bluuebunny/crossref_metadata_2025 huggingface dataset:
A test of the relationship between sequence and structure in proteins: Excision of the heme binding site in apocytochrome b5
Authors: Constans Aileen J. , Mayer Michael R. , Sukits Steven E , Lecomte Juliette T.J.
Published: February 2009

The water‐soluble domain of rat hepatic holocytochrome b5 is anαβ protein containing elements of secondary structure in the sequence βl ‐α l ‐β4‐β3‐α2‐α3‐β5‐α4‐α5‐β2‐α6. The heme group is enclosed by four helices,α2,α3,α4, andα5. To test the hypothesis that a small b hemoprotein can be constructed in two parts, one forming the heme site, the other an organizing scaffold, a protein fragment corresponding to βl‐αl‐β4‐β3‐Δ‐β2‐α6 was prepared, where Δ is a seven‐residue linker bypassing the heme binding site. The fragment (“abridged b5”) was found to containα and β secondary structure by circular dichroism spectroscopy and tertiary structure by Trp fluorescence emission spectroscopy. NMR data revealed a species with spectral properties similar to those of the full‐length apoprotein. This folded form is in slow equilibrium on the chemical shift time scale with other less folded species. Thermal denaturation, as monitored by circular dichroism, absorption, and fluorescence spectroscopy, as well as size‐exclusion chromatography‐fast protein liquid chromatography (SEC‐FPLC), confirmed the coexistence of at least two distinct conformational ensembles. It was concluded that the protein fragment is capable of adopting a specific fold likely related to that of cytochrome b5, but does not achieve high thermodynamic stability and cooperativity. Abridged b5 demonstrates that the spliced sequence contains the information necessary to fold the protein. It suggests that the dominating influence to restrict the conformational space searched by the chain is structural propensities at a local level rather than internal packing. The sequence also holds the properties necessary to generate a barrier to unfolding.
Amide proton exchange measurements as a probe of the stability and dynamics of the n‐terminal domain of the ribosomal protein L9: Comparison with the intact protein
Authors: Vugmeyster Liliya , Kuhlman Brian , Raleigh Daniel P.
Published: February 2009

Amide H/D exchange rates have been measured for the N‐terminal domain of the ribosomal protein L9, residues 1‐56. The rates were measured at pD 3.91, 5.03, and 5.37. At pD 5.37, 18 amides exchange slowly enough to give reliable rate measurements. At pD 3.91, seven additional residues could be followed. The exchange is shown to occur by the EX2 mechanism for all conditions studied. The rates for the N‐terminal domain are very similar to those previously measured for the corresponding region in the full‐length protein (Lillemoen J et al., 1997, J Mol Biol 268:482‐493). In particular, the rates for the residues that we have shown to exchange via global unfolding in the N‐terminal domain agree within the experimental error with the rates measured by Hoffman and coworkers, suggesting that the structure of the domain is preserved in isolation and that the stability of the isolated domain is comparable to the stability of this domain in intact L9.
Distance geometry generates native‐like folds for small helical proteins using the consensus distances of predicted protein structures
Authors: Huang Enoch S. , Ponder Jay W. , Samudrala Ram
Published: February 2009

For successful ab initio protein structure prediction, a method is needed to identify native‐like structures from a set containing both native and non‐native protein‐like conformations. In this regard, the use of distance geometry has shown promise when accurate inter‐residue distances are available. We describe a method by which distance geometry restraints are culled from sets of 500 protein‐like conformations for four small helical proteins generated by the method of Simons et al. (1997). A consensus‐based approach was applied in which every inter‐Cα distance was measured, and the most frequently occurring distances were used as input restraints for distance geometry. For each protein, a structure with lower coordinate root‐mean‐square (RMS) error than the mean of the original set was constructed; in three cases the topology of the fold resembled that of the native protein. When the fold sets were filtered for the best scoring conformations with respect to an all‐atom knowledge‐based scoring function, the remaining subset of 50 structures yielded restraints of higher accuracy. A second round of distance geometry using these restraints resulted in an average coordinate RMS error of 4.38 Å.
Electrostatic coupling to pH‐titrating sites as a source of cooperativity in protein‐ligand binding
Authors: Spassov Velin , Bashford Donald
Published: February 2009

