bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural and functional consequences of the presence of a fourth disulfide bridge in the scorpion short toxins: Solution structure of the potassium channel inhibitor HsTX1
Authors: Savarin Philippe , Romi‐Lebrun Régine , Zinn‐Justin Sophie , Gilquin Bernard , Ménez André , Lebrun Bruno , Nakajima Terumi
Published: February 2009

We have determined the three‐dimensional structure of the potassium channel inhibitor HsTX1, using nuclear magnetic resonance and molecular modeling. This protein belongs to the scorpion short toxin family, which essentially contains potassium channel blockers of 29 to 39 amino acids and three disulfide bridges. It is highly active on voltage‐gated Kv1.3 potassium channels. Furthermore, it has the particularity to possess a fourth disulfide bridge. We show that HsTX1 has a fold similar to that of the three‐disulfide‐bridged toxins and conserves the hydrophobic core found in the scorpion short toxins. Thus, the fourth bridge has no influence on the global conformation of HsTX1. Most residues spatially analogous to those interacting with voltage‐gated potassium channels in the three‐disulfide‐bridged toxins are conserved in HsTX1. Thus, we propose that Tyr21, Lys23, Met25, and Asn26 are involved in the biological activity of HsTX1. As an additional positively charged residue is always spatially close to the aromatic residue in toxins blocking the voltage‐gated potassium channels, and as previous mutagenesis experiments have shown the critical role played by the C‐terminus in HsTX1, we suggest that Arg33 is also important for the activity of the four disulfide‐bridged toxin. Docking calculations confirm that, if Lys23 and Met25 interact with the GYGDMH motif of Kv1.3, Arg33 can contact Asp386 and, thus, play the role of the additional positively charged residue of the toxin functional site. This original configuration of the binding site of HsTX1 for Kv1.3, if confirmed experimentally, offers new structural possibilities for the construction of a molecule blocking the voltage‐gated potassium channels.
Inhibition of the HIV‐1 and HIV‐2 proteases by a monoclonal antibody
Authors: Lescar Julien , Brynda Jiri , Bentley Graham A. , Riottot Marie‐Madeleine , Chitarra Véronique , Rezacova Pavlina , Stouracova Renata , Fabry Milan , Horejsi Magda , Sedlacek Juraj
Published: February 2009

The monoclonal antibody 1696, directed against the HIV‐1 protease, displays strong inhibitory effects toward the catalytic activity of the enzyme of both the HIV‐1 and HIV‐2 isolates. This antibody cross‐reacts with peptides that include the N‐terminus of the enzyme, a region that is well conserved in sequence among different viral strains and which, furthermore, is crucial for homodimerization to the active enzymatic form. This observation, as well as antigenbinding studies in the presence of an active site inhibitor, suggest that 1696 inhibits the HIV protease by destabilizing its active homodimeric form. To characterize further how the antibody 1696 inhibits the HIV‐1 and HIV‐2 proteases, we have solved the crystal structure of its Fab fragment by molecular replacement and refined it at 3.0 Å resolution. The antigen binding site has a deep cavity at its center, which is lined mainly by acidic and hydrophobic residues, and is large enough to accommodate several antigen residues. The structure of the Fab 1696 could form a starting basis for the design of alternative HIV protease‐inhibiting molecules of broad specificity.
Robustness of protein folding kinetics to surface hydrophobic substitutions
Authors: Gu H. , Doshi N. , Kim D.E. , Simons K.T. , Santiago J.V. , Nauli S. , Baker David
Published: February 2009

We use both combinatorial and site‐directed mutagenesis to explore the consequences of surface hydrophobic substitutions for the folding of two small single domain proteins, the src SH3 domain, and the IgG binding domain of Peptostreptococcal protein L. We find that in almost every case, destabilizing surface hydrophobic substitutions have much larger effects on the rate of unfolding than on the rate of folding, suggesting that nonnative hydrophobic interactions do not significantly interfere with the rate of core assembly.
Structural determinants in domain II of human glutathione transferase M2–2 govern the characteristic activities with aminochrome, 2‐cyano‐1, 3‐dimethyl‐1‐nitrosoguanidine, and 1, 2‐dichloro‐4‐nitrobenzene
Authors: Hansson Lars O. , Widersten Mikael , Mannervik Bengt , Bolton‐Grob Robyn
Published: February 2009

