Energy Minimization Model Research Articles

Structure and Assembly of the Heterotrimeric and Homotrimeric C-Propeptides of Type I Collagen: Significance of the α2(I) Chain

Assembly of the type I procollagen molecule begins with interactions among the C-pro alpha1(I) and C-pro alpha2(I) domains. The C-propeptide domains themselves have subdomains of distinct structures. The important questions are where chain association begins and the basis of the chain selectivity which leads to the preferential formation of the [C-pro alpha1(I)]2[C-pro alpha2(I)] heterotrimer. These questions are addressed by energy minimization modeling of the individual C-propeptide structures, study of their docking interactions, and comparison of the heterotrimeric and homotrimeric C-pro structures and stability. The comparisons show the remarkable impact of the C-pro alpha2 chain on the structure of the assembled trimeric C-propeptide. In the modeling, the three chains were anchored and registered by a short C-terminal collagen triple-helical segment followed by the C-telopeptides in their docked conformation, and then the remaining C-propeptide chains were allowed to interact and dock. Surprisingly, propeptide trimerization did not proceed through the previously proposed N-terminal "oligomerization domain" of the C-propeptide [McAlinden et al. (2003) J. Biol. Chem. 278, 42200] but rather in the most C-terminal domains of type I procollagen chains. Molecular dynamics showed heterotrimer assembly to begin with dimer formation between globular G2alpha2 and the G2alpha1(2) domains followed by trimerization at the G1 domains. Assembly initiation in the putative oligomerization coiled-coil domain is not possible because of the Pro residues at positions 3, 7, and 11 at the N-terminus of the alpha2 C-propeptide chain. To confirm the computations and proposed assembly pathway, the G2alpha1 and G2alpha2 domains were prepared recombinantly as the maltose binding protein constructs, and their interactions were studied by dynamic light scattering and gel filtration chromatography. Under the conditions examined MBP remained as monomer, MBP-G2alpha1 and MBP-G2alpha2 alone formed dimers, but a 2:1 mixture of MBP-G2alpha1 and MBP-G2alpha2 favored trimer formation. Thus, the C-terminal globular domains (G2) of the type I collagen C-propeptides play a crucial role in the initiation of intermolecular assembly and heterotrimer selectivity.

Water content of ambient aerosol during the Pittsburgh Air Quality Study

The aerosol water content and volumetric growth factors of fine particulate matter were measured during July–August 2001 and January–June 2002 in an urban park about 6 km from downtown Pittsburgh, Pennsylvania. Most of the aerosol during the study was transported to the region from other areas, and its composition and concentration were characteristic of the regional particulate matter in the northeastern United States. During the summer months the ambient aerosol practically always contained water even when the relative humidity (RH) was as low as 30%. In contrast, during the winter the aerosol was dry below 60% RH. The spring months were characterized by a transitional behavior between these two states. The observed seasonal behavior can be explained by the aerosol acidity. The summer aerosol was acidic and retained water at low RH. The winter aerosol was neutral and became wet when the relative humidity reached the deliquescence point of ammonium nitrate. The observations during July 2001 were compared with the predictions of the thermodynamic Gibbs Free Energy Minimization (GFEMN) model and the aerosol inorganics model (AIM), neglecting the organic aerosol contribution to water absorption. The models under‐predicted water concentrations by about 35%, but no clear correlation between organic mass and the excess water was observed. On average, the contribution of the organics to water absorption appeared to be higher during the afternoon hours and when the aerosol was presumably more oxidized.

Energy Minimization Model for Pattern Extraction of the Movement Behavior of Animals

Recently, patterning and analyzing complex data of behaviors of animals in response to external stimuli such as toxic chemicals has become focus of attentions. In this paper, an energy minimization model to extract the features of response behavior of chironomids under toxic treatment is proposed, which is applied on the image of velocity vectors. The model is based on the improved active contour model and the variation of the energy values produced by the evolving active contour. We attempt to implement an adaptive computational method to characterize the changes in response behaviors of chironomids after treatment with an insecticide, diazinon. Active contour is formed around each collection of velocities to gradually evolve to find the optimal boundaries of velocity collections through processes of energy minimization. The energy minimization model effectively reveals characteristic patterns of behavior for the treatment versus no treatment, and identifies changes in behavioral states as the time progressed.

