Structure determination of proteins and peptides in solution: simulation, chirality and NMR studies

The study of protein fold is a central problem in life science, leading in the last years to several attempts for improving our knowledge of the protein structures. In this thesis this challenging problem is tackled by means of molecular dynamics, chirality and NMR studies. In the last decades, many algorithms were designed for the protein secondary structure assignment, which reveals the local protein shape adopted by segments of amino acids. In this regard, the use of local chirality for the protein secondary structure assignment was demonstreted, trying to correlate as well the propensity of a given amino acid for a particular secondary structure. The protein fold can be studied also by Nuclear Magnetic Resonance (NMR) investigations, finding the average structure adopted from a protein. In this context, the effect of Residual Dipolar Couplings (RDCs) in the structure refinement was shown, revealing a strong improvement of structure resolution. A wide extent of this thesis is devoted to the study of avian prion protein. Prion protein is the main responsible of a vast class of neurodegenerative diseases, known as Bovine Spongiform Encephalopathy (BSE), present in mammals, but not in avian species and it is caused from the conversion of cellular prion protein to the pathogenic misfolded isoform, accumulating in the brain in form of amiloyd plaques. In particular, the N-terminal region, namely the initial part of the protein, is quite different between mammal and avian species but both of them contain multimeric sequences called Repeats, octameric in mammals and hexameric in avians. However, such repeat regions show differences in the contained amino acids, in particular only avian hexarepeats contain tyrosine residues. The chirality analysis of avian prion protein configurations obtained from molecular dynamics reveals a high stiffness of the avian protein, which tends to preserve its regular secondary structure. This is due to the presence of prolines, histidines and especially tyrosines, which form a hydrogen bond network in the hexarepeat region, only possible in the avian protein, and thus probably hampering the aggregation.

High-resolution NMR of encapsulated proteins dissolved in low-viscosity fluids

The majority of known proteins are too large to be comprehensively examined by solution NMR methods, primarily because they tumble too slowly in solution. Here we introduce an approach to making the NMR relaxation properties of large proteins amenable to modern solution NMR techniques. The encapsulation of a protein in a reverse micelle dissolved in a low-viscosity fluid allows it to tumble as fast as a much smaller protein. The approach is demonstrated and validated with the protein ubiquitin encapsulated in reverse micelles prepared in a variety of alkane solvents.

Solution structures by H-1 NMR of the novel cyclic trypsin inhibitor SFTI-1 from sunflower seeds and an acyclic permutant

SFTI-1 is a recently discovered cyclic peptide trypsin inhibitor from sunflower seeds comprising 14 amino acid residues. It is the most potent known Bowman-Birk inhibitor and the only naturally occurring cyclic one. The solution structure of SFTI-1 has been determined by H-1-NMR spectroscopy and compared with a synthetic acyclic permutant. The solution structures of both are remarkably similar. The lowest energy structures from each family of 20 structures of cyclic and acyclic SFTI-1 have an rmsd over the backbone and heavy atoms of 0.29 Angstrom and 0.66 Angstrom, respectively. The structures consist of two short antiparallel beta -strands joined by an extended loop containing the active site at one end. Cyclic SFTI-1 also has a hairpin turn completing the cycle. Both molecules contain particularly stable arrangements of cross-linking hydrogen bonds between the beta -strands and a single disulfide bridge, making them rigid and well defined in solution. These stable arrangements allow both the cyclic and acyclic variants of SFTI-1 to inhibit trypsin with very high potencies (0.5 nM and 12.1 nM, respectively). The cyclic nature of SFTI-1 appears to have evolved to provide higher trypsin inhibition as well as higher stability. The solution structures are similar to the crystal structure of the cyclic inhibitor in complex with trypsin. The lack of a major conformational change upon binding suggests that the structure of SFTI-1 is rigid and already pre-organized for maximal binding due to minimization of entropic losses compared to a more flexible ligand. These properties make SFTI-1 an ideal platform for the design of small peptidic pharmaceuticals or pesticides. (C) 2001 Academic Press.

Protein loop compaction and the origin of the effect of arginine and glutamic acid mixtures on solubility, stability and transient oligomerization of proteins

Addition of a 50 mM mixture of l-arginine and l-glutamic acid (RE) is extensively used to improve protein solubility and stability, although the origin of the effect is not well understood. We present Small Angle X-ray Scattering (SAXS) and Nuclear Magnetic Resonance (NMR) results showing that RE induces protein compaction by collapsing flexible loops on the protein core. This is suggested to be a general mechanism preventing aggregation and improving resistance to proteases and to originate from the polyelectrolyte nature of RE. Molecular polyelectrolyte mixtures are expected to display long range correlation effects according to dressed interaction site theory. We hypothesize that perturbation of the RE solution by dissolved proteins is proportional to the volume occupied by the protein. As a consequence, loop collapse, minimizing the effective protein volume, is favored in the presence of RE.