Two human Mu class glutathione transferases, hGST M1–1 and hGST M2–2, with high sequence identity (84%) exhibit a 100‐fold difference in activities with the substrates aminochrome, 2‐cyano‐1, 3‐dimethyl‐1‐nitrosoguanidine (cyanoDMNG), and 1, 2‐dichloro‐4‐nitrobenzene (DCNB), hGST M2–2 being more efficient. A sequence alignment with the rat Mu class GST M3–3, an enzyme also showing high activities with aminochrome and DCNB, demonstrated an identical structural cluster of residues 164–168 in the α6‐helices of rGST M3–3 and hGST M2–2, a motif unique among known sequences of human, rat, and mouse Mu class GSTs. A putative electrostatic network Arg107‐Asp161‐Arg165‐Glu164(‐Gln167) was identified based on the published three‐dimensional structure of hGST M2–2. Corresponding variant residues of hGSTM1–1 (Leu165, Asp164, and Arg167) as well as the active site residue Ser209 were targeted for point mutations, introducing hGST M2–2 residues to the framework of hGST M1–1, to improve the activities with substrates characteristic of hGST M2–2. In addition, chimeric enzymes composed of hGST M1–1 and hGST M2–2 sequences were analyzed. The activity with 1‐chloro‐2, 4‐dinitrobenzene (CDNB) was retained in all mutant enzymes, proving that they were catalytically competent, but none of the point mutations improved the activities with hGST M2–2 characteristic substrates. The chimeric enzymes showed that the structural determinants of these activities reside in domain II and that residue Arg 165 in hGST M2–2 appears to be important for the reactions with cyanoDMNG and DCNB. A mutant, which contained all the hGST M2–2 residues of the putative electrostatic network, was still lacking one order of magnitude of the activities with the characteristic substrates of wild‐type hGST M2–2. It was concluded that a limited set of point mutations is not sufficient, but that indirect secondary structural affects also contribute to the hGST M2–2 characteristic activities with aminochrome, cyanoDMNG, and DCNB.
Human liver mitochondrial aldehyde dehydrogenase: Three‐dimensional structure and the restoration of solubility and activity of chimeric forms
Authors: Ni Li , Zhou Jianzhong , Weiner Henry , Hurley Thomas D.
Published: February 2009

Human liver cytosolic and mitochondrial isozymes of aldehyde dehydrogenase share 70% sequence identity. However, the first 21 residues are not conserved between the human isozymes (15% identity). The three‐dimensional structures of the beef mitochondrial and sheep cytosolic forms have virtually identical three‐dimensional structures. Here, we solved the structure of the human mitochondrial enzyme and found it to be identical to the beef enzyme. The first 21 residues are found on the surface of the enzyme and make no contact with other subunits in the tetramer. A pair of chimeric enzymes between the human isozymes was made. Each chimera had the first 21 residues from one isozyme and the remaining 479 from the other. When the first 21 residues were from the mitochondrial isozyme, an enzyme with cytosolic‐like properties was produced. The other was expressed but was insoluble. It was possible to restore solubility and activity to the chimera that had the first 21 cytosolic residues fused to the mitochondrial ones by making point mutations to residues at the N‐terminal end. When residue 19 was changed from tyrosine to a cysteine, the residue found in the mitochondrial form, an active enzyme could be made though the Km for NAD+ was 35 times higher than the native mitochondrial isozyme and the specific activity was reduced by 75%. This residue interacts with residue 203, a nonconserved, nonactive site residue. A mutation of residue 18, which also interacts with 203, restored solubility, but not activity. Mutation to residue 15, which interacts with 104, also restored solubility but not activity. It appears that to have a soluble or active enzyme a favorable interaction must occur between a residue in a surface loop and a residue elsewhere in the molecule even though neither make contact with the active site region of the enzyme.
Feasibility in the inverse protein folding protocol
Authors: Ota Motonori , Nishikawa Ken
Published: February 2009

Methods for protein structure (3D)–sequence (1D) compatibility evaluation (threading) have been developed during the past decade. The protocol in which a sequence can recognize its compatible structure in the structural library (i.e., the fold recognition or the forward‐folding search) is available for the structure prediction of new proteins. However, the reverse protocol, in which a structure recognizes its homologous sequences among a sequence database, named the inverse‐folding search, is a more difficult application. In this study, we have investigated the feasibility of the latter approach. A structural library, composed of about 400 well‐resolved structures with mutually dissimilar sequences, was prepared, and 163 of them had remote homologs in the library. We examined whether they could correctly seek their homologs by both forward‐and inverse‐folding searches. The results showed that the inverse‐folding protocol is more effective than the forward‐folding protocol, once the reference states of the compatibility functions are appropriately adjusted. This adjustment only slightly affects the ability of the forward‐folding search. We noticed that the scoring, in which a given sequence is re‐mounted onto a structure according to the 3D–1D alignment determined by the dynamic programming method, is only effective in the forward‐folding protocol and not in the inverse‐folding protocol. Namely, the inverse‐folding search works significantly better with the score given by the 3D–1D alignment per se, rather than that obtained by the re‐mounting. The implications of these results are discussed.
Macromolecular docking of a three‐body system: The recognition of human growth hormone by its receptor
Authors: Hendrix Donna K. , Klein Teri E. , Kuntz Irwin D.
Published: February 2009

Human growth hormone (hGH) binds to its receptor (hGHr) in a three‐body interaction: one molecule of the hormone and two identical monomers of the receptor form a trimer. Curiously, the hormone‐receptor interactions in the trimer are not equivalent and the formation of the complex occurs in a specific kinetic order (Cunningham BC, Ultsch M, De Vos AM, Mulkerrin MG, Clauser KR, Wells JA, 1991, Science 254:821–825). In this paper, we model the recognition of hGH to the hGHr using shape complementarity of the three‐dimensional structures and macromolecular docking to explore possible binding modes between the receptor and hormone. The method, reported previously (Hendrix DK, Kuntz ID, 1998, Pacific symposium on biocomputing 1998, pp 1234–1244), is based upon matching complementary‐shaped strategic sites on the molecular surface. We modify the procedure to examine three‐body systems. We find that the order of binding seen experimentally is also essential to our model. We explore the use of mutational data available for hGH to guide our model. In addition to docking hGH to the hGHr, we further test our methodology by successfully reproducing 16 macromolecular complexes from X‐ray crystal structures, including enzyme‐inhibitor, antibody‐antigen, protein dimer, and protein‐DNA complexes.
Ternary complex structure of human hgprtase, prpp, mg2+, and the inhibitor HPP reveals the involvement of the flexible loop in substrate binding
Authors: Balendiran Ganesaratnam K. , Molina JOSÉ A. , Xu Yiming , Torres‐Martinez Jan , Stevens Robert , Focia Pamela J. , Eakin Ann E. , Sacchettini James C. , Craig Sydney P.
Published: February 2009