Inhibitory effects of flavonoids on the reduction of progesterone to 20α-hydroxyprogesterone in rat liver

The first aim of this study is to characterize the reduction of progesterone in rat liver. Progesterone was mainly reduced to 20α-hydroxyprogesterone in the cytosolic fraction of rat liver. The amount of 20α-hydroxyprogesterone formed from progesterone in the cytosolic fraction was significantly larger in the males than in the females and this enzyme reaction proceeded not only in the presence of NADPH, but also in the presence of NADH. Furthermore, we attempted to evaluate the inhibitory effects of 15 flavonoids on the NADPH-dependent reduction of progesterone to 20α-hydroxyprogesterone in liver cytosol of male rats. The order of the inhibitory potencies was luteolin > apigenin > quercetin > myricetin = fisetin = kaempferol. Other flavonoids exhibited lower inhibitory potencies. Energy-minimized molecular models demonstrated that a planar benzopyrone ring (A and C rings) with a coplanar phenyl ring (B ring) is a structural characteristic determining the inhibitory effects of flavonoids other than isoflavones.

Image Decomposition Using Total Variation and div(BMO)

This paper is devoted to the decomposition of an image f into u + v, with u a piecewise-smooth or "cartoon" component, and v an oscillatory component (texture or noise), in a variational approach. Meyer [Oscillating Patterns in Image Processing and Nonlinear Evolution Equations, Univ. Lecture Ser. 22, AMS, Providence, RI, 2001] proposed refinements of the total variation model (Rudin, Osher, and Fatemi [Phys. D, 60 (1992), pp. 259--268]) that better represent the oscillatory part v: the spaces of generalized functions $G = {\rm div}(L^\infty)$ and $F = {\rm div}(BMO)$ (this last space arises in the study of Navier--Stokes equations; see Koch and Tataru [Adv. Math., 157 (2001), pp. 22--35]) have been proposed to model v, instead of the standard L2 space, while keeping u a function of bounded variation. Mumford and Gidas [Quart. Appl. Math., 59 (2001), pp. 85--111] also show that natural images can be seen as samples of scale-invariant probability distributions that are supported on distributions only and not on sets of functions. However, there is no simple solution to obtain in practice such decompositions f = u + v when working with G or F. In earlier works [L. Vese and S. Osher, J. Sci. Comput., 19 (2003), pp. 553--572], [L. A. Vese and S. J. Osher, J. Math. Imaging Vision, 20 (2004), pp. 7--18], [S. Osher, A. Solé, and L. Vese, Multiscale Model. Simul., 1 (2003), pp. 349--370], the authors have proposed approximations to the (BV,G) decomposition model, where the $L^\infty$ space has been substituted by Lp, $1 \leq p < \infty$. In the present paper, we introduce energy minimization models to compute (BV,F) decompositions, and as a by-product we also introduce a simple model to realize the (BV,G) decomposition. In particular, we investigate several methods for the computation of the BMO norm of a function in practice. Theoretical, experimental results and comparisons to validate the proposed new methods are presented.

Structural Characterization of Modular Supramolecular Architectures in Solution

Structures of modular supramolecular architectures consisting of a hexameric, diphenylethyne-linked porphyrin macrocyclic array and the corresponding host-guest complex formed by inclusion of a tripyridyl guest molecule were characterized in solution using high-angle X-ray scattering. Scattering measurements made to 6 A resolution coupled with pair distance function (PDF) analyses demonstrated that (1) the porphyrin architectures are not rigid but are distributed across a conformational ensemble with a mean diameter that is 1.5 A shorter than the diameter of a symmetric, energy-minimized model structure, (2) the conformational envelope has limits of 3 A positional dispersion and full rotational freedom for all six porphyrin groups, and (3) insertion of the tripyridyl guest molecule expands the diameter of the host conformer by 0.6 A and decreases the configurational dispersion by approximately 2-fold. These results validate the molecular design, provide a new measure of conformational ensembles in solution that cannot be obtained by other techniques, and establish a structural basis for understanding the photophysical and guest-hosting functions of the hexameric porphyrin architectures in liquids.

