A restraint molecular dynamics and simulated annealing approach for protein homology modeling utilizing mean angles

BACKGROUND: We have developed the program PERMOL for semi-automated homology modeling of proteins. It is based on restrained molecular dynamics using a simulated annealing protocol in torsion angle space. As main restraints defining the optimal local geometry of the structure weighted mean dihedral angles and their standard deviations are used which are calculated with an algorithm described earlier by Döker et al. (1999, BBRC, 257, 348–350). The overall long-range contacts are established via a small number of distance restraints between atoms involved in hydrogen bonds and backbone atoms of conserved residues. Employing the restraints generated by PERMOL three-dimensional structures are obtained using standard molecular dynamics programs such as DYANA or CNS. RESULTS: To test this modeling approach it has been used for predicting the structure of the histidine-containing phosphocarrier protein HPr from E. coli and the structure of the human peroxisome proliferator activated receptor γ (Ppar γ). The divergence between the modeled HPr and the previously determined X-ray structure was comparable to the divergence between the X-ray structure and the published NMR structure. The modeled structure of Ppar γ was also very close to the previously solved X-ray structure with an RMSD of 0.262 nm for the backbone atoms. CONCLUSION: In summary, we present a new method for homology modeling capable of producing high-quality structure models. An advantage of the method is that it can be used in combination with incomplete NMR data to obtain reasonable structure models in accordance with the experimental data

NMR derived changes of lipoprotein particle concentrations related to impaired fasting glucose, impaired glucose tolerance, or manifest type 2 diabetes mellitus

Background Type 2 diabetes mellitus (T2D) and corresponding borderline states, impaired fasting glucose (IFG) and/or glucose tolerance (IGT), are associated with dyslipoproteinemia. It is important to distinguish between factors that cause T2D and that are the direct result of T2D. Methods The lipoprotein subclass patterns of blood donors with IFG, IGT, with IFG combined with IGT, and T2D are analyzed by nuclear magnetic resonance (NMR) spectroscopy. The development of lipoprotein patterns with time is investigated by using samples retained for an average period of 6 years. In total 595 blood donors are classified by oral glucose tolerance test (oGTT) and their glycosylated hemoglobin (HbA1c) concentrations. Concentrations of lipoprotein particles of 15 different subclasses are analyzed in the 10,921 NMR spectra recorded under fasting and non-fasting conditions. The subjects are assumed healthy according to the strict regulations for blood donors before performing the oGTT. Results Under fasting conditions manifest T2D exhibits a significant concentration increase of the smallest HDL particles (HDL A) combined with a decrease in all other HDL subclasses. In contrast to other studies reviewed in this paper, a general concentration decrease of all LDL particles is observed that is most prominent for the smallest LDL particles (LDL A). Under normal nutritional conditions a large, significant increase of the concentrations of VLDL and chylomicrons is observed for all groups with IFG and/or IGT and most prominently for manifest T2D. As we show it is possible to obtain an estimate of the concentrations of the apolipoproteins Apo-A1, Apo-B100, and Apo-B48 from the NMR data. In the actual study cohort, under fasting conditions the concentrations of the lipoproteins are not increased significantly in T2D, under non-fasting conditions only Apo-B48 increases significantly. Conclusion In contrast to other studies, in our cohort of “healthy” blood donors the T2D associated dyslipoproteinemia does not change the total concentrations of the lipoprotein particles produced in the liver under fasting and non-fasting conditions significantly but only their subclass distributions. Compared to the control group, under non-fasting conditions participants with IGT and IFG or T2D show a substantial increase of plasma concentrations of those lipoproteins that are produced in the intestinal tract. The intestinal insulin resistance becomes strongly observable

Intrinsic disorder in the common N-terminus of human adenovirus 5 E1B-55K and its related E1BN proteins indicated by studies on E1B-93R

AbstractThe E1B transcription unit of human adenovirus encodes at least five different proteins generated by alternative splicing of a common E1B precursor mRNA. E1B-156R, -93R and -84R contain individual carboxy termini but share a common amino terminus. To acquire data on the structure of the amino terminus we performed biophysical analyses on E1B-93R. We show that E1B-93R is mostly unstructured and fulfills the criteria of an intrinsically disordered protein (IDP). The intrinsic disorder in the amino terminus of these proteins is evolutionary conserved in all seven human adenovirus species. As IDPs comprise a rapidly growing family of proteins which, despite their lack of a well defined structure, often fulfill essential regulatory functions, the observations described here might open up a new avenue for the understanding of the regulation and functions of E1B proteins, in particular the multifunctional E1B-55K oncoprotein

Solution structure and pressure response of thioredoxin-1 of Plasmodium falciparum

We present here the solution structures of the protein thioredoxin-1 from Plasmodium falciparum (PfTrx-1), in its reduced and oxidized forms. They were determined by high-resolution NMR spectroscopy at 293 K on uniformly 13C-, 15N-enriched, matched samples allowing to identification of even small structural differences. PfTrx-1 shows an α/β-fold with a mixed five-stranded β-sheet that is sandwiched between 4 helices in a β1 α1 β2 α2 β3 α3 β4 β5 α4 topology. The redox process of the CGPC motif leads to significant structural changes accompanied by larger chemical shift changes from residue Phe25 to Ile36, Thr70 to Thr74, and Leu88 to Asn91. By high-field high-pressure NMR spectroscopy, rare conformational states can be identified that potentially are functionally important and can be used for targeted drug development. We performed these experiments in the pressure range from 0.1 MPa to 200 MPa. The mean combined, random-coil corrected B1* values of reduced and oxidized thioredoxin are quite similar with -0.145 and -0.114 ppm GPa-1, respectively. The mean combined, random-coil corrected B2* values in the reduced and oxidized form are 0.179 and 0.119 ppm GPa-2, respectively. The mean ratios of the pressure coefficients B2/B1 are -0.484 and -0.831 GPa-1 in the reduced and oxidized form respectively. They differ at some points in the structure after the formation of the disulfide bond between C30 and C33. The thermodynamical description of the pressure dependence of chemical shifts requires the assumption of at least three coexisting conformational states of PfTrx-1. These three conformational states were identified in the reduced as well as in the oxidized form of the protein, therefore, they represent sub-states of the two main oxidation states of PfTrx-1

