Surface topology of Minibody by selective chemical modifications and mass spectrometry.

The surface topology of the Minibody, a small de novo-designed beta-protein, has been probed by a strategy that combines selective chemical modification with a variety of reagents and mass spectrometric analysis of the modified fragments. Under appropriate conditions, the susceptibility of individual residues primarily depends on their surface accessibility so that their relative reactivities can be correlated with their position in the tertiary structure of the protein. Moreover, this approach provides information on interacting residues, since intramolecular interactions might greatly affect the reactivity of individual side chains by altering their pKa values. The results of this study indicate that, while overall the Minibody model is correct, the beta-sheet formed by the N- and C-terminal segments is most likely distorted. This is also in agreement with previous results that were obtained using a similar approach where mass spectrometry was used to identify Minibody fragments from limited proteolysis (Zappacosta F, Pessi A, Bianchi E, Venturini S, Sollazzo M, Tramontano A. Marino G, Pucci P. 1996. Probing the tertiary structure of proteins by limited proteolysis and mass spectrometry: The case of Minibody. Protein Sci 5:802-813). The chemical modification approach, in combination with limited proteolysis procedures, can provide useful, albeit partial, structural information to complement simulation techniques. This is especially valuable when, as in the Minibody case, an NMR and/or X-ray structure cannot be obtained due to insufficient solubility of the molecule.     

Prediction of the tertiary structure of parathyroid-hormone-related protein (residues 1–34) by the island model

Prediction of the tertiary structure of a 34 residue N-terminal fragment of parathyroid-hormone-related protein was carried out by the island model. This peptide is known as a major causative agent of humoral hypercalcemia of malignancy, but structural information studied by X-ray diffraction has not been reported. We adopted the secondary structure determined by NMR and packed on the basis of island model of protein folding developed by us. Predicted structure is discussed in connection with the interaction of active sites.     

Tritium planigraphy: Differences in the spatial structures of the influenza virus M1 protein in crystal,solution, and virion

The structure of the M1 protein of the influenza virus A/Puerto Rico/8/34 (PR8, subtype H1N1) in solution at acidic pH and in the composition of the virion has been studied by the tritium planigraphy method. A model of the spatial structure was constructed using a special algorithm simulating the experiment and a set of algorithms for predicting the secondary structure and disordered regions in proteins. The tertiary structure was refined using the Rosetta program. For a comparison of the structures in solution and inside the virion, the data of X-ray diffraction analysis for the NM domain were also used. The main difference in the structures of the protein in solution and the crystalline state is observed in the region of contact of N and M domains, which in the crystalline state is packed more densely. The regions of the maximum label incorporation almost completely coincide with unstructured regions in the protein that were predicted by the bioinformatics analysis. These regions are concentrated in the C domain and in loop regions between M, N, and C domains. The data were confirmed by analytical centrifugation and dynamic light scattering. Anomalous hydrodynamic dimensions and a low structuration of the M1 protein in solution were found. The polyfunctionality of the protein in the cell is probably related to its flexible tertiary structure, which, owing to unstructured regions, provides contact with various partner molecules.     

Knowledge based modelling of homologous proteins, Part I: Three-dimensional frameworks derived from the simultaneous superposition of multiple structures

An approach is described for modelling the three-dimensional structure of a protein from the tertiary structures of several homologous proteins that have been determined by X-ray analysis. A method is developed for the simultaneous superposition of several protein molecules and for the calculation of an 'average structure' or 'framework'. Investigation of the convergence properties of this method, in the case of both weighted and unweighted least squares, demonstrates that both give a unique answer and the latter is robust for an homologous family of proteins. Multi-dimensional scaling is used to subgroup of the proteins with respect to structural homology. The framework calculated on the basis of the family of homologous proteins, or of an appropriate subgroup, is used to align fragments of the known protein structures of high sequence homology with the unknown. This alignment provides a basis for model building the tertiary structure. Different techniques for using the framework to model the mainchain of various globins and an immunoglobulin domain in the structurally conserved regions are investigated.     

