidence, an atomistic model detailing the nucleation-initiated pathogenesis of PD is proposed. Once misfolded, the proteins can assemble into fibrils, the primary structural components of the deleterious PD plaques. Our model identifies a process of structural modifications to an initially unfolded $\alpha$-synuclein that results in a partially folded intermediate with a well defined nucleation site as a precursor to the fully misfolded protein. The identified pathway can enable studies of reversal mechanisms and inhibitory agents, potentially leading to the development of effective therapies. [Preview Abstract] |
Thursday, March 19, 2009 5:06PM - 5:18PM |
X39.00010: ESR Spectroscopy Provides Direct Evidence of Cu$^{2+}$ Coordination by Three Histidine Residues in A$\beta _{1-16}$ Byong-kyu Shin, Sunil Saxena We provide direct evidence that all three histidine residues in amyloid-$\beta _{1-16}$ (A$\beta _{1-16})$ coordinate to Cu$^{2+}$. In our approach, we generate three A$\beta _{1-16}$ analogues, in each of which a selected histidine residue is isotopically enriched with $^{15}$N. Pulsed electron spin resonance (ESR) experiments such as electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) clearly show that each of the three histidine imidazole rings at position 6, 13, and 14 in A$\beta _{1-16}$ binds to Cu$^{2+}$ as each of the three Cu$^{2+}$--$^{15}$N-labeled A$\beta _{1-16 }$complexes displays ESEEM and HYSCORE spectra which are distinctively different from those of the Cu$^{2+}$--nonlabeled A$\beta _{1-16}$ complex. The method employed here does not require either chemical side-chain modification or amino acid residue replacement, each of which is traditionally used to determine whether an amino acid residue in a protein binds to a metal ion. We also find that the histidine coordination in A$\beta _{1-16}$ is independent of the Cu$^{2+}$-to-peptide ratio, which is in contrast to the case of A$\beta _{1-40}$. The ESR results suggest tight binding between the histidine residues and the Cu$^{2+}$ ion, which is likely the reason of the high binding affinity of A$\beta $ peptide to Cu$^{2+}$. [Preview Abstract] |
Thursday, March 19, 2009 5:18PM - 5:30PM |
X39.00011: Force Spectroscopy of Iron in Nitrosylated Hemes J.T. Sage, A. Barabanschikov, W. Zeng, N.J. Silvernail, W.R. Scheidt Nitric oxide (NO) regulates important physiological processes by interacting with the Fe atom in heme proteins. We investigate the effect of NO binding on the local structure and dynamics of $^{57}$Fe by determining its vibrational density of states (VDOS), both experimentally, using nuclear resonance vibrational spectroscopy (NRVS) and computationally, using density functional theory (DFT). All Fe–-ligand modes contribute to the VDOS, which provides uniquely quantitative information on the frequency, amplitude, and direction of the Fe motion. The VDOS also yields an experimental value for the stiffness, an effective force constant that probes nearest-neighbor interactions by measuring the force required to displace the Fe with the surrounding atoms fixed. Although vibrational mixing between Fe–-NO stretching and FeNO bending character complicates structural interpretations of FeNO vibrations observed near 450 and 560 cm$^{-1}$, we find that the former mode contributes more strongly to the stiffness, indicating its sensitivity to the strength of the Fe-–N bond. Comparison with DFT predictions identifies a feature observed near 130 cm$^{-1}$ in the VDOS of nitrosylated myoglobin as a vibration of the covalent link to the protein. We find that NO binding alters the interaction of the heme Fe with its local environment, and may facilitate NO recognition by heme proteins. [Preview Abstract] |
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