Analysis on Multiresolution Mosaic Images

Image mosaicing is the act of combining two or more images and is used in many applications in computer vision, image processing, and computer graphics. It aims to combine images such that no obstructive boundaries exist around overlapped regions and to create a mosaic image that exhibits as little distortion as possible from the original images. In the proposed technique, the to-be-combined images are first projected into wavelet subspaces. The images projected into the same wavelet space are then blended. Our blending function is derived from an energy minimization model which balances the smoothness around the overlapped region and the fidelity of the blended image to the original images. Experiment results and subjective comparison with other methods are given.

행동궤적의 패턴 분류를 위한 에너지 최소화 모델

본 논문은 외부 자극에 대한 생물 행동의 복잡하고 다양한 특징들을 추출하고 분석하기 위한 방법을 제안한다. 이를 위해 물 속 생물인 깔따구의 행동궤적으로부터 얻어 낸 속도 벡터의 위상영상에 적응적이고 수리적인 방법인 에너지 최소화 모델을 적용한다. 즉, 다이아지논이라는 약물이 처리되기 전과 후의 깔따구의 행동궤적의 특징을 위상영상으로부터 찾아내어 행동 패턴을 분류하고 이 약물에 대한 깔따구의 적응적 행동 특징을 추출하는 것이다. 특징추출을 위해 도입한 방법은 T. Chan과 L. Vese에 의해 제안된 개선 Active Contour 모델에 근거한 것으로 Active Contour를 진화시키는 과정에서 생성되는 에너지함수 값의 변화를 이용한 것이다. Active Contour 모델이란 주어진 영상에 놓인 커브를 그 커브에 의해 분할된 부분영상들의 에너지 값들의 합을 최소화하는 방향으로 변화하게 함으로써 영상 내 객체의 경계를 찾는 영상분할 방법이다. 깔따구의 행동궤적 데이터는 CCD 카메라를 통해 0.25초 간격으로 약물을 처리하기 전과 후 4일 간을 관찰하여 획득하고, 이 행동궤적 데이터에서 행동의 특징 요소가 되는 속도벡터 성분을 15-20분 간격으로 추출하여 위상영상을 만든다. 그리고 이 위상영상에 Active Contour를 적용함으로써 시간에 따라 감소하는 에너지 함수 값의 그래프에서 구해진 기울기 변화에 대한 수리적 계산과 분석을 통해 깔따구 행동궤적의 특징을 찾고 행동 패턴을 분류한다. 또한, 에너지 최소화 모델은 약물 처리된 깔따구의 반응적인 행동이 이에 적응하고 있음을 효과적으로 보여준다. In order to extract and analyze complex features of the behavior of animals in response to external stimuli such as toxic chemicals, we implemented an adaptive computational method to characterize changes in the behavior of chironomids in response to treatment with the insecticide, diazinon. In this paper, we propose an energy minimization model to extract the features of response behavior of chironomids under toxic treatment, which is applied on the image of velocity vectors. It is based on the improved active contour model and the variations of the energy functional, which are produced by the evolving active contour. The movement tracks of individual chironomid larvae were continuously measured in 0.25 second intervals during the survey period of 4 days before and after the treatment. Velocity on each sample track at 0.25 second intervals was collected in 15-20 minute periods and was subsequently checked to effectively reveal behavioral states of the specimens tested. Active contour was formed around each collection of velocities to gradually evolve to find the optimal boundaries of velocity collections through processes of energy minimization. The active contour which is improved by T. Chan and L. Vese is used in this paper. The energy minimization model effectively revealed characteristic patterns of behavior for the treatment versus no treatment, and identified changes in behavioral states .is the time progressed.