Site‐directed mutagenesis was used to replace Lys68 of the human hypoxanthine phosphoribosyltransferase (HGPRTase) with alanine to exploit this less reactive form of the enzyme to gain additional insights into the structure activity relationship of HGPRTase. Although this substitution resulted in only a minimal (one‐to threefold) increase in the Km values for binding pyrophosphate or phosphoribosylpyrophosphate, the catalytic efficiencies (kcat/Km) of the forward and reverse reactions were more severely reduced (6‐ to 30‐fold), and the mutant enzyme showed positive cooperativity in binding of α‐D‐5‐phosphoribosyl‐1‐pyrophosphate (PRPP) and nucleotide. The K68A form of the human HGPRTase was cocrystallized with 7‐hydroxy [4, 3‐d] pyrazolo pyrimidine (HPP) and Mg PRPP, and the refined structure reported. The PRPP molecule built into the [(Fo – Fc)ϕcalc] electron density shows atomic interactions between the Mg PRPP and enzyme residues in the pyrophosphate binding domain as well as in a long flexible loop (residues Leu101 to Gly111) that closes over the active site. Loop closure reveals the functional roles for the conserved SY dipeptide of the loop as well as the molecular basis for one form of gouty arthritis (S103R). In addition, the closed loop conformation provides structural information relevant to the mechanism of catalysis in human HGPRTase.
Prediction of the location and type of β‐turns in proteins using neural networks
Authors: Shepherd Adrian J. , Gorse Denise , Thornton Janet M.
Published: February 2009

A neural network has been used to predict both the location and the type of β‐turns in a set of 300 nonhomologous protein domains. A substantial improvement in prediction accuracy compared with previous methods has been achieved by incorporating secondary structure information in the input data. The total percentage of residues correctly classified as β‐turn or not‐β‐turn is around 75% with predicted secondary structure information. More significantly, the method gives a Matthews correlation coefficient (MCC) of around 0.35, compared with a typical MCC of around 0.20 using other β‐turn prediction methods. Our method also distinguishes the two most numerous and well‐defined types of β‐turn, types I and II, with a significant level of accuracy (MCCs 0.22 and 0.26, respectively).
Expression, purification, and crystallography of the conserved methionine‐rich domain of human signal recognition particle 54 kda protein
Authors: Gowda Krishne , Clemons William M. , Zwieb Christian , Black Shaun D.
Published: February 2009

Protein SRP54 is an essential component of eukaryotic signal recognition particle (SRP). The methionine‐rich M‐domain (SRP54M or 54M) interacts with the SRP RNA and is also involved in the binding to signal peptides of secretory proteins during their targeting to cellular membranes. To gain insight into the molecular details of SRP‐mediated protein targeting, we studied the human 54M polypeptide. The recombinant human protein was expressed successfully in Escherichia coli and was purified to homogeneity. Our studies determined the sites that were susceptible to limited proteolysis, with the goal to design smaller functional mutant derivatives that lacked nonessential amino acid residues from both termini. Of the four polypeptides produced by V8 protease or chymotrypsin, 54MM‐2 was the shortest (120 residues; Mr = 13, 584.8), but still contained the conserved amino acids suggested to associate with the signal peptide or the SRP RNA. 54MM‐2 was cloned, expressed, purified to homogeneity, and was shown to bind human SRP RNA in the presence of protein SRP19, indicating that it was functional. Highly reproducible conditions for the crystallization of 54MM‐2 were established. Examination of the crystals by X‐ray diffraction showed an orthorhombic unit cell of dimensions a = 29.127 Å, b = 63.693 Å, and c = 129.601 Å, in space group P212121, with reflections extending to at least 2.0 Å.
Energetic analysis of an antigen/antibody interface: Alanine scanning mutagenesis and double mutant cycles on the hyhel‐10/lysozyme interaction
Authors: Pons Jaume , Rajpal Arvind , Kirsch Jack F.
Published: February 2009

