Research Article
A helix-turn motif in the C-terminal domain of histone H1
- ROGER VILA, IMMA PONTE, M. ANGELES JIMÉNEZ, MANUEL RICO, PEDRO SUAU
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- 01 April 2000, pp. 627-636
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The structural study of peptides belonging to the terminal domains of histone H1 can be considered as a step toward the understanding of the function of H1 in chromatin. The conformational properties of the peptide Ac-EPKRSVAFKKTKKEVKKVATPKK (CH-1), which belongs to the C-terminal domain of histone H1° (residues 99–121) and is adjacent to the central globular domain of the protein, were examined by means of 1H-NMR and circular dichroism. In aqueous solution, CH-1 behaved as a mainly unstructured peptide, although turn-like conformations in rapid equilibrium with the unfolded state could be present. Addition of trifluoroethanol resulted in a substantial increase of the helical content. The helical limits, as indicated by (i, i + 3) nuclear Overhauser effect (NOE) cross correlations and significant up-field conformational shifts of the Cα protons, span from Pro100 to Val116, with Glu99 and Ala117 as N- and C-caps. A structure calculation performed on the basis of distance constraints derived from NOE cross peaks in 90% trifluoroethanol confirmed the helical structure of this region. The helical region has a marked amphipathic character, due to the location of all positively charged residues on one face of the helix and all the hydrophobic residues on the opposite face. The peptide has a TPKK motif at the C-terminus, following the α-helical region. The observed NOE connectivities suggest that the TPKK sequence adopts a type (I) β-turn conformation, a σ-turn conformation or a combination of both, in fast equilibrium with unfolded states. Sequences of the kind (S/T)P(K/R)(K/R) have been proposed as DNA binding motifs. The CH-1 peptide, thus, combines a positively charged amphipathic helix and a turn as potential DNA-binding motifs.
Elucidation of the solution structure of cardiotoxin analogue V from the Taiwan cobra (Naja naja atra)—Identification of structural features important for the lethal action of snake venom cardiotoxins
- GURUNATHAN JAYARAMAN, THALLAMPURANAM KRISHNASWAMY SURESH KUMAR, CHUNG-CHU TSAI, SAMPATH SRISAILAM, SHAN-HO CHOU, CHEWN-LANG HO, CHIN YU
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- 01 April 2000, pp. 637-646
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The aim of the present study is to understand the structural features responsible for the lethal activity of snake venom cardiotoxins. Comparison of the lethal potency of the five cardiotoxin isoforms isolated from the venom of Taiwan cobra (Naja naja atra) reveals that the lethal potency of CTX I and CTX V are about twice of that exhibited by CTX II, CTX III, and CTX IV. In the present study, the solution structure of CTX V has been determined at high resolution using multidimensional proton NMR spectroscopy and dynamical simulated annealing techniques. Comparison of the high resolution solution structures of CTX V with that of CTX IV reveals that the secondary structural elements in both the toxin isoforms consist of a triple and double-stranded antiparallel β-sheet domains. Critical examination of the three-dimensional structure of CTX V shows that the residues at the tip of Loop III form a distinct “finger-shaped” projection comprising of nonpolar residues. The occurrence of the nonpolar “finger-shaped” projection leads to the formation of a prominent cleft between the residues located at the tip of Loops II and III. Interestingly, the occurrence of a backbone hydrogen bonding (Val27CO to Leu48NH) in CTX IV is found to distort the “finger-shaped” projection and consequently diminish the cleft formation at the tip of Loops II and III. Comparison of the solution structures and lethal potencies of other cardiotoxin isoforms isolated from the Taiwan cobra (Naja naja atra) venom shows that a strong correlation exists between the lethal potency and occurrence of the nonpolar “finger-shaped” projection at the tip of Loop III. Critical analysis of the structures of the various CTX isoforms from the Taiwan cobra suggest that the degree of exposure of the cationic charge (to the solvent) contributed by the invariant lysine residue at position 44 on the convex side of the CTX molecules could be another crucial factor governing their lethal potency.