Biophysical characterization and molecular modeling of the coordinative-intercalative DNA monoadduct of a platinum-acridinylthiourea agent in a site-specifically modified dodecamer

The guanine- N7 monoadduct of [Pt(en)Cl(ACRAMTU)](NO(3))(2) (PT-ACRAMTU; en=ethane-1,2-diamine, ACRAMTU=1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea), a dual metalating/intercalating cytotoxic agent, was generated in a double-stranded dodecamer, d(CCTCTCG*TCTCC/GGAGACGAGAGG) (III*), and isolated by preparative reverse-phase high-performance liquid chromatography (HPLC). The adduct was characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), circular-dichroism spectropolarimetry (CD), UV-melting curves, and NMR spectroscopy. In addition, a molecular mechanics/restrained molecular dynamics (MM/rMD) study was performed for this adduct using the AMBER force field. Monoadduction of the sequence leads to a pronounced increase in melting temperature, Delta T(m)= T(m)(III*)- T(m)(III)=9.7 degrees C. Because there is complete enthalpy-entropy compensation, binding occurs without noticeable thermodynamic destabilization. This feature and the CD (induced-ligand circular dichroism) and NMR (upfield shifts of aromatic acridine proton signals) data are indicative of a unique, nondenaturing dual-binding mode that involves partial intercalation of the acridine chromophore. An energy-minimized AMBER model ofIII* demonstrates that platination of G7- N7 of guanine in the major groove and partial insertion of the acridine moiety into the C6G19/G7C18 base step on the 5' face of the modified purine base is feasible and supportive of the experimental results. Differences in the biophysical properties betweenIII* and duplexes containing adducts of the clinical-drug cisplatin are outlined, and possible biological consequences are discussed.

Structural characterization of a pentacene monolayer on an amorphous SiO2 substrate with grazing incidence x-ray diffraction.

Grazing incidence X-ray diffraction reveals that a pentacene monolayer, grown on an amorphous SiO2 substrate that is commonly used as a dielectric layer in organic thin film transistors (OTFTs), is crystalline. A preliminary energy-minimized model of the monolayer, based on the GIXD data, reveals that the pentacene molecules adopt a herringbone arrangement with their long axes tilted slightly from the substrate normal. Although this arrangement resembles the general packing features of the (001) layer in single crystals of bulk pentacene, the monolayer lattice parameters and crystal structure differ from those of the bulk. Because carrier transport in pentacene OTFTs is presumed to occur in the semiconductor layers near the dielectric interface, the discovery of a crystalline monolayer structure on amorphous SiO2 has important implications for transport in OTFTs.

Structure of a Hydroxyproline (Hyp)-Arabinogalactan Polysaccharide from Repetitive Ala-Hyp Expressed in Transgenic Nicotiana tabacum

A synthetic gene encoding the fusion protein (Ala-Hyp)(51)-enhanced green fluorescent protein expressed in Nicotiana tabacum cells produced a fusion glycoprotein with all proline residues hydroxylated and substituted with an arabinogalactan polysaccharide. Alkaline hydrolysis of the fusion glycoprotein yielded a population of hydroxyproline (Hyp)-arabinogalactan polysaccharides ranging in size from 13 to 26 saccharide residues/Hyp, with a median size of 15-17 residues. We isolated a 15-residue Hyp-arabinogalactan for structure determination by sugar analyses and one- and two-dimensional nuclear magnetic resonance techniques that provided the assignment of proton and carbon signals of a small polysaccharide O-linked to the hydroxyl group of Hyp. The polysaccharide consisted of a 1,3-linked beta-D-Galp backbone with a single 1,6-linked beta-D-Galp "kink." The backbone had two side chains of Galp substituted at position 3 with an arabinose di- or trisaccharide and at position 6 with glucuronic acid or rhamnosyl glucuronic acid. Energy-minimized space-filling molecular models showed hydrogen bonding within polysaccharides attached to repetitive Ala-Hyp and also between polysaccharides and the peptide backbone. Polysaccharides distorted the peptide Ramachandran angles consistent with the circular dichroic spectra of isolated (Ala-Hyp)(51) and its reversion to a polyproline II-like helix after deglycosylation. This first complete structure of a Hyp-arabinogalactan polysaccharide shows that computer-based molecular modeling of Hyp-rich glycoproteins is now feasible and supports the suggestion that small repetitive subunits comprise larger arabinogalactan polysaccharides.