Alanine scanning mutagenesis of the HyHEL‐10 paratope of the HyHEL‐10/HEWL complexdemonstrates that the energetically important side chains (hot spots) of both partners are in contact.A plot of ΔΔGHyHEL‐10_mutant vs.ΔΔGhewl_mutant for the five of six interacting side‐chain hydrogen bonds is linear (Slope = 1).Only 3 of the 13 residues in the HEWL epitope contribute >4 kcal/mol to the free energy of formation of the complexwhen replaced by alanine, but 6 of the 12 HyHEL‐10 paratope amino acids do.Double mutant cycle analysis of the single crystallographically identified salt bridge, D32H/K97, shows that there is a significant energetic penalty when either partner is replaced with a neutral side‐chain amino acid, but the D32HN/K97M complex is as stable as the WT.The role of the disproportionately high number of Tyr residues in the CDR was evaluated by comparing theΔΔG values of the Tyr Phe vs. the corresponding Tyr Ala mutations.The nonpolar contacts in the light chain contribue only about one‐half of the totalΔΔG observed for the Tyr Ala mutation, while they are significantly more important in the heavy chain.Replacement of the N31L/K96 hydrogen bond with a salt bridge, N31DL/K96, destabilizes the complex by 1.4 kcal/mol. The free energy of interaction,ΔΔGint, obtained from double mutant cycle analysis showed that ΔΔGint for any complex for which the HEWL residue probed is a major immunodeterminant is very close to the loss of free energy observed for the HyHEL‐10 single mutant.Error propagation analysis of double mutant cycles shows that data of atypically high precision are required to use this method meaningfully, except where large ΔΔG values are analyzed.
The variable and conserved interfaces of modeled olfactory receptor proteins
Authors: Pilpel Yitzhak , Lancet Doron
Published: February 2009

The accumulation of hundreds of olfactory receptor (OR) sequences, along with the recent availability of detailed models of other G‐protein‐coupled receptors, allows us to analyze the OR amino acid variability patterns in a structural context. A Fourier analysis of 197 multiply aligned olfactory receptor sequences showed an α‐helical periodicity in the variability profile. This was particularly pronounced in the more variable transmembranal segments 3, 4, and 5. Rhodopsin‐based homology modeling demonstrated that the inferred variable helical faces largely point to the interior of the receptor barrel. We propose that a set of 17 hypervariable residues, which point to the barrel interior and are more extracellularly disposed, constitute the odorant complementarity determining regions. While 12 of these residues coincide with established ligand‐binding contact postions in other G‐protein‐coupled receptors, the rest are suggested to form an olfactory‐unique aspect of the binding pocket. Highly conserved olfactory receptor‐specific sequence motifs, found in the second and third intracellular loops, may comprise the G‐protein recognition epitope. The prediction of olfactory receptor functional sites provides concrete suggestions of site‐directed mutagenesis experiments for altering ligand and G‐protein specificity.
ChloroP, a neural network‐based method for predicting chloroplast transit peptides and their cleavage sites
Authors: Emanuelsson Olof , Nielsen Henrik , Heijne Gunnar Von
Published: February 2009

We present a neural network based method (ChloroP) for identifying chloroplast transit peptides and their cleavage sites. Using cross‐validation, 88% of the sequences in our homology reduced training set were correctly classified as transit peptides or nontransit peptides. This performance level is well above that of the publicly available chloroplast localization predictor PSORT. Cleavage sites are predicted using a scoring matrix derived by an automatic motif‐finding algorithm. Approximately 60% of the known cleavage sites in our sequence collection were predicted to within 62 residues from the cleavage sites given in SWISS‐PROT. An analysis of 715 Arabidopsis thaliana sequences from SWISS‐PROT suggests that the ChloroP method should be useful for the identification of putative transit peptides in genome‐wide sequence data. The ChloroP predictor is available as a web‐server at http://www.cbs.dtu.dk/services/ChloroP/.
Polymer principles and protein folding
Authors: Dill Ken A.
Published: February 2009

This paper surveys the emerging role of statistical mechanics and polymer theory in protein folding. In the polymer perspective, the folding code is more a solvation code than a code of local ØΨ propensities. The polymer perspective resolves two classic puzzles: (1) the Blind Watchmaker' Paradox that biological proteins could not have originated from random sequences, and (2) Levinthal' Paradox that the folded state of a protein cannot be found by random search. Both paradoxes are traditionally framed in terms of random unguided searches through vast spaces, and vastness is equated with impossibility. But both processes are partly guided. The searches are more akin to balls rolling down funnels than balls rolling aimlessly on flat surfaces. In both cases, the vastness of the search is largely irrelevant to the search time and success. These ideas are captured by energy and fitness landscapes. Energy landscapes give a language for bridging between microscopics and macroscopics, for relating folding kinetics to equilibrium fluctuations, and for developing new and faster computational search strategies.
Folding funnels, binding funnels, and protein function
Authors: Tsai Chung‐Jung , Kumar Sandeep , Ma Buyong , Nussinov Ruth
Published: February 2009

Folding funnels have been the focus of considerable attention during the last few years. These have mostly been discussed in the general context of the theory of protein folding. Here we extend the utility of the concept of folding funnels, relating them to biological mechanisms and function. In particular, here we describe the shape of the funnels in light of protein synthesis and folding; flexibility, conformational diversity, and binding mechanisms; and the associated binding funnels, illustrating the multiple routes and the range of complexed conformers. Specifically, the walls of the folding funnels, their crevices, and bumps are related to the complexity of protein folding, and hence to sequential vs. nonsequential folding. Whereas the former is more frequently observed in eukaryotic proteins, where the rate of protein synthesis is slower, the latter is more frequent in prokaryotes, with faster translation rates. The bottoms of the funnels reflect the extent of the flexibility of the proteins. Rugged floors imply a range of conformational isomers, which may be close on the energy landscape. Rather than undergoing an induced fit binding mechanism, the conformational ensembles around the rugged bottoms argue that the conformers, which are most complementary to the ligand, will bind to it with the equilibrium shifting in their favor. Furthermore, depending on the extent of the ruggedness, or of the smoothness with only a few minima, we may infer nonspecific, broad range vs. specific binding. In particular, folding and binding are similar processes, with similar underlying principles. Hence, the shape of the folding funnel of the monomer enables making reasonable guesses regarding the shape of the corresponding binding funnel. Proteins having a broad range of binding, such as proteolytic enzymes or relatively nonspecific endonucleases, may be expected to have not only rugged floors in their folding funnels, but their binding funnels will also behave similarly, with a range of complexed conformations. Hence, knowledge of the shape of the folding funnels is biologically very useful. The converse also holds: If kinetic and thermodynamic data are available, hints regarding the role of the protein and its binding selectivity may be obtained. Thus, the utility of the concept of the funnel carries over to the origin of the protein and to its function.
The 2.1 Å structure of an elicitin‐ergosterol complex: A recent addition to the Sterol Carrier Protein family
Authors: Boissy Guillaume , O'Donohue Michael , Gaudemer Odile , Perez Valérie , Pernollet Jean‐Claude , Brunie Simone
Published: February 2009