Mutational analysis of the major coat protein of M13 identifies residues that control protein display
- GREGORY A. WEISS, JAMES A. WELLS, SACHDEV S. SIDHU
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- 01 April 2000, pp. 647-654
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We have reported variants of the M13 bacteriophage major coat protein (P8) that enable high copy display of monomeric and oligomeric proteins, such as human growth hormone and steptavidin, on the surface of phage particles (Sidhu SS, Weiss GA, Wells JA. 2000. High copy display of large proteins on phage for functional selections. J Mol Biol 296:487–495). Here, we explore how an optimized P8 variant (opti-P8) could evolve the ability to efficiently display a protein fused to its N-terminus. Reversion of individual opti-P8 residues back to the wild-type P8 residue identifies a limited set of hydrophobic residues responsible for the high copy protein display. These hydrophobic amino acids bracket a conserved hydrophobic face on the P8 alpha helix thought to be in contact with the phage coat. Mutations additively combine to promote high copy protein display, which was further enhanced by optimization of the linker between the phage coat and the fusion protein. These data are consistent with a model in which protein display-enhancing mutations allow for better packing of the fusion protein into the phage coat. The high tolerance for phage coat protein mutations observed here suggests that filamentous phage coat proteins could readily evolve new capabilities.
A method to predict residues conferring functional differences between related proteins: Application to MAP kinase pathways
- DANIEL R. CAFFREY, LUKE A.J. O'NEILL, DENIS C. SHIELDS
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- 01 April 2000, pp. 655-670
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Physicochemical properties are potentially useful in predicting functional differences between aligned protein subfamilies. We present a method that considers physicochemical properties from ancestral sequences predicted to have given rise to the subfamilies of interest by gene duplication. Comparison between two map kinases subfamilies, p38 and ERK, revealed a region that had an excess of change in properties after gene duplication followed by conservation within the two subfamilies. This region corresponded to that experimentally defined as important for substrate and pathway specificity. The derived scores for the region of interest were found to differ significantly in their distribution compared to the rest of the protein when the Kolmogorov–Smirnov test was applied (p = 0.005). Thus, the incorporation of ancestral physicochemical properties is useful in predicting functional differences between protein subfamilies. In addition, the method was applied to the MKK and MAPK components of the p38 and JNK pathways. These proteins showed a similar pattern in their evolution and regions predicted to confer functional differences are discussed.
Backbone dynamics measurements on leukemia inhibitory factor, a rigid four-helical bundle cytokine
- SHENGGEN YAO, DAVID K. SMITH, MARK G. HINDS, JIAN-GUO ZHANG, NICOS A. NICOLA, RAYMOND S. NORTON
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- 01 April 2000, pp. 671-682
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The backbone dynamics of the four-helical bundle cytokine leukemia inhibitory factor (LIF) have been investigated using 15N NMR relaxation and amide proton exchange measurements on a murine–human chimera, MH35-LIF. For rapid backbone motions (on a time scale of 10 ps to 100 ns), as probed by 15N relaxation measurements, the dynamics parameters were calculated using the model-free formalism incorporating the model selection approach. The principal components of the inertia tensor of MH35-LIF, as calculated from its NMR structure, were 1:0.98:0.38. The global rotational motion of the molecule was, therefore, assumed to be axially symmetric in the analysis of its relaxation data. This yielded a diffusion anisotropy D∥/D⊥ of 1.31 and an effective correlation time (4D⊥ + 2D∥)−1 of 8.9 ns. The average values of the order parameters (S2) for the four helices, the long interhelical loops, and the N-terminus were 0.91, 0.84, and 0.65, respectively, indicating that LIF is fairly rigid in solution, except at the N-terminus. The S2 values for the long interhelical loops of MH35-LIF were higher than those of their counterparts in short-chain members of the four-helical bundle cytokine family. Residues involved in LIF receptor binding showed no consistent pattern of backbone mobilities, with S2 values ranging from 0.71 to 0.95, but residues contributing to receptor binding site III had relatively lower S2 values, implying higher amplitude motions than for the backbone of sites I and II. In the relatively slow motion regime, backbone amide exchange measurements showed that a number of amides from the helical bundle exchanged extremely slowly, persisting for several months in 2H2O at 37 °C. Evidence for local unfolding was considered, and correlations among various structure-related parameters and the backbone amide exchange rates were examined. Both sets of data concur in showing that LIF is one of the most rigid four-helical bundle cytokines.