Normal coordinate structural decomposition of the heme distortions of hemoglobin in various quaternary states and bound to allosteric effectors.

The distortions of the alpha1, alpha2, beta1, and beta2 hemes of human hemoglobin (HbA) in various quaternary states and as affected by the presence of allosteric effectors was investigated by subjecting CHARMM energy-minimized models to normal coordinate structural decomposition (NSD) analysis. NSD was applied to the individual hemes extracted from the R, T, and R2-state models of HbA and to HbA bound to DPG and to IHP. Overall, NSD results are indicative of characteristic distortions, not only for the hemes of the different HbA quaternary states, but also for the hemes of the HbA models bound to allosteric effectors. Comparing the distortions of the inequivalent alpha and beta hemes in T-state HbA, we show good correlation between NSD and the experimentally observed low-frequency nu52 (Eg) and gamma7 (A2u) modes reported in the literature for alpha and beta HbA hemes while noting substantial differences between these types for B2u and B1u distortions. For the R2 hemes, NSD yields heme distortions that are more comparable to those of the R-state, especially in magnitude. However, the R2 hemes do not exhibit inequivalence of alpha and beta heme distortions, a result that may contribute to an understanding of the functional importance of this state. Relative to T-state heme distortions, NSD results on the effector-bound hemes show that tertiary changes induced in T-state HbA as a result of binding DPG and IHP drastically affect heme distortions. In the alpha hemes extracted from the HbA-DPG model, most noteworthy are the increased wav(x) and wav(y) distortions and enhancement of ruf and dom deformations. In the beta hemes, the wav(y) is the most affected distortion with increase in sad. The NSD results are also different for the hemes of the HbA-IHP model, in that the beta sad and ruf deformations are more enhanced with increase of doming in the alpha hemes. Our results describe the effect of the subtle protein-induced changes on the nonplanarity of the HbA hemes that may play a role in the regulation of their oxygen affinity.

Structural analysis of the DNA-binding domain of alternatively spliced steroid receptors.

We have generated 24 DNA-binding domain structure models of alternatively spliced or mutated steroid receptor variants by homology-based modeling. Members of the steroid receptor family dispose of a DNA-binding domain which is built from two zinc fingers with a preserved sequence homology of about 90%. Data from crystallographic analysis of the glucocorticoid receptor DNA-binding domain are therefore appropriate to serve as a template structure. We inserted or deleted amino acid residues in order to study the structural details of the glucocorticoid, mineralocorticoid, and androgen receptor splice variants. The receptor variants are created by QUANTA- and MODELLER-based modeling. Subsequently, the resulting energy-minimized models were compared with each other and with the wild-type receptor respectively. A prediction for the receptor function based mainly on the preservation or destruction of secondary structures has been carried out. The simulations showed that amino acid insertions of one, four or nine additional residues of existing steroid receptor splice variants were tolerated without destruction of the secondary structure. In contrast, a deletion of four amino acids at the splice site junction leads to modifications in the secondary structure of the DNA-recognition helix which apparently disturb the receptor-DNA interaction. Furthermore, an insertion of 23 amino acid residues between the zinc finger of the androgen receptor leads to a large loop with an additional alpha-helical structure which seems to disconnect a specific contact from its hormone response element. Thereafter, the prediction of receptor function based on the molecular models was compared with the available experimental results from the in vitro function tests. We obtained a close correspondence between the molecular modeling-based predictions and the conclusions of receptor function drawn from in vitro studies.

Reversal mechanism in permanent magnetic materials

The phenomenon of magnetization reversal in a hard magnet is studied analytically within a framework of a simplified energy minimization model for a two grain system. The transition region (a domain wall-like moment structure) is found to play a crucial role in nucleating a reversed domain and in the subsequent formation of a domain wall near the grain boundary. This is shown to result in a considerable reduction in coercivity. Temperature effects are taken into account by considering thermally activated jumps over energy barriers that hinder the motion of the transition region. We found that thermal fluctuations at room temperature can substantially reduce coercivity provided the grains are sufficiently small.