Elicitins, produced by most of the phytopathogenic fungi of the genus Phytophthora, provoke in tobacco both remote leaf necrosis and the induction of a resistance against subsequent attack by various microorganisms. Despite the recent description of the three‐dimensional crystal structure of cryptogein (CRY), the molecular basis of the interactions between Phytophthora and plants largely remains unknown. The X‐ray crystal structure, refined at 2.1 Å, of a ligand complexed, mutated CRY, K13H, is reported. Analysis of this structure reveals that CRY is able to encapsulate a ligand that induces only a minor conformational change in the protein structure. The ligand has been identified as an ergosterol by gas chromatographic analysis coupled with mass spectrometry analysis. This result is consistent with biochemical data that have shown that elicitins are a distinct class of Sterol Carrier Proteins (SCP). Data presented here provide the first structural description of the pertinent features of the elicitin sterol interaction and permit a reassessment of the importance of both the key residue 13 and the mobility of the omega loop for the accessibility of the sterol to the cavity. The biological implications thereof are discussed. This paper reports the first structure of a SCP/sterol complex.
Identifying the structural boundaries of independent folding domains in the a subunit of tryptophan synthase, a β/α barrel protein
Authors: Zitzewitz Jill A. , Gualfetti Peter J. , Perkons Ieva A. , Wasta Stacey A. , Matthews C. Robert
Published: February 2009

Two equilibrium intermediates have previously been observed in the urea denaturation of the α subunit of tryptophan synthase (αTS) fromEscherichia coli, an eight‐stranded β/α barrel protein. In the current study, a series of amino‐terminal fragments were characterized to probe the elementary folding units that may be in part responsible for this complex behavior. Stop‐codon mutagenesis was used to produce eight fragments ranging in size from 105–214 residues and containing incremental elements of secondary structure. Equilibrium studies by circular dichroism indicate that all of these fragments are capable of adopting secondary structure. All except for the shortest fragment fold cooperatively. The addition of the fourth, sixth, and eighth β‐strands leads to distinct increases in structure, cooperativity, and/or stability, suggesting that folding involves the modular assembly of βαβ supersecondary structural elements. One‐dimensional NMR titrations at high concentrations of urea, probing the environment around His92, were also performed to test for the presence of residual structure in the fragments. All fragments that contained the first four βα units of structure exhibited a cooperative unfolding transition at high concentrations of urea with significant but reduced stability relative to the full‐length protein. These results suggest that the residual structure in αTS requires the participation of hydrophobic residues in multiple β‐strands that span the entire sequence.
Sulfolobus acidocaldarius inorganic pyrophosphatase: Structure, thermostability, and effect of metal ion in an archael pyrophosphatase
Authors: Leppänen Veli‐Matti , Nummelin Heli , Hansen Thomas , Lahti Reijo , Schäfer Günter , Goldman Adrian
Published: February 2009

The first crystal structure of an inorganic pyrophosphatase (S‐PPase) from an archaebacterium, the thermophile Sulfolobus acidocaldarius, has been solved by molecular replacement and refined to an R‐factor of 19.7% at 2.7 Å. S‐PPase is a D3 homohexameric protein with one Mg2+ per active site in a position similar to, but not identical with, the first activating metal in mesophilic pyrophosphatases (PPase). In mesophilic PPases, Asp65, Asp70, and Asp102 coordinate the Mg2+ while only Asp65 and Asp102 do in S‐PPase, and the Mg2+ moves by 0.7 Å. S‐PPase may therefore be deactivated at low temperature by mispositioning a key metal ion.

The monomer S‐PPase structure is very similar to that of Thermus thermophilus (T‐PPase) and Escherichia coli (E‐PPase), root‐mean‐square deviations around 1 Å/Cα. But the hexamer structures of S‐and T‐PPase are more tightly packed and more similar to each other than they are to that of E‐PPase, as shown by the increase in surface area buried upon oligomerization. In T‐PPase, Arg116 creates an interlocking ionic network to both twofold and threefold related monomers; S‐PPase has hydrophilic interactions to threefold related monomers absent in both E‐and T‐PPase. In addition, the thermostable PPases have about 7% more hydrogen bonds per monomer than E‐PPase, and, especially in S‐PPase, additional ionic interactions anchor the C‐terminus to the rest of the protein. Thermostability in PPases is thus due to subtle improvements in both monomer and oligomer interactions.
The molecular structure of an unusual cytochrome c2 determined at 2.0 å; the cytochrome ch from methylobacterium extorquens
Authors: Read Jon , Gill Raj , Dales Simon L. , Cooper Jon B. , Wood Steve P. , Anthony Chris
Published: February 2009