Comparison of the effects of 2,2,2-trifluoroethanol on peptide and protein structure and function

The co-solvent 2,2,2-trifluoroethanol (TFE) has been often used to aid formation of secondary structure in solution peptides or alternately as a denaturant within protein folding studies. Hen egg white lysozyme (HEWL) and a synthetic model peptide defining HEWL helix-4 were used as comparative model systems to systematically investigate the effect of increasing TFE concentrations on the structure of proteins and peptides. HEWL was analyzed using NMR, far-UV CD and fluorescence spectroscopy; with correlation of these results towards changes in enzymatic activity and the helix-4 peptide was analysed using NMR. Data illustrates two conflicting modes of interaction: Low TFE concentrations stabilize tertiary structure, observed from an increase in the number of NMR NOE contacts. Higher TFE concentrations denatured HEWL with the loss of lysozyme tertiary structure. The effects of TFE upon secondary structural elements within HEWL are distinct from those observed for the helix-4 peptide. This illustrates a dissimilar interaction of TFE towards both protein and peptide at equivalent TFE concentrations. The concentration that TFE promotes stabilization over denaturation is likely to be protein dependent although the structural action can be extrapolated to other protein systems with implications for the use of TFE in structural stability studies.     

Crystal structure of a protein-toxin alpha 1-purothionin at 2.5A and a comparison with predicted models

Alpha 1-Purothionin (alpha 1-P), a wheatgerm protein and lytic toxin, has a secondary and tertiary structure similar to that of crambin as revealed by CD and NMR studies. alpha 1-P crystallizes in the tetragonal space group 1422 with unit cell dimensions: a = b = 53.59 and c = 69.79 A. X-ray diffraction data have been measured to 2.5 A Bragg spacing. The crystal structure has been determined by molecular replacement methods, using an energy-minimized alpha 1-P model structure derived from crambin (Whitlow and Teeter: Journal of Biomolecular Structure and Dynamics 2:831-848, 1985, Journal of the American Chemical Society 108:7163-7172, 1986). The energy-minimized model gives a slightly cleaner rotation solution and better refinement against the x-ray data than do the crambin or unminimized alpha 1-P structures. The final crystallographic residual with the data in the 10-2.5 A resolution range is 0.216. The refined alpha 1-P structure has a backbone rms difference of 0.74 A from crambin and 0.55 A from the energy-minimized alpha 1-P model. A low resolution NMR model of alpha 1-P calculated from metric matrix distance geometry and restrained molecular dynamics differs from crambin's backbone by 2.3 A rms deviation (Clore et al.: EMBO Journal 5:2729-2735, 1986). Backbone dihedral angles for our predicted model differ from the refined alpha 1-P structure in only one region (at a turn where there is a deletion relative to crambin). The NMR model had differences in four regions.     

Characterization of an artificial dimer of ribonuclease H using 1H NMR spectroscopy

Summary The protein fusion technique was applied in the synthesis of an artificial dimer of ribonuclease H (305 residues). ¹H NMR spectroscopy was used to analyze the structure of this dimer. Spectral profiles and pK_a values of the histidine residues obtained using ¹H NMR indicate that the dimer retains the secondary and tertiary structures of the intact monomer. Selective spin-lattice relaxation measurements suggest that the two monomeric units in the dimer are in tight contact. Furthermore, the 2D ¹H NMR and paramagnetic relaxation filter results show that the two monomers bind together through interactions between the N- and C-terminal sites of the linked regions.     

Hydrogen-deuterium exchange strategy for delineation of contact sites in protein complexes

We use NMR spectra to determine protein-protein contact sites by observing differences in amide proton hydrogen-deuterium exchange in the complex compared to the free protein in solution. Aprotic organic solvents are used to preserve H/D labeling patterns that would be scrambled in water solutions. The binding site between the mammalian co-chaperone Aha1 with the middle domain of the chaperone Hsp90 obtained by our H/D exchange method corresponds well with that in the X-ray crystal structure of the homologous complex from yeast, even to the observation of a secondary binding site. This method can potentially provide data for complexes with unknown structure and for large or dynamic complexes inaccessible via NMR and X-ray methods.     

A new electron transport mechanism in mitochondrial steroid hydroxylase systems based on structural changes upon the reduction of adrenodoxin