Modeling of the Ion Exchange in Pulp Suspensions by Gibbs Energy Minimization

The ion exchange processes, which occur when two compartments of aqueous solutions separated by a semipermeable interface are placed in aqueous electrolyte solutions, were modeled using the multiphase Gibbs energy-minimization method. The Gibbs energy-minimization technique was applied for the ion-exchange system consisting of pulp fibers and the surrounding aqueous bulk solution. In such a system, the anionic acid groups inside the fibers cause an uneven distribution of ionic species between the solution within the fiber walls and the solution external to the fibers. The method was tested with four cation concentrations, which are naturally present in the fibers and whose partitioning between the fiber phase and the external solution phase has been described earlier. Although the Donnan distribution constant is not explicitly calculated in the Gibbs energy-minimization model, the results are consistent with the Donnan equilibrium theory. With the Gibbs energy-minimization multiphase model, the formation of solid precipitates can also be calculated.

Mechanism of chitosanase-oligosaccharide interaction: subsite structure of Streptomyces sp. N174 chitosanase and the role of Asp57 carboxylate.

We have investigated the mechanism of the interaction of Streptomyces sp. N174 chitosanase with glucosamine hexasaccharide [(GlcN)(6)] by site-directed mutagenesis, thermal unfolding, and (GlcN)(6) digestion experiments, followed by theoretical calculations. From the energy-minimized model of the chitosanase-(GlcN)(6) complex structure (Marcotte et al., 1996), Asp57, which is present in all known chitosanases, was proposed to be one of the amino acid residues that interacts with the oligosaccharide substrate. The chitosanase gene was mutated at Asp57 to Asn (D57N) and Ala (D57A), and the relative activities of the mutated chitosanases were found to be 72 and 0.5% of that of the wild type, respectively. The increase in the transition temperature of thermal unfolding (T(m)), usually observed upon the addition of (GlcN)(n) to chitosanase mutants unaffected in terms of substrate binding, was considerably suppressed in the D57A mutant. These data suggest that Asp57 is important for substrate binding. The experimental time-courses of [(GlcN)(6)] degradation were analyzed by a theoretical model in order to obtain the binding free energy values of the individual subsites of the chitosanases. A (-3, -2, -1, +1, +2, +3) subsite model agreed best with the experimental data. This analysis also indicated that the mutation of Asp57 affects substrate affinity at subsite (-2), suggesting that Asp57 most likely participates in the substrate binding at this subsite.

Heat-flux induced changes to multicrystallineD2surfaces

Phase-shifting interferometry reveals that a heat flux normal to the gas-solid interface reduces the surface roughness of thick (10--300 \ensuremath{\mu}m) multicrystalline ${\mathrm{D}}_{2}$ films. The initial roughness, caused by misaligned crystals and grain boundaries produced during the initial random nucleation and rapid crystal growth used in the experiment decreases with increasing heat flux. A simple energy minimization model quantitatively explains the functional relationship between surface roughness and heat flux.

Flexibility of the Kir6.2 inward rectifier K + channel pore

Interactions of sulfhydryl reagents with introduced cysteines in the pore-forming (Kir6.2) subunits of the K(ATP) channel were examined. 2-Aminoethyl methanethiosulfonate (MTSEA(+)) failed to modify Cd(2+)-insensitive control-Kir6.2 channels, but rapidly and irreversibly modified Kir6.2[L164C] (L164C) channels. Although a single Cd(2+) ion is coordinated by L164C, four MTSEA(+) "hits" can occur, each sequentially reducing the single-channel current. A dimeric fusion of control-Kir6.2 and L164C subunits generates Cd(2+)-insensitive channels, confirming that at least three cysteines are required for coordination, but MTSEA(+) modification of the dimer occurs in two hits. L164C channels were not modified by bromotrimethyl ammoniumbimane (qBBr(+)), even though qBBr(+) caused voltage-dependent block (as opposed to modification) that was comparable to that of MTSEA(+) or 3-(triethylammonium)propyl methanethiosulfonate (MTSPTrEA(+)), implying that qBBr(+) can also enter the inner cavity but does not modify L164C residues. The Kir channel pore structure was modeled by homology with the KcsA crystal structure. A stable conformation optimally places the four L164C side chains for coordination of a single Cd(2+) ion. Modification of these cysteines by up to four MTSEA(+) (or three MTSPTrEA(+), or two qBBr(+)) does not require widening of the cavity to accommodate the derivatives within it. However, like the KcsA crystal structure, the energy-minimized model shows a narrowing at the inner entrance, and in the Kir6.2 model this narrowing excludes all ions. To allow entry of ions as large as MTSPTrEA(+) or qBBr(+), the entrance must widen to >8 A, but this widening is readily accomplished by minimal M2 helix motion and side-chain rearrangement.