Cytochrome cH is the electron donor to the oxidase in methylotrophic bacteria. Its amino acid sequence suggests that it is a typical Class I cytochrome c, but some features of the sequence indicated that its structure might be of special interest. The structure of oxidized cytochrome cH has been solved to 2.0 Å resolution by X‐ray diffraction. It has the classical tertiary structure of the Class 1 cytochromes c but bears a closer gross resemblance to mitochondrial cytochrome c than to the bacterial cytochrome c2. The left‐hand side of the haem cleft is unique; in particular, it is highly hydrophobic, the usual water is absent, and the “conserved” Tyr67 is replaced by tryptophan. A number of features of the structure demonstrate that the usual hydrogen bonding network involving water in the haem channel is not essential and that other mechanisms may exist for modulation of redox potentials in this cytochrome.
A functional protein pore with a “retro” transmembrane domain
Authors: Cheley Stephen , Braha Orit , Lu Xiaofeng , Conlan Sean , Bayley Hagan
Published: February 2009

Extended retro (reversed) peptide sequences have not previously been accommodated within functional proteins. Here, we show that the entire transmembrane portion of the β‐barrel of the pore‐forming protein α‐hemolysin can be formed by retrosequences comprising a total of 175 amino acid residues, 25 contributed by the central sequence of each subunit of the heptameric pore. The properties of wild‐type and retro heptamers in planar bilayers are similar. The single‐channel conductance of the retro pore is 15% less than that of the wild‐type heptamer and its current‐voltage relationship denotes close to ohmic behavior, while the wild‐type pore is weakly rectifying. Both wild‐type and retro pores are very weakly anion selective. These results and the examination of molecular models suggest that β‐barrels may be especially accepting of retro sequences compared to other protein folds. Indeed, the ability to form a retro domain could be diagnostic of a β‐barrel, explaining, for example, the activity of the retro forms of many membrane‐permeabilizing peptides. By contrast with the wild‐type subunits, monomeric retro subunits undergo premature assembly in the absence of membranes, most likely because the altered central sequence fails to interact with the remainder of the subunit, thereby initiating assembly. Despite this difficulty, a technique was devised for obtaining heteromeric pores containing both wild‐type and retro subunits. Most probably as a consequence of unfavorable interstrand side‐chain interactions, the heteromeric pores are less stable than either the wild‐type or retro homoheptamers, as judged by the presence of subconductance states in single‐channel recordings. Knowledge about the extraordinary plasticity of the transmembrane β‐barrel of α‐hemolysin will be very useful in the de novo design of functional membrane proteins based on the β‐barrel motif.
Real‐time nmr studies on a transient folding intermediate of barstar
Authors: Killick Thomas R. , Freund STEFAN M. V. , Fersht Alan R.
Published: February 2009

The refolding of barstar, the intracellular inhibitor of barnase, is dominated by the slow formation of a cis peptidyl prolyl bond in the native protein. The triple mutant C40/82A P27A in which two cysteine residues and one trans proline were replaced by alanine was used as model system to investigate the kinetics and structural consequences of the trans/cis interconversion of Pro48. One‐ and two‐dimensional real‐time NMR spectroscopy was used to follow the trans/cis interconversion after folding was initiated by rapid dilution of the urea denatured protein. Series of 1H, 15N HSQC spectra acquired with and without the addition of peptidyl prolyl isomerase unambiguously revealed the accumulation of a transient trans‐Pro48 intermediate within the dead time of the experiment. Subtle chemical shift differences between the native state and the intermediate spectra indicate that the intermediate is predominantly native‐like with a local rearrangement in the Pro48 loop and in the β‐sheet region including residues Tyr47, Ala82, Thr85, and Val50.
The paradox between m values and δCp' for denaturation of ribonuclease T1 with disulfide bonds intact and broken
Authors: Baskakov Ilia V. , Bolen D.W.
Published: February 2009

Urea‐induced denaturations of RNase T1 and reduced and carboxyamidated RNase T1 (RTCAM) as a function of temperature were analyzed using the linear extrapolation method, and denaturation m values, ΔCp, ΔH, ΔS, and ΔG quantities were determined. Because both ΔCp and m values are believed to reflect the protein surface area newly exposed on denaturation, the prediction is that the ratio of m values for RNase T1 and RTCAM should equal the ΔCp ratio for the two proteins. This is not the case, for it is found that the m value of RTCAM is 1.5 times that of RNase T1, while the denaturation ΔCp' for the two proteins are identical. The paradox of why the two parameters, m and ΔCp, are not equivalent in their behavior is of importance in the interpretations of their respective molecular‐level meanings. It is found that the measured denaturation ΔCp' are consistent with ΔCp' calculated on the basis of empirical relationships between the change in surface area on denaturation (ΔASA), and that the measured m value of RNase T1 agrees with m calculated from empirical data relating m to ΔASA. However, the measured m of RTCAM is so much out of line with its calculated m as to call into question the validity of always equating m with surface area newly exposed on denaturation.
Autonomous folding of a peptide corresponding to the N‐terminal β‐hairpin from ubiquitin
Authors: Zerella Rosa , Evans Philip A. , Ionides JOHN M.C. , Packman Len C. , Trotter B. Wesley , Mackay Joel P. , Williams Dudley H.
Published: February 2009