Oxygen binding by α(Fe2+)2β(Ni2+)2 hemoglobin crystals
- STEFANO BRUNO, STEFANO BETTATI, MICHELE MANFREDINI, ANDREA MOZZARELLI, MARTINO BOLOGNESI, DANIELA DERIU, CAMILLO ROSANO, ANTONIO TSUNESHIGE, TAKASHI YONETANI, ERIC R. HENRY
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- 01 April 2000, pp. 683-692
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Oxygen binding by hemoglobin fixed in the T state either by crystallization or by encapsulation in silica gels is apparently noncooperative. However, cooperativity might be masked by different oxygen affinities of α and β subunits. Metal hybrid hemoglobins, where the noniron metal does not bind oxygen, provide the opportunity to determine the oxygen affinities of α and β hemes separately. Previous studies have characterized the oxygen binding by α(Ni2+)2 β(Fe2+)2 crystals. Here, we have determined the three-dimensional (3D) structure and oxygen binding of α(Fe2+)2 β(Ni2+)2 crystals grown from polyethylene glycol solutions. Polarized absorption spectra were recorded at different oxygen pressures with light polarized parallel either to the b or c crystal axis by single crystal microspectrophotometry. The oxygen pressures at 50% saturation (p50s) are 95 ± 3 and 87 ± 4 Torr along the b and c crystal axes, respectively, and the corresponding Hill coefficients are 0.96 ± 0.06 and 0.90 ± 0.03. Analysis of the binding curves, taking into account the different projections of the α hemes along the optical directions, indicates that the oxygen affinity of α1 hemes is 1.3-fold lower than α2 hemes. Inspection of the 3D structure suggests that this inequivalence may arise from packing interactions of the Hb tetramer within the monoclinic crystal lattice. A similar inequivalence was found for the β subunits of α(Ni2+)2 β(Fe2+)2 crystals. The average oxygen affinity of the α subunits (p50 = 91 Torr) is about 1.2-fold higher than the β subunits (p50 = 110 Torr). In the absence of cooperativity, this heterogeneity yields an oxygen binding curve of Hb A with a Hill coefficient of 0.999. Since the binding curves of Hb A crystals exhibit a Hill coefficient very close to unity, these findings indicate that oxygen binding by T-state hemoglobin is noncooperative, in keeping with the Monod, Wyman, and Changeux model.