Site-specific labelling of proteins with a rigid lanthanide-binding tag

This paper describes a generic method for the site-specific attachment of lathanide complexes to proteins through a disulfide bond. The method is demonstrated by the attachment of a lanthanide-binding peptide tag to the single cysteine residue present in the N-terminal DNA-binding domain of the Echerichia coli arginine repressor. Complexes with Y3+, Tb3+, Dy3+, Ho3+, Er3+, Tm3+ and Yb3+ ions were formed and analysed by NMR spectroscopy. Large pseudocontact shifts and residual dipolar couplings were induced by the lanthanide-binding tag in the protein NMR spectrum, a result indicating that the tag was rigidly attached to the protein. The axial components of the magnetic susceptibility anisostropy tensors determined for the different lanthanide ions were similarly but not identically oriented. A single tag with a single protein attachment site can provide different pseudocontact shifts from different magnetic susceptibility tensors and thus provide valuable nondegenerate long-range structure information in the determination of 3D protein structures by NMR spectroscopy.

Resistively detected NMR of the nu=1 quantum Hall state: A tilted magnetic field study

Previous resistively detected NMR (RDNMR) studies on the nu approximate to 1 quantum Hall state have reported a ""dispersionlike"" line shape and extremely short nuclear-spin-lattice relaxation times, observations which have been attributed to the formation of a skyrme lattice. Here we examine the evolution of the RDNMR line shape and nuclear-spin relaxation for Zeeman: Coulomb energy ratios ranging from 0.012 to 0.036. According to theory, suppression of the skyrme crystal, along with the associated Goldstone mode nuclear-spin-relaxation mechanism, is expected at the upper end of this range. However, we find that the anomalous line shape persists at high Zeeman energy, and only a modest decrease in the RDNMR-detected nuclear-spin-relaxation rate is observed.

Determination of the molecular weight of proteins in solution from a single small-angle X-ray scattering measurement on a relative scale

This paper describes a new and simple method to determine the molecular weight of proteins in dilute solution, with an error smaller than similar to 10%, by using the experimental data of a single small-angle X-ray scattering (SAXS) curve measured on a relative scale. This procedure does not require the measurement of SAXS intensity on an absolute scale and does not involve a comparison with another SAXS curve determined from a known standard protein. The proposed procedure can be applied to monodisperse systems of proteins in dilute solution, either in monomeric or multimeric state, and it has been successfully tested on SAXS data experimentally determined for proteins with known molecular weights. It is shown here that the molecular weights determined by this procedure deviate from the known values by less than 10% in each case and the average error for the test set of 21 proteins was 5.3%. Importantly, this method allows for an unambiguous determination of the multimeric state of proteins with known molecular weights.

Evaluation of the effect of ammonium carbamate on the stability of proteins

BACKGROUND: The use of the volatile salt ammonium carbamate in protein downstream processing has recently been proposed. The main advantage of using volatile salts is that they can be removed from precipitates and liquid effluents through pressure reduction or temperature increase. Although previous studies showed that ammonium carbamate is efficient as a precipitant agent, there was evidence of denaturation in some enzymes. In this work, the effect of ammonium carbamate on the stability of five enzymes was evaluated. RESULTS: Activity assays showed that alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1), lysozyme (1,4-beta-N-acetylmuramoylhydrolase, EC 3.2.1.17) and lipase (triacyl glycerol acyl hydrolase, EC 3.1.1.3) did not undergo activity loss in ammonium carbamate solutions with concentrations from 1.0 to 5.0 mol kg(-1), whereas cellulase complex (1,4-(1,3 : 14)-beta-D-glucan 4-glucano-hydrolase, EC 3.2.1.4) and peroxidase (hydrogen peroxide oxidoreductase, EC 1.11.1.7) showed an average activity loss of 55% and 44%, respectively. Precipitation assays did not show enzyme denaturation or phase separation for alpha-amylase and lipase, while celullase and peroxidase precipitated with some activity reduction. Analysis of similar experiments with ammonium and sodium sulfate did not affect the activity of enzymes. CONCLUSION: Celullase and peroxidase were denatured by ammonium carbamate. While more systematic studies are not available, care must be taken in designing a protein precipitation with this salt. The results suggest that the generally accepted idea that salts that denature proteins tend to solubilize them does not hold for ammonium carbamate. (C) 2010 Society of Chemical Industry

On the solubility of proteins as a function of pH: Mathematical development and application

Thermodynamic relations between the solubility of a protein and the solution pH are presented in this work. The hypotheses behind the development are that the protein chemical potential in liquid phase can be described by Henry`s law and that the solid-liquid equilibrium is established only between neutral molecules. The mathematical development results in an analytical expression of the solubility curve, as a function of the ionization equilibrium constants, the pH and the solubility at the isoelectric point. It is shown that the same equation can be obtained either by directly calculating the fraction of neutral protein molecules or by integrating the curve of the protein average charge. The methodology was successfully applied to the description of the solubility of porcine insulin as a function of pH at three different temperatures and of bovine beta-lactoglobulin at four different ionic strengths. (C) 2011 Elsevier B.V. All rights reserved.