The N‐terminal 17 residues of ubiquitin have been shown by 1H NMR to fold autonomously into a β‐hairpin structure in aqueous solution. This structure has a specific, native‐like register, though side‐chain contacts differ in detail from those observed in the intact protein. An autonomously folding hairpin has previously been identified in the case of streptococcal protein G, which is structurally homologous with ubiquitin, but remarkably, the two are not in topologically equivalent positions in the fold. This suggests that the organization of folding may be quite different for proteins sharing similar tertiary structures. Two smaller peptides have also been studied, corresponding to the isolated arms of the N‐terminal hairpin of ubiquitin, and significant differences from simple random coil predictions observed in the spectra of these subfragments, suggestive of significant limitation of the backbone conformational space sampled, presumably as a consequence of the strongly β‐structure favoring composition of the sequences. This illustrates the ability of local sequence elements to express a propensity for β‐structure even in the absence of actual sheet formation. Attempts were made to estimate the population of the folded state of the hairpin, in terms of a simple two‐state folding model. Using published “random coil” values to model the unfolded state, and values derived from native ubiquitin for the putative unique, folded state, it was found that the apparent population varied widely for different residues and with different NMR parameters. Use of the spectra of the subfragment peptides to provide a more realistic model of the unfolded state led to better agreement in the estimates that could be obtained from chemical shift and coupling constant measurements, while making it clear that some other approaches to population estimation could not give meaningful results, because of the tendency to populate the β‐region of conformational space even in the absence of the hairpin structure.
Formation of amyloid fibrils by peptides derived from the bacterial cold shock protein CspB
Authors: Groß Michael , Wilkins Deborah K. , Pitkeathly Maureen C. , Chung Evonne W. , Higham Claire , Clark Anne , Dobson Christopher M.
Published: February 2009

Three peptides covering the sequence regions corresponding to the first two (CspB‐1), the first three (CspB‐2), and the last two (CspB‐3) β‐strands of CspB, the major cold shock protein of Bacillus subtilis, have been synthesized and analyzed for their conformations in solution and for their precipitation behavior. The peptides are nearly insoluble in water, but highly soluble in aqueous solutions containing 50% acetonitrile (pH 4.0). Upon shifts of the solvent condition toward lower or higher acetonitrile concentrations, the peptides all form fibrils resembling those observed in amyloid associated diseases. These fibrils have been identified and characterized by electron microscopy, binding of the dye congo red, and X‐ray fiber diffraction. Characterization of the peptides in solution by circular dichroism and NMR spectroscopy shows that the formation of these fibrils does not require specific preformed secondary structure in the solution state species. While the majority of the soluble fraction of each peptide is monomeric and unstructured, different types of structures including α‐helical, β‐sheet, and random coil conformations are observed under conditions that eventually lead to fibril formation. We conclude that the absence of tertiary contacts under solution conditions where binding interactions between peptide units are still favorable is a crucial requirement for amyloid formation. Thus, fragmentation of a sequence, like partial chemical denaturation or mutation, can enhance the capacity of specific protein sequences to form such fibrils.
Simple sequence is abundant in eukaryotic proteins
Authors: Golding G.B.
Published: February 2009

All proteins of Saccharomyces cerevisiae have been compared to determine how frequently segments from one protein are present in other proteins. Proteins that are recently evolutionarily related were excluded. The most frequently present protein segments are long, tandem repetitions of a single amino acid. For some of these segments, up to 14% of all proteins in the genome were found to have similar peptides within them. These peptide segments may not be functional protein domains. Although they are the most common shared feature of yeast proteins, their ubiquity and simplicity argue that their probable function may be to simply serve as spacers between other protein motifs.
Centrosymmetric bilayers in the 0.75 å resolution structure of a designed alpha‐helical peptide, D, L‐Alpha‐1
Authors: Patterson William R. , Anderson Daniel H. , Degrado William F. , Cascio Duilio , Eisenberg David
Published: February 2009

We report the 0.75 Å crystal structure of a racemic mixture of the 12‐residue designed peptide “Alpha‐1” (Acetyl‐ELLKKLLEELKG), the L‐enantiomer of which is described in the accompanying paper. Equivalent solutions of the centrosymmetric bilayers were determined by two direct phasing programs in space groups P1 and P1. The unit cell contains two L‐alpha‐helices and two D‐alpha‐helices. The columnar‐sheet bilayer motif seen in L‐Alpha‐1 is maintained in the D, L‐Alpha‐1 structure except that each sheet of head‐to‐tail helices is composed of one enantiomer and is related to its neighboring sheets by inversion symmetry. Comparison to the L‐Alpha‐1 structure provides further insight into peptide design. The high resolution and small asymmetric unit allowed building an intricate model (R = 13.1%, Rfree = 14.5%) that incorporates much of the discrete disorder of peptide and solvent. Ethanolamine and 2‐methyl‐2, 4‐pentanediol (MPD) molecules bind near helix termini. Rigid body analysis identifies sites of restricted displacements and torsions. Side‐chain discrete disorder propagates into the backbone of one helix but not the other. Although no side chain in Alpha‐1 is rigid, the environments in the crystal restrict some of them to no or only one active torsion.
Pressure‐induced unfolding of lysozyme in aqueous guanidinium chloride solution
Authors: Sasahara Kenji , Nitta Katsutoshi
Published: February 2009