15N and 1H NMR study of histidine containing protein (HPr) from Staphylococcus carnosus at high pressure
- HANS ROBERT KALBITZER, ADRIAN GÖRLER, HUA LI, PETER V. DUBOVSKII, WOLFGANG HENGSTENBERG, CLAUDIA KOWOLIK, HIROAKI YAMADA, KAZUYUKI AKASAKA
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- 01 April 2000, pp. 693-703
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The pressure-induced changes in 15N enriched HPr from Staphylococcus carnosus were investigated by two-dimensional (2D) heteronuclear NMR spectroscopy at pressures ranging from atmospheric pressure up to 200 MPa. The NMR experiments allowed the simultaneous observation of the backbone and side-chain amide protons and nitrogens. Most of the resonances shift downfield with increasing pressure indicating generalized pressure-induced conformational changes. The average pressure-induced shifts for amide protons and nitrogens are 0.285 ppm GPa−1 at 278 K and 2.20 ppm GPa−1, respectively. At 298 K the corresponding values are 0.275 and 2.41 ppm GPa−1. Proton and nitrogen pressure coefficients show a significant but rather small correlation (0.31) if determined for all amide resonances. When restricting the analysis to amide groups in the β-pleated sheet, the correlation between these coefficients is with 0.59 significantly higher. As already described for other proteins, the amide proton pressure coefficients are strongly correlated to the corresponding hydrogen bond distances, and thus are indicators for the pressure-induced changes of the hydrogen bond lengths. The nitrogen shift changes appear to sense other physical phenomena such as changes of the local backbone conformation as well. Interpretation of the pressure-induced shifts in terms of structural changes in the HPr protein suggests the following picture: the four-stranded β-pleated sheet of HPr protein is the least compressible part of the structure showing only small pressure effects. The two long helices a and c show intermediary effects that could be explained by a higher compressibility and a concomitant bending of the helices. The largest pressure coefficients are found in the active center region around His15 and in the regulatory helix b which includes the phosphorylation site Ser46 for the HPr kinase. This suggests that this part of the structure occurs in a number of different structural states whose equilibrium populations are shifted by pressure. In contrast to the surrounding residues of the active center loop that show large pressure effects, Ile14 has a very small proton and nitrogen pressure coefficient. It could represent some kind of anchoring point of the active center loop that holds it in the right place in space, whereas other parts of the loop adapt themselves to changing external conditions.
Crystal structure of adenosine kinase from Toxoplasma gondii at 1.8 Å resolution
- WILLIAM J. COOK, LAWRENCE J. DeLUCAS, DEBASISH CHATTOPADHYAY
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- 01 April 2000, pp. 704-712
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Human infection with Toxoplasma gondii is an important cause of morbidity and mortality. Protozoan parasites such as T. gondii are incapable of de novo purine biosynthesis and must acquire purines from their host, so the purine salvage pathway offers a number of potential targets for antiparasitic chemotherapy. In T. gondii tachyzoites, adenosine is the predominantly salvaged purine nucleoside, and thus adenosine kinase is a key enzyme in the purine salvage pathway of this parasite. The structure of T. gondii adenosine kinase was solved using molecular replacement and refined by simulated annealing at 1.8 Å resolution to an R-factor of 0.214. The overall structure and the active site geometry are similar to human adenosine kinase, although there are significant differences. The T. gondii adenosine kinase has several unique features compared to the human sequence, including a five-residue deletion in one of the four linking segments between the two domains, which is probably responsible for a major change in the orientation of the two domains with respect to each other. These structural differences suggest the possibility of developing specific inhibitors of the parasitic enzyme.
Slow conformational dynamics of an endonuclease persist in its complex with its natural protein inhibitor
- SARA B.-M. WHITTAKER, MICHAEL CZISCH, RAINER WECHSELBERGER, ROBERT KAPTEIN, ANDREW M. HEMMINGS, RICHARD JAMES, COLIN KLEANTHOUS, GEOFFREY R. MOORE
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- 01 April 2000, pp. 713-720
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The bacterial toxin colicin E9 is secreted by producing Escherichia coli cells with its 9.5 kDa inhibitor protein Im9 bound tightly to its 14.5 kDa C-terminal DNase domain. Double- and triple-resonance NMR spectra of the isolated DNase domain uniformly labeled with 13C/15N bound to unlabeled Im9 contain more signals than expected for a single DNase conformer, consistent with the bound DNase being present in more than one form. The presence of chemical exchange cross peaks in 750 MHz 15N–1H–15N HSQC–NOESY–HSQC spectra for backbone NH groups of Asp20, Lys21, Trp22, Leu23, Lys69, and Asn70 showed that the bound DNase was in dynamic exchange. The rate of exchange from the major to the minor form was determined to be 1.1 ± 0.2 s−1 at 298 K. Previous NMR studies have shown that the free DNase interchanges between two conformers with a forward rate constant of 1.61 ± 0.11 s−1 at 288 K, and that the bound Im9 is fixed in one conformation. The NMR studies of the bound DNase show that Im9 binds similarly to both conformers of the DNase and that the buried Trp22 is involved in the dynamic process. For the free DNase, all NH groups within a 9 Å radius of any point of the Trp22 ring exhibit heterogeneity suggesting that a rearrangement of the position of this side chain is connected with the conformational interchange. The possible functional significance of this feature of the DNase is discussed.