Partial characterization of proteins from baru (Dipteryx alata Vog) seeds

BACKGROUND: Baru (Dipteryx alata Vog.) is a fruit distributed throughout the Brazilian savanna and contains a seed with a high protein content, whose properties have been rarely explored. The purpose of this study was to characterize this protein, especially by isolation and quantifying its fractions and measuring some of its molecular properties. RESULTS: Baru seeds contain 244 g kg(-1) protein on a dry weight basis. Solubility profiles showed a preponderance of globulins. This fraction dominated the seed composition, with 61.7 wt% of the total soluble proteins. Albumins and glutelins accounted for 14 and 3.3 wt%, respectively. SDS-PAGE resolution of albumin and globulin showed main bands with molecular weights of 84 kDa and 64,66 and 73 kDa, respectively. The total protein of the flour and the globulin showed values of in vitro digestibility of 85.59% and 90.54%, relative to casein. Total globulin produced only one chromatographic peak, both on Sepharose CL-6B gel filtration and on DEAE-cellulose ion-exchange columns, eluted at a concentration of 0.12 mol L(-1) NaCl. CONCLUSION: The baru seed had high protein content with large quantities of storage proteins. The chromatographic and solubility profiles indicate the predominance of a fraction with characteristics of a legumin-type protein. (C) 2011 Society of Chemical Industry

Solution structure of a defensin-like peptide from platypus venom

Three defensin-like peptides (DLPs) were isolated from platypus venom and sequenced. One of these peptides, DLP-1, was synthesized chemically and its three-dimensional structure was determined using NMR spectroscopy. The main structural elements of this 42-residue peptide were an anti-parallel beta-sheet comprising residues 15-18 and 37-40 and a small 3(10) helix spanning residues 10-12. The overall three-dimensional fold is similar to that of beta-defensin-12, and similar to the sodium-channel neurotoxin ShI (Stichodactyla helianthus neurotoxin I). However, the side chains known to be functionally important in beta-defensin-12 and ShI are not conserved in DLP-1, suggesting that it has a different biological function. Consistent with this contention, we showed that DLP-1 possesses no anti-microbial properties and has no observable activity on rat dorsal-root-ganglion sodium-channel currents.

Expression of proteins in the extracellular matrix of pulp tissue in human primary teeth during physiologic root resorption

Objectives: To analyze the expression of tenascin, fibronectin, collagens I and III, osteonectin, and bone morphogenetic protein 4 (BMP4) in the extracellular matrix of pulp tissue in primary teeth during physiologic root resorption. Method and Materials: Eighteen teeth were decalcified and equally distributed into 3 groups (group I, teeth with two-thirds root length; group II, teeth with one-third root length; and group III, teeth lacking the root). Results: Immunohistochemical analysis showed that all the proteins were expressed. Tenascin, collagen I, and osteonectin showed strong and broad reactivity in group I, with weaker and rare reactivity in groups II and III. The expression of fibronectin, collagen III, and BMP4 did not vary with root resorption phase. Conclusion: The expression of tenascin, collagen I, and osteonectin was reduced in the extracellular matrix and odontoblasts during root resorption. This fact may be related to the decreasing pulp response to damage and treatment during the progression of root resorption. (Quintessence Int 2009; 40: 553-558)

N4WBP5, a potential target for ubiquitination by the Nedd4 family of proteins, is a novel golgi-associated protein

Nedd4 belongs to a family of ubiquitin-protein ligases that is characterized by 2-4 WW domains, a carboxyl-terminal Hect ((h) under bar omologous to (E) under bar6-AP (C) under bar arboxyl (t) under bar erminus)-domain and in most cases an amino-terminal C2 domain. We had previously identified a series of proteins that associates with the WW domains of Nedd4. In this paper, we demonstrate that one of the Nedd4-binding proteins, N4WBP5, belongs to a small group of evolutionarily conserved proteins with three transmembrane domains. N4WBP5 binds Nedd4 WW domains via the two PPXY motifs present in the amino terminus of the protein. In addition to Nedd4, N4WBP5 can interact with the WW domains of a number of Nedd4 family members and is ubiquitinated. Endogenous N4WBP5 localizes to the Golgi complex. Ectopic expression of the protein disrupts the structure of the Golgi, suggesting that N4WBP5 forms part of a family of integral Golgi membrane proteins. Based on previous observations in yeast, we propose that N4WBP5 may act as an adaptor for Nedd4-like proteins and their putative targets to control ubiquitin-dependent protein sorting and trafficking.