The pressure‐induced unfolding of lysozyme was investigated in an aqueous guanidinium chloride solution by means of ultraviolet spectroscopy. Assuming a two‐state transition model, volume changes were calculated from the slope of free energy vs. pressure plots over a temperature range of 10 to 60 °C. Between 25 and 60 °C, almost constant volume changes were observed in the transition region, which was reflected in almost identical slopes of the free energy change vs. pressure plots. On the other hand, the different slopes were observed in the pressure dependence of free energy change at temperatures lower than 25 °C. These data were interpreted as suggesting that a two‐state model is not appropriate at low temperature, but instead one or more intermediates are present under these conditions. The volume changes for unfolding became less negative at temperatures higher than 25 °C.
Equilibrium unfolding of a small low‐potential cytochrome, cytochrome c553 from Desulfovibrio vulgaris
Authors: Wittung‐Stafshede Pernilla
Published: February 2009

To understand general aspects of stability and folding of c‐type cytochromes, we have studied the folding characteristics of cytochrome c553 from Desulfovibrio vulgaris (Hildenborough). This cytochrome is structurally similar but lacks sequence homology to other heme proteins; moreover, it has an abnormally low reduction potential. Unfolding of oxidized and reduced cytochrome c553 by guanidine hydrochloride (GuHCl) was monitored by circular dichroism (CD) and Soret absorption; the same unfolding curves were obtained with both methods supporting that cytochrome c553 unfolds by an apparent two‐state process. Reduced cytochrome c553 is 7(3) kJ/mol more stable than the oxidized form; accordingly, the reduction potential of unfolded cytochrome c553 is 100(20) mV more negative than that of the folded protein. In contrast to many other unfolded cytochrome c proteins, upon unfolding at pH 7.0 both oxidized and reduced heme in cytochrome c553 become high‐spin. The lack of heme misligation in unfolded cytochrome c553 implies that its unfolded structure is less constrained than those of cytochromes c with low‐spin, misligated hemes.
Effects of tryptophan to phenylalanine substitutions on the structure, stability, and enzyme activity of the IIABMan subunit of the mannose transporter of Escherichia coli
Authors: Markovic‐Housley Zora , Stolz Beat , Lanz Regina , Erni Bernhard
Published: February 2009

The hydrophilic subunit of the mannose transporter (IIABMan) of Escherichia coli is a homodimer that contains four tryptophans per monomer, three in the N‐terminal domain (Trp12, Trp33, and Trp69) and one in the C‐terminal domain (Trp182). Single and double Trp‐Phe mutants of IIABMan and of the IIA domain were produced. Fluorescence emission studies revealed that Trp33 and Trp12 are the major fluorescence emitters, Trp69 is strongly quenched in the native protein and Trp182 strongly blue shifted, indicative of a hydrophobic environment. Stabilities of the Trp mutants of dimeric IIAMan and IIABMan were estimated from midpoints of the GdmHCl‐induced unfolding transitions and from the amount of dimers that resisted dissociation by SDS (sodium dodecyl sulfate), respectively. W12F exhibited increased stability, but only 6% of the wild‐type phosphotransferase activity, whereas W33F was marginally and W69F significantly destabilized, but fully active. Second site mutations W33F and W69F in the background of the W12F mutation reduced protein stability and suppressed the functional defect of W12F. These results suggest that flexibility is required for the adjustments of protein–protein contacts necessary for the phosphoryltransfer between the phosphorylcarrier protein HPr, IIAMan, IIBMan, and the incoming mannose bound to the transmembrane IICMan–IIDMan complex.
Structural studies by X‐ray diffraction on metal substituted desulforedoxin, a rubredoxin‐type protein
Authors: Archer M. , Carvalho A.L. , Teixeira S. , Moura I. , Moura J.J.G. , Rusnak F. , Romão M.J.
Published: February 2009

Desulforedoxin (Dx), isolated from the sulfate reducing bacterium Desulfovibrio gigas, is a small homodimeric (2 × 36 amino acids) protein. Each subunit contains a high‐spin iron atom tetrahedrally bound to four cysteinyl sulfur atoms, a metal center similar to that found in rubredoxin (Rd) type proteins. The simplicity of the active center in Dx and the possibility of replacing the iron by other metals make this protein an attractive case for the crystallographic analysis of metal‐substituted derivatives. This study extends the relevance of Dx to the bioinorganic chemistry field and is important to obtain model compounds that can mimic the four sulfur coordination of metals in biology. Metal replacement experiments were carried out by reconstituting the apoprotein with In3+, Ga3 +, Cd2+, Hg2+, and Ni2+ salts. The In3 + and Ga3+ derivatives are isomorphous with the iron native protein; whereas Cd2+, Hg2+, and Ni2+ substituted Dx crystallized under different experimental conditions, yielding two additional crystal morphologies; their structures were determined by the molecular replacement method. A comparison of the three‐dimensional structures for all metal derivatives shows that the overall secondary and tertiary structures are maintained, while some differences in metal coordination geometry occur, namely, bond lengths and angles of the metal with the sulfur ligands. These data are discussed in terms of the entatic state theory.