NMR solution structure of the θ subunit of DNA polymerase III from Escherichia coli
- MAX A. KENIRY, HILARY A. BERTHON, JI YEON YANG, CAROLINE S. MILES, NICHOLAS E. DIXON
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- 01 April 2000, pp. 721-733
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The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits (α, ε, and θ). The θ subunit is the smallest, but the least understood of the three. As a first step in a program aimed at understanding its function, the structure of the θ subunit has been determined by triple-resonance multidimensional NMR spectroscopy. Although only a small protein, θ was difficult to assign fully because approximately one-third of the protein is unstructured, and some sections of the remaining structured parts undergo intermediate intramolecular exchange. The secondary structure was deduced from the characteristic nuclear Overhauser effect patterns, the 3JHNα coupling constants and the consensus chemical shift index. The C-terminal third of the protein, which has many charged and hydrophilic amino acid residues, has no well-defined secondary structure and exists in a highly dynamic state. The N-terminal two-thirds has three helical segments (Gln10–Asp19, Glu38–Glu43, and His47–Glu54), one short extended segment (Pro34–Ala37), and a long loop (Ala20–Glu29), of which part may undergo intermediate conformational exchange. Solution of the three-dimensional structure by NMR techniques revealed that the helices fold in such a way that the surface of θ is bipolar, with one face of the protein containing most of the acidic residues and the other face containing most of the long chain basic residues. Preliminary chemical shift mapping experiments with a domain of the ε subunit have identified a loop region (Ala20–Glu29) in θ as the site of association with ε.
An enigmatic peptide ligation reaction: Protease-catalyzed oligomerization of a native protein segment in neat aqueous solution
- SANGARALINGAM KUMARAN, DEBJANI DATTA, RAJENDRA P. ROY
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- 01 April 2000, pp. 734-741
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We report an enigmatic peptide ligation reaction catalyzed by Glu-specific Staphylococcus aureus V8 protease that occurs in neat aqueous solution around neutral pH utilizing a totally unprotected peptide substrate containing free α-carboxyl and α-amino groups. V8 protease catalyzed a chain of ligation steps between pH 6 and 8 at 4 °C, producing a gamut of covalent oligomers (dimer through octamer or higher) of a native protein segment TAAAKFE (S39) derived from ribonuclease A (RNAse A). Size-exclusion chromatography suggested the absence of strong interaction between the reacting peptides. The circular dichroism spectra of monomer through pentamer showed length-dependent enhancement of secondary structure in the oligomers, suggesting that protease-catalyzed ligation of a monomer to an oligomer resulted in a product that was more structured than its precursor. The relative conformational stability of the oligomers was reflected in their ability to resist proteolysis, indicating that the oligomerization reaction was facilitated as a consequence of the “conformational trapping” of the product. The ligation reaction proceeded in two phases—slow formation and accumulation of the dimer followed by a fast phase of oligomerization, implying that the conformational trap encountered in the oligomerization reaction was a two-step process. The Gly substitution at any position of the TAAAKFE sequence was deleterious, suggesting that the first step of the conformational trap, namely the dimerization reaction, that proceeded very slowly even with the parent peptide, was quite sensitive to amino acid sequence. In contrast, the oligomerization reaction of an Ala analog, AAAAKFE, occurred in much the same way as S39, albeit with faster rate, suggesting that Ala substitution stabilized the overall conformational trapping process. The results suggest the viability of the product-directed “conformational trap” as a mechanism to achieve peptide ligation of totally unprotected peptide fragments in neat aqueous solution. Further, the study projects the presence of considerable innate synthetic potential in V8 protease, baring rich possibilities of protein engineering of this enzyme to generate a “V8 peptide ligase.”