The adrenal ferredoxin (adrenodoxin, Adx) is an acidic 14.4-kDa [2Fe-2S] ferredoxin that belongs to the vertebrate ferredoxin family. It is involved in the electron transfer from the flavoenzyme NADPH-adrenodoxin-reductase to cytochromes P-450(scc) and P-450(11)(beta). The interaction between the redox partners during electron transport has not yet been fully established. Determining the tertiary structure of an electron-transfer protein may be very helpful in understanding the transport mechanism. In the present work, we report a structural study on the oxidized and reduced forms of bovine adrenodoxin (bAdx) in solution using high-resolution NMR spectroscopy. The protein was produced in Escherichia coli and singly or doubly labeled with (15)N or (13)C/(15)N, respectively. Approximately 70 and 75% of the (15)N, (13)C, and (1)H resonances could be assigned for the reduced and the oxidized bAdx, respectively. The secondary and tertiary structures of the reduced and oxidized states were determined using NOE distance information. (1)H(N)-T(1) relaxation times of certain residues were used to obtain additional distance constraints to the [2Fe-2S] cluster. The results suggest that the solution structure of oxidized Adx is quite similar to the X-ray structure. However, structural changes occur upon reduction of the [2Fe-2S] cluster, as indicated by NMR measurements. It could be shown that these conformational changes, especially in the C-terminal region, cause the dissociation of the Adx dimer upon reduction. A new electron transport mechanism proceeding via a modified shuttle mechanism, with both monomers and dimers acting as electron carriers, is proposed.     

Adaptability of restrained molecular dynamics for tertiary structure prediction: application to Crotalus atrox venom phospholipase A2

In order to assess the adaptability and/or applicability of the restrained molecular dynamics (RMD) simulation for building a possible tertiary structure of a protein from the X-ray crystal structure of a family reference protein, the tertiary structure prediction of Crotalus atrox venom phospholipase A2 (PLA2) was attempted based on the X-ray crystal structure of bovine pancreatic PLA2. For the formation of secondary and tertiary structures from the fully extended starting structure, the RMD simulation with interatomic distance restraints and torsion angle restraints, which were derived from homologous amino acid sequence regions in the reference protein, was carried out until the molecular system was fully equilibrated. The predicted tertiary structure of C. atrox venom PLA2 was compared with its X-ray crystal structure, and furthermore the utility of this method was discussed by reference to the similar tertiary structure prediction of beta-trypsin from the X-ray crystal structure of an elastase.     

Metal-dependent stabilization of an active HMG protein

Using a cloned single domain of the high mobility group protein 1 (HMGB1), we evaluated the effect of introducing metal binding site(s) on protein stability and function. An HMG domain is a conserved sequence of approximately 80 amino acids rich in basic, aromatic and proline residues that is active in binding DNA in a sequence- or structure-specific manner. The design strategy focuses on anchoring selected regions of the protein, specifically loops and turns in the molecule, using His-metal ligands. Changes in secondary structure, thermostability and DNA binding properties of a series of such mutants were evaluated. The two most stable mutant constructs contain three surface histidine replacements (two metal binding sites) in the regions encompassing both turns of the molecule. On ligation with the divalent nickel cation, the stability of these two triple histidine mutants (I38H/N51H/D55H and G39H/N51H/D55H) increases by 1.3 and 1.6 kcal/mol, respectively, relative to the wild-type protein, although the creation of binding sites per se destabilizes the protein. The DNA-binding properties of the modified proteins are not impaired by the introduction of the metal binding motifs. These results indicate that it is feasible to stabilize protein tertiary structure using appropriate placement of surface His-metal bonds without loss of function.     

Complete sequence and model for the C1 subunit of the carotenoprotein, crustacyanin, and model for the dimer, beta-crustacyanin, formed from the C1 and A2 subunits with astaxanthin

The complete sequence has been determined for the C1 subunit of crustacyanin, an astaxanthin-binding protein from the carapace of the lobster Homarus gammarus (L.). The polypeptide, 181 residues long, is similar (38% identity) to the other main subunit, A2 and to plasma retinol-binding protein. The tertiary structure of the C1 subunit has been modelled on that derived for the A2 subunit from the coordinates of retinol-binding protein. Residues lining the putative binding cavities and at the putative carotenoid binding sites of the two subunits are highly conserved. The carotenoid environments are characterized by a preponderance of aromatic and polar residues and the absence of charged side-chains. A tentative model for the dimer, beta-crustacyanin, formed between the two subunits with their associated carotenoid ligands, is discussed. The model is based on the crystal structure of the dimer of bilin-binding protein, a member of the same superfamily. This structure has enabled us to examine mechanisms for the bathochromic spectral shift of the protein-bound carotenoid and to identify likely contact regions between dimers in octameric alpha-crustacyanin.     