The Effect of Mutating Arginine-469 on the Substrate Binding and Refolding Activities of 70-kDa Heat Shock Cognate Protein

On the basis of the X-ray structure of DnaK, we obtained an energy-minimized model for the C-terminal domain of rat 70-kDa heat shock cognate protein (hsc70). The model suggests that Arg-469 may play an important role in maintaining the substrate-bound conformation of hsc70. To verify this hypothesis, we substituted cysteine for Arg-469 and generated the hsc70(R469C) mutant. Compared to the wild-type hsc70, the mutant was more accessible to cleavage by endopeptidase Lys-C, implying that the overall structure of hsc70(R469C) is relatively loose. Moreover, hsc70(R469C) did not form tightly associated complexes with S-carboxymethyl-α-lactalbumin, an unfolded protein. The amount of heptapeptide FYQLALT bound to hsc70(R469C) was also decreased as determined by gel filtration. Thus, the affinity of hsc70(R469C) for polypeptide substrates is reduced. In the presence of DnaJ, the capability of hsc70(R469C) to refold the denatured luciferase was decreased by 50%. Therefore, for hsc70, reduction in affinity for substrates may affect its DnaJ-dependent refolding activity.

Synthesis and conformational features of human salivary mucin C-terminal derived peptide epitope carrying Thomsen-Friedenreich antigen: implications for its role in self-association.

The conformational features of a chemically synthesized 23-residue glycopeptide construct (II) carrying Gal-beta-(1,3)-alpha-GalNAc and its deglycosylated counterpart (I; Gal: galactose; GalNAc: N-acetyl galactosamine) derived from the C-terminal domain of human salivary mucin (MUC7) were investigated using CD spectroscopy as well as molecular dynamic simulation studies. The corresponding deglycosylated peptide (I) was essentially used to compare and study the influence of the sugar moiety on peptide backbone conformation. CD measurements in aqueous medium revealed that the apopeptide (I) contains significant populations of beta-strand conformation while the glycopeptide (II) possess, partly, helical structure. This transition in the secondary structure upon glycosylation from beta-strand to helical conformation clearly demonstrates that the carbohydrate moiety exerts significant influence on the peptide backbone. On the other hand, upon titrating structure stabilizing organic cosolvent, trifluoroethanol (TFE), both the peptides showed pronounced helical structure. However, the propensity for helical structure formation is less pronounced in glycopeptide compared to apopeptide suggesting that the bulky carbohydrate moiety possibly posing steric hindrance to the formation of TFE-induced secondary structure in II. Energy-minimized molecular model for the glycopeptide revealed that the preferred helix conformation in aqueous medium appears to be stabilized by the hydrogen-bonded salt bridge like interaction between carbohydrate --OH and Lys-10 side--N(+)H(3) group. Size exclusion chromatographic analysis of both (glyco)peptides I and II showed an apparent Kd of 2.3 and 0.52 microM, respectively, indicating that glycopeptide (II) has greater tendency for self-association. Due to high amphipathic character as well as due to the presence of a leucine zipper motif ( approximately LLYMKNLL approximately ), which is known to increase the stability at the coiled-coil interface via hydrophobic interactions, we propose therefore that, this domain could be one of the key elements involved in the self-association of intact MUC7 in vivo. Profound conformational effects governed by glycosylation exemplified herein could have implications in determining structure-function relationships of mucin glycoproteins.