Insect peptides with improved protease-resistance protect mice against bacterial infection
- LASZLO OTVOS, KRISZTINA BOKONYI, ISTVAN VARGA, BALINT I. OTVOS, RALF HOFFMANN, HILDEGUND C.J. ERTL, JOHN D. WADE, AILSA M. McMANUS, DAVID J. CRAIK, PHILIPPE BULET
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- 01 April 2000, pp. 742-749
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At a time of the emergence of drug-resistant bacterial strains, the development of antimicrobial compounds with novel mechanisms of action is of considerable interest. Perhaps the most promising among these is a family of antibacterial peptides originally isolated from insects. These were shown to act in a stereospecific manner on an as-yet unidentified target bacterial protein. One of these peptides, drosocin, is inactive in vivo due to the rapid decomposition in mammalian sera. However, another family member, pyrrhocoricin, is significantly more stable, has increased in vitro efficacy against Gram-negative bacterial strains, and if administered alone, as we show here, is devoid of in vitro or in vivo toxicity. At low doses, pyrrhocoricin protected mice against Escherichia coli infection, but at a higher dose augmented the infection of compromised animals. Analogs of pyrrhocoricin were, therefore, synthesized to further improve protease resistance and reduce toxicity. A linear derivative containing unnatural amino acids at both termini showed high potency and lack of toxicity in vivo and an expanded cyclic analog displayed broad activity spectrum in vitro. The bioactive conformation of native pyrrhocoricin was determined by nuclear magnetic resonance spectroscopy, and similar to drosocin, reverse turns were identified as pharmacologically important elements at the termini, bridged by an extended peptide domain. Knowledge of the primary and secondary structural requirements for in vivo activity of these peptides allows the design of novel antibacterial drug leads.
Determination of α-helix N1 energies after addition of N1, N2, and N3 preferences to helix/coil theory
- JIA KE SUN, SIMON PENEL, ANDREW J. DOIG
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- 01 April 2000, pp. 750-754
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Surveys of protein crystal structures have revealed that amino acids show unique structural preferences for the N1, N2, and N3 positions in the first turn of the α-helix. We have therefore extended helix-coil theory to include statistical weights for these locations. The helix content of a peptide in this model is a function of N-cap, C-cap, N1, N2, N3, C1, and helix interior (N4 to C2) preferences. The partition function for the system is calculated using a matrix incorporating the weights of the fourth residue in a hexamer of amino acids and is implemented using a FORTRAN program. We have applied the model to calculate the N1 preferences of Gln, Val, Ile, Ala, Met, Pro, Leu, Thr, Gly, Ser, and Asn, using our previous data on helix contents of peptides Ac-XAKAAAAKAAGY-CONH2. We find that Ala has the highest preference for the N1 position. Asn is the most unfavorable, destabilizing a helix at N1 by at least 1.4 kcal mol−1 compared to Ala. The remaining amino acids all have similar preferences, 0.5 kcal mol−1 less than Ala. Gln, Asn, and Ser, therefore, do not stabilize the helix when at N1.
Improving protein crystal quality by decoupling nucleation and growth in vapor diffusion
- EMMANUEL SARIDAKIS, NAOMI E. CHAYEN
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- 01 April 2000, pp. 755-757
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A simple method for growing protein crystals in the metastable zone using the vapor diffusion technique is described. The coverslips holding the hanging drops are transferred, after being incubated for some time at conditions normally giving many small crystals, over reservoirs at concentrations that normally yield clear drops. Fewer, much larger and better diffracting crystals are obtained, compared with conventional crystallization at similar conditions. To our knowledge, this is the first report of a significant crystal improvement due to “backing off” from nucleation conditions, using the hanging drop method.