Comparison of Composer and ORCHESTRAR

Although the number of known protein structures is increasing, the number of protein sequences without determined structures is still much larger. Three-dimensional (3D) protein structure information helps in the understanding of functional mechanisms, but solving structures by X-ray crystallography or NMR is often a lengthy and difficult process. A relatively fast way of determining a protein's 3D structure is to construct a computer model using homologous sequence and structure information. Much work has gone into algorithms that comprise the ORCHESTRAR homology modeling program in the SYBYL software package. This novel homology modeling tool combines algorithms for modeling conserved cores, variable regions, and side chains. The paradigm of using existing knowledge from multiple templates and the underlying protein environment knowledgebase is used in all of these algorithms, and will become even more powerful as the number of experimentally derived protein structures increases. To determine how ORCHESTRAR compares to Composer (a broadly used, but an older tool), homology models of 18 proteins were constructed using each program so that a detailed comparison of each step in the modeling process could be carried out. Proteins modeled include kinases, dihydrofolate reductase, HIV protease, and factor Xa. In almost all cases ORCHESTRAR produces models with lower root-mean-squared deviation (RMSD) values when compared with structures determined by X-ray crystallography or NMR. Moreover, ORCHESTRAR produced a homology model for three target sequences where Composer failed to produce any. Data for RMSD comparisons between structurally conserved cores, structurally variable regions, side-chain conformations are presented, as well as analyses of active site and protein-protein interface configurations.     

Conformational studies on muscular parvalbumins cooperative binding of calcium (II) to parvalbumins.

1H NMR and ORD were used to characterize the respective variations of tertiary structure and secondary structure of parvalbumins with calcium content ((Pa(O), without calcium and PaCa2 calcium saturated) and temperature. It has been observed that the tertiary structure can be lost without significant variation of the helical content. Cooperative binding of calcium to Pa(O) has been shown by NMR spectroscopy under low ionic strength conditions and at neutral pH. The present study shows that the calcium binding affinity of parvalbumin is dependent on the tertiary structure. Calcium binding and calcium release functions of parvalbumins in the muscle may be controlled by their tertiary structure.     

Accessibility and mobility of lysine residues in beta-lactoglobulin

N epsilon-[2H6]Isopropyllysyl-beta-lactoglobulin was prepared by reductive alkylation of beta-lactoglobulin with [2H6]acetone and NaBH4 to provide a 2H (NMR) probe for the study of lysine involvement in lipid-protein interactions. Amino acid analysis showed 80% of the protein's 15 lysine residues to be labeled. Unmodified lysine residues were located through peptide maps produced from CNBr, tryptic, and chymotryptic digests of the labeled protein. Lys47 was not modified; Lys135,138,141, located along an amphipathic helical rod, were each partially unmodified. All other lysine residues were at least 90% modified. Average correlation times calculated from 2H NMR spectra were 20 and 320 ps for 8.7 and 3.3 residues, respectively, in 6 M guanidine hydrochloride; in nondenaturing solution, values of 70 and 320 ps were obtained for 6.5 and 3.2 residues, respectively, with the remaining 2.3 modified residues not observed, suggesting that side chains of lysine residues in unordered or flexible regions were more mobile than those in stable periodic structures. 2H NMR spectra of the protein complexed with dipalmitoylphosphatidylcholine confirmed the extrinsic membrane protein type behavior of beta-lactoglobulin previously reported from 31P NMR studies of the phospholipids complexed with beta-lactoglobulin. Although no physiological function has yet been identified, comparison of these results with the X-ray structure [Papiz et al. (1986) Nature (London) 324, 383-385] supports the hypothesis that residues not accessible for modification may help to stabilize the cone-shaped beta-barrel thought to contain binding sites for small lipid-soluble molecules.     

Effect of pressure on the tertiary structure and dynamics of folded basic pancreatic trypsin inhibitor

The on-line high-pressure cell NMR technique was used to study pressure-induced changes in the tertiary structure and dynamics of a globular protein, basic pancreatic trypsin inhibitor (BPTI). Practically all the proton signals of BPTI were observed with (1)H two-dimensional NMR spectroscopy at 750 MHz at variable pressure between 1 and 2000 bar. Chemical shifts, nuclear Overhauser effect (NOE), and line shapes were used to analyze conformational and dynamic changes of the protein as functions of pressure. Linear, reversible, but nonuniform pressure-induced chemical shift changes of practically all the C(alpha) protons and side chain protons showed that the entire secondary and tertiary structures are altered by pressure within the folded ensemble of BPTI. The high field shift tendency of most side chain proton signals and the increase in NOE intensities of some specific side chain protons indicated a site-specific compaction of the tertiary structure. Pressure dependence of ring flip rates was deduced from resonance line shapes of the slices of the two-dimensional NMR spectrum for ring proton signals of Tyr-35 and Phe-45. The rates of the flip-flop motions were considerably reduced at high pressure, from which activation volumes were determined to be 85 +/- 20 A(3) (or 51.2 ml/mol) and 46 +/- 9 A(3) (or 27.7 ml/mol) for Tyr-35 and Phe-45, respectively, at 57 degrees C. The present experiments confirm that pressure affects the entire secondary and tertiary structures of a globular protein with specific compaction of a core, leading to quite significant changes in slow internal dynamics of a globular protein.     

