bluuebunny/crossref_metadata_2025_split huggingface dataset:
Folding funnels, binding funnels, and protein function
Authors: Tsai Chung‐Jung , Kumar Sandeep , Ma Buyong , Nussinov Ruth 
Published: February 2009

AbstractFolding 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

AbstractElicitins, 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

AbstractTwo 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

AbstractThe 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

AbstractCytochrome 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

AbstractExtended 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

AbstractThe 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

AbstractUrea‐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

AbstractThe 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

AbstractThree 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

AbstractAll 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

AbstractWe 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

AbstractThe 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

AbstractTo 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

AbstractThe 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.