A correlation of the transfer time with published results for vapor diffusion equilibration of poly(ethylene glycol) solutions is also presented.
Specificity in substrate binding by protein folding catalysts: Tyrosine and tryptophan residues are the recognition motifs for the binding of peptides to the pancreas-specific protein disulfide isomerase PDIp
- LLOYD W. RUDDOCK, ROBERT B. FREEDMAN, PETER KLAPPA
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- 01 April 2000, pp. 758-764
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Using a cross-linking approach, we recently demonstrated that radiolabeled peptides or misfolded proteins specifically interact in vitro with two luminal proteins in crude extracts from pancreas microsomes. The proteins were the folding catalysts protein disulfide isomerase (PDI) and PDIp, a glycosylated, PDI-related protein, expressed exclusively in the pancreas. In this study, we explore the specificity of these proteins in binding peptides and related ligands and show that tyrosine and tryptophan residues in peptides are the recognition motifs for their binding by PDIp. This peptide-binding specificity may reflect the selectivity of PDIp in binding regions of unfolded polypeptide during catalysis of protein folding.
Analysis of knowledge-based protein–ligand potentials using a self-consistent method
- J. SHIMADA, A.V. ISHCHENKO, E.I. SHAKHNOVICH
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- 01 April 2000, pp. 765-775
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We propose a self-consistent approach to analyze knowledge-based atom–atom potentials used to calculate protein–ligand binding energies. Ligands complexed to actual protein structures were first built using the SMoG growth procedure (DeWitte & Shakhnovich, 1996) with a chosen input potential. These model protein–ligand complexes were used to construct databases from which knowledge-based protein–ligand potentials were derived. We then tested several different modifications to such potentials and evaluated their performance on their ability to reconstruct the input potential using the statistical information available from a database composed of model complexes. Our data indicate that the most significant improvement resulted from properly accounting for the following key issues when estimating the reference state: (1) the presence of significant nonenergetic effects that influence the contact frequencies and (2) the presence of correlations in contact patterns due to chemical structure. The most successful procedure was applied to derive an atom–atom potential for real protein–ligand complexes. Despite the simplicity of the model (pairwise contact potential with a single interaction distance), the derived binding free energies showed a statistically significant correlation (∼0.65) with experimental binding scores for a diverse set of complexes.
Novel disulfide engineering in human carbonic anhydrase II using the PAIRWISE side-chain geometry database
- RANDALL E. BURTON, JENNIFER A. HUNT, CAROL A. FIERKE, TERRENCE G. OAS
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- 01 April 2000, pp. 776-785
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An analysis of the pairwise side-chain packing geometries of cysteine residues observed in high-resolution protein crystal structures indicates that cysteine pairs have pronounced orientational preferences due to the geometric constraints of disulfide bond formation. A potential function was generated from these observations and used to evaluate models for novel disulfide bonds in human carbonic anhydrase II (HCAII). Three double-cysteine variants of HCAII were purified and the effective concentrations of their thiol groups were determined by titrations with glutathione and dithiothreitol. The effects of the cysteine mutations on the native state structure and stability were characterized by circular dichroism, enzymatic activity, sulfonamide binding, and guanidine hydrochloride titration. These analyses indicate that the PAIRWISE potential is a good predictor of the strength of the disulfide bond itself, but the overall structural and thermodynamic effects on the protein are complicated by additional factors. In particular, the effects of cysteine substitutions on the native state and the stabilization of compact nonnative states by the disulfide can override any stabilizing effect of the cross-link.