Hydrophobic photolabeling as a new method for structural characterization of molten globule and related protein folding intermediates.

Recent advances in attempts to unravel the protein folding mechanism have indicated the need to identify the folding intermediates. Despite their transient nature, in a number of cases it has been possible to detect and characterize some of the equilibrium intermediates, for example, the molten globule (MG) state. The key features of the MG state are retention of substantial secondary structure of the native state, considerable loss of tertiary structure leading to increased hydrophobic exposure, and a compact structure. NMR, circular dichroism, and fluorescence spectroscopies have been most useful in characterizing such intermediates. We report here a new method for structural characterization of the MG state that involves probing the exposed hydrophobic sites with a hydrophobic photoactivable reagent--2[3H]diazofluorene. This carbene-based reagent binds to hydrophobic sites, and on photolysis covalently attaches itself to the neighboring amino acid side chains. The reagent photolabels alpha-lactalbumin as a function of pH (3-7.4), the labeling at neutral pH being negligible and maximal at pH 3. Chemical and proteolytic fragmentation of the photolabeled protein followed by peptide sequencing permitted identification of the labeled residues. The results obtained indicate that the sequence corresponding to B (23-34) and C (86-98) helix of the native structure are extensively labeled. The small beta-domain (40-50) is poorly labeled, Val42 being the only residue that is significantly labeled. Our data, like NMR data, indicate that in the MG state of alpha-lactalbumin, the alpha-domain has a greater degree of persistent structure than the beta-domain. However, unlike the NMR method, the photolabeling method is not limited by the size of the protein and can provide information on several new residues, for example, Leu115. The current method using DAF thus allows identification of stable and hydrophobic exposed regions in folding intermediates as the reagent binds and on photolysis covalently links to these regions.     

Modeling and docking the endothelin G-protein-coupled receptor

A model of the endothelin G-protein-coupled receptor (ET(A)) has been constructed using a segmented approach. The model was produced using a bovine rhodopsin model as a template for the seven transmembrane alpha-helices. The three cytoplasmic loop regions and the C-terminal region were modeled on NMR structures of corresponding segments from bovine rhodopsin. The three extracellular loops were modeled on homologous loop regions in other proteins of known structure. The N-terminal region was modeled as a three-helix domain based on its homology with a hydrolase protein. To test the model, the FTDOCK algorithm was used to predict the ligand-binding site for the crystal structure of human endothelin. The site of docking is consistent with mutational and biochemical data. The principal sites of interaction in the endothelin ligand all lie on one face of a helix that has been implicated by structure-activity relationship studies as being essential for binding. As further support for the model, attempts to dock bigET, an inactive precursor to endothelin that does not bind to the receptor, found no sites for tight binding. The model of the receptor-ligand complex produced forms a basis for rational drug design of agonists and antagonists for this G-protein-coupled receptor.     

Structure of lysozyme dissolved in neat organic solvents as assessed by NMR and CD spectroscopies

The structure of the model protein hen egg-white lysozyme dissolved in water and in five neat organic solvents (ethylene glycol, methanol, dimethylsulfoxide (DMSO), formamide, and dimethylformamide (DMF)) has been examined by means of 1H NMR and circular dichroism (CD) spectroscopies. The NMR spectra of lysozyme reveal the lack of a defined tertiary structure in all five organic solvents, although the examination of line widths suggests the possibility of some ordered structure in ethylene glycol and in methanol. The near-UV CD spectra of the protein suggest no tertiary structure in lysozyme dissolved in DMSO, formamide, and DMF, while a distinctive (albeit less pronounced than in water) tertiary structure is seen in ethylene glycol and a drastically changed one in methanol. A highly developed secondary structure was observed by far-UV CD in ethylene glycol and methanol; interestingly, the alpha-helix content of the protein in both was greater than in water, while the beta-structure content was lower. (Solvent absorbance in the far-UV region prevents conclusions about the secondary structure in DMSO, formamide and DMF.) Copyright 1999 John Wiley & Sons, Inc.