Human Immunodeficiency Virus Type 1 Nef protein modulates the lipid composition of virions and host cell membrane microdomains

BACKGROUND: The Nef protein of Human Immunodeficiency Viruses optimizes viral spread in the infected host by manipulating cellular transport and signal transduction machineries. Nef also boosts the infectivity of HIV particles by an unknown mechanism. Recent studies suggested a correlation between the association of Nef with lipid raft microdomains and its positive effects on virion infectivity. Furthermore, the lipidome analysis of HIV-1 particles revealed a marked enrichment of classical raft lipids and thus identified HIV-1 virions as an example for naturally occurring membrane microdomains. Since Nef modulates the protein composition and function of membrane microdomains we tested here if Nef also has the propensity to alter microdomain lipid composition. RESULTS: Quantitative mass spectrometric lipidome analysis of highly purified HIV-1 particles revealed that the presence of Nef during virus production from T lymphocytes enforced their raft character via a significant reduction of polyunsaturated phosphatidylcholine species and a specific enrichment of sphingomyelin. In contrast, Nef did not significantly affect virion levels of phosphoglycerolipids or cholesterol. The observed alterations in virion lipid composition were insufficient to mediate Nef's effect on particle infectivity and Nef augmented virion infectivity independently of whether virus entry was targeted to or excluded from membrane microdomains. However, altered lipid compositions similar to those observed in virions were also detected in detergent-resistant membrane preparations of virus producing cells. CONCLUSION: Nef alters not only the proteome but also the lipid composition of host cell microdomains. This novel activity represents a previously unrecognized mechanism by which Nef could manipulate HIV-1 target cells to facilitate virus propagation in vivo

Macrophage cholesterol efflux correlates with lipoprotein subclass distribution and risk of obstructive coronary artery disease in patients undergoing coronary angiography

<p>Abstract</p> <p>Background</p> <p>Studies in patients with low HDL have suggested that impaired cellular cholesterol efflux is a heritable phenotype increasing atherosclerosis risk. Less is known about the association of macrophage cholesterol efflux with lipid profiles and CAD risk in normolipidemic subjects. We have therefore measured macrophage cholesterol efflux in142 normolipidemic subjects undergoing coronary angiography.</p> <p>Methods</p> <p>Monocytes isolated from blood samples of patients scheduled for cardiac catheterization were differentiated into macrophages over seven days. Isotopic cholesterol efflux to exogenously added apolipoprotein A-I and HDL2 was measured. Quantitative cholesterol efflux from macrophages was correlated with lipoprotein subclass distribution in plasma from the same individuals measured by NMR-spectroscopy of lipids and with the extent of coronary artery disease seen on coronary angiography.</p> <p>Results</p> <p>Macrophage cholesterol efflux was positively correlated with particle concentration of smaller HDL and LDL particles but not with total plasma concentrations of HDL or LDL-cholesterol. We observed an inverse relationship between macrophage cholesterol efflux and the concntration of larger and triglyceride rich particles (VLDL, chylomicrons). Subjects with significant stenosis on coronary angiography had lower cholesterol efflux from macrophages compared to individuals without significant stenosis (adjusted p = 0.02).</p> <p>Conclusion</p> <p>Macrophage cholesterol efflux is inversely correlated with lipoprotein particle size and risk of CAD.</p

Ceramic cells for high pressure NMR spectroscopy of proteins

Application of high pressure to biological macromolecules can be used to find new structural states with a smaller specific volume of the system. High pressure NMR spectroscopy is a most promising analytical tool for the study of these states at atomic resolution. High pressure quartz cells are difficult to handle, high quality sapphire high pressure cells are difficult to obtain commercially. In this work, we describe the use of high pressure ceramic cells produced from yttrium stabilized ZrO2 that are capable of resisting pressures up to 200 MPa. Since the new cells should also be usable in the easily damageable cryoprobes a completely new autoclave for these cells has been constructed, including an improved method for pressure transmission, an integrated safety jacket, a displacement body, and a fast self-closing emergency valve.DF

Structural transitions in full-length human prion protein detected by xenon as probe and spin labeling of the N-terminal domain

Fatal neurodegenerative disorders termed transmissible spongiform encephalopathies (TSEs) are associated with the accumulation of fibrils of misfolded prion protein PrP. The noble gas xenon accommodates into four transiently enlarged hydrophobic cavities located in the well-folded core of human PrP(23-230) as detected by [H-1, N-15]-HSQC spectroscopy. In thermal equilibrium a fifth xenon binding site is formed transiently by amino acids A120 to L125 of the presumably disordered N-terminal domain and by amino acids K185 to T193 of the well-folded domain. Xenon bound PrP was modelled by restraint molecular dynamics. The individual microscopic and macroscopic dissociation constants could be derived by fitting the data to a model including a dynamic opening and closing of the cavities. As observed earlier by high pressure NMR spectroscopy xenon binding influences also other amino acids all over the N-terminal domain including residues of the AGAAAAGA motif indicating a structural coupling between the N-terminal domain and the core domain. This is in agreement with spin labelling experiments at positions 93 or 107 that show a transient interaction between the N-terminus and the start of helix 2 and the end of helix 3 of the core domain similar to that observed earlier by Zn2+-binding to the octarepeat motif