Structure of an analog of fusion peptide from hemagglutinin
- PETER V. DUBOVSKII, HUA LI, SHO TAKAHASHI, ALEXANDER S. ARSENIEV, KAZUYUKI AKASAKA
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- 01 April 2000, pp. 786-798
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A 20-residue peptide E5 containing five glutamates, an analog of the fusion peptide of influenza virus hemagglutinin (HA) exhibiting fusion activity at acidic pH lower than 6.0–6.5 was studied by circular dichroism (CD), Fourier transform infrared, and 1H-NMR spectroscopy in water, water/trifluoroethanol (TFE) mixtures, dodecylphosphocholine (DPC) micelles, and phospholipid vesicles. E5 became structurally ordered at pH ≤6 and the helical content in the peptide increased in the row: water < water/TFE < DPC ∼ phospholipid vesicle while the amount of β-structure was approximately reverse. 1H-NMR data and line-broadening effect of 5-, 16-doxylstearates on proton resonances of DPC bound peptide showed E5 forms amphiphilic α-helix in residues 2–18, which is flexible in 11–18 part. The analysis of the proton chemical shifts of DPC bound and CD intensity at 220 nm of phospholipid bound E5 showed that the pH dependence of helical content is characterized by the same pKa ≈5.6. Only Glu11 and Glu15 in DPC bound peptide showed such elevated pKas, presumably due to transient hydrogen bond(s) Glu11 (Glu15) δCOO−(H+) … HN Glu15 that dispose(s) the side chain of Glu11 (Glu15) residue(s) close to the micelle/water interface. These glutamates are present in the HA-fusion peptide and the experimental half-maximal pH of fusion for HA and E5 peptides is ∼5.6. Therefore, a specific anchorage of these peptides onto membrane necessary for fusion is likely driven by the protonation of the carboxylate group of Glu11 (Glu15) residue(s) participating in transient hydrogen bond(s).
Folding of a dimeric β-barrel: Residual structure in the urea denatured state of the human papillomavirus E2 DNA binding domain
- YU-KEUNG MOK, LEONARDO G. ALONSO, L. MAURICIO T.R. LIMA, MARK BYCROFT, GONZALO DE PRAT-GAY
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- 01 April 2000, pp. 799-811
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The dimeric β-barrel is a characteristic topology initially found in the transcriptional regulatory domain of the E2 DNA binding domain from papillomaviruses. We have previously described the kinetic folding mechanism of the human HPV-16 domain, and, as part of these studies, we present a structural characterization of the urea-denatured state of the protein. We have obtained a set of chemical shift assignments for the C-terminal domain in urea using heteronuclear NMR methods and found regions with persistent residual structure. Based on chemical shift deviations from random coil values, 3JNHNα coupling constants, heteronuclear single quantum coherence peak intensities, and nuclear Overhauser effect data, we have determined clusters of residual structure in regions corresponding to the DNA binding helix and the second β-strand in the folded conformation. Most of the structures found are of nonnative nature, including turn-like conformations. Urea denaturation at equilibrium displayed a loss in protein concentration dependence, in absolute parallel to a similar deviation observed in the folding rate constant from kinetic experiments. These results strongly suggest an alternative folding pathway in which a dimeric intermediate is formed and the rate-limiting step becomes first order at high protein concentrations. The structural elements found in the denatured state would collide to yield productive interactions, establishing an intermolecular folding nucleus at high protein concentrations. We discuss our results in terms of the folding mechanism of this particular topology in an attempt to contribute to a better understanding of the folding of dimers in general and intertwined dimeric proteins such as transcription factors in particular.
Scoring functions in protein folding and design
- RUXANDRA I. DIMA, JAYANTH R. BANAVAR, AMOS MARITAN
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- 01 April 2000, pp. 812-819
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We present an analysis of the assumptions behind some of the most commonly used methods for evaluating the goodness of the fit between a sequence and a structure. Our studies on a lattice model show that methods based on statistical considerations are easy to use and can capture some of the features of protein-like sequences and their corresponding native states, but unfortunately are incapable of recognizing, with certainty, the native-like conformation of a sequence among a set of decoys. Meanwhile, an optimization method, entailing the determination of the parameters of an effective free energy of interaction, is much more reliable in recognizing the native state of a sequence. However, the statistical method is shown to perform quite well in tests of protein design.