This paper describes an alternative mechanism for the cooperative binding of charged ligands to proteins. The ligand‐binding sites are electrostatically coupled to protein side chains that can undergo protonation and deprotonation. The binding of one ligand alters the protein's protonation equilibrium in a manner that makes the the binding of the second ligand more favorable. This mechanism requires no conformational change to produce a cooperative effect, although it is not exclusive of conformational change. We present a theoretical description of the mechanism, and calculations on three kinds of systems: A model system containing one protonation site and two ligand‐binding sites; a model system containing two protonation sites and two ligand‐binding sites; and calbindin D9k, which contains two Ca2+‐binding sites and 30 protonation sites. For the one‐protonation‐site model, it is shown that the influence of the protonation site can only be cooperative. The competition of this effect with the anticooperative effect of ligand‐ligand repulsion is studied in detail. For the two‐protonation site model, the effect can be either cooperative or, in special cases, anticooperative. For calbindin D9k, the calculations predict that six protonation sites in or near the ligand‐binding sites make a cooperative contribution that approximately cancels the anticooperative effect of Ca2+‐Ca2+ repulsion, accounting for more than half of the total cooperative effect that is needed to overcome repulsion and produce the net cooperativity observed experimentally. We argue that cooperative mechanisms of the kind described here are likely when there is more than one ligand‐binding site in a protein domain.
Characterization of recombinant human cathepsin B expressed at high levels in baculovirus
Authors: Steed Paul M. , Lasala Daniel , Liebman Jeffrey , Wigg Andrew , Clark Kirk , Knap Ania K.
Published: February 2009

The lysosomal cysteine protease cathepsin B has been studied intensely for many years because of its unique characteristics and its potential involvement in disease states. A reproducible, high yield expression system for active recombinant protein is key to biochemical and biophysical studies as well as rational drug design. Although several microbial and mammalian expression systems for recombinant human cathepsin B have been described, these have been limited by low or variable yields. Further, in some of these systems hyper‐glycosylation of the enzyme near the active site affects its activity. We describe a baculovirus expression system and purification scheme that solve all of these problems. Yields of active, protected enzyme were reproducibly in excess of 25 mg/L. Since this protein was not hyper‐glycosylated, it had greater activity than cathepsin B produced in yeast systems as indicated by a threefold increase in Kcat. In addition, the biophysical properties of the baculovirus‐expressed cathepsin B, as measured by dynamic light scattering, were more amenable to crystallographic study since the data indicated proteins of more uniform size. Therefore, this system for the production of recombinant human cathepsin B constitutes a major improvement in both quantity and quality over those previously reported. Further, we demonstrate that the manner of expression and purification of this enzyme has profound effects on its kinetic and physical parameters.
Crystal structure of spinach plastocyanin at 1.7 Å resolution
Authors: Xue Yafeng , Ökvist Mats , Hansson Örjan , Young Simon
Published: February 2009

The crystal structure of plastocyanin from spinach has been determined using molecular replacement, with the structure of plastocyanin from poplar as a search model. Successful crystallization was facilitated by site‐directed mutagenesis in which residue Gly8 was substituted with Asp. The region around residue 8 was believed to be too mobile for the wild‐type protein to form crystals despite extensive screening. The current structure represents the oxidized plastocyanin, copper (II), at low pH (≈4.4). In contrast to the similarity in the core region as compared to its poplar counterpart, the structure shows some significant differences in loop regions. The most notable is the large shift of the 59‐61 loop where the largest shift is 3.0 Å for the Cα atom of Glu59. This results in different patterns of electrostatic potential around the acidic patches for the two proteins.
Structure of betaine aldehyde dehydrogenase at 2.1 Å resolution
Authors: Johansson Kenth , Ramaswamy S. , Eklund Hans , El‐Ahmad Mustapha , Hjelmqvist Lars , Jörnvall Hans
Published: February 2009

The three‐dimensional structure of betaine aldehyde dehydrogenase, the most abundant aldehyde dehydrogenase (ALDH) of cod liver, has been determined at 2.1 Å resolution by the X‐ray crysallographic method of molecular replacement. This enzyme represents a novel structure of the highly multiple ALDH, with at least 12 distinct classes in humans. This betaine ALDH of class 9 is different from the two recently determined ALDH structures (classes 2 and 3). Like these, the betaine ALDH structure has three domains, one coenzyme binding domain, one catalytic domain, and one oligomerization domain. Crystals grown in the presence or absence of NAD+ have very similar structures and no significant conformational change occurs upon coenzyme binding. This is probably due to the tight interactions between domains within the subunit and between subunits in the tetramer. The oligomerization domains link the catalytic domains together into two 20‐stranded pleated sheet structures. The overall structure is similar to that of the tetrameric bovine class 2 and dimeric rat class 3 ALDH, but the coenzyme binding with the nicotinamide in anti conformation, resembles that of class 2 rather than of class 3.
Effects of proline cis‐trans isomerization on TB domain secondary structure
Authors: Yuan Xuemei , Werner Jorn M. , Campbell Iain D. , Downing A. Kristina , Knott Vron , Handford Penny A.
Published: February 2009

The transforming growth factor β (TGF‐β) binding protein‐like (TB) domain is found principally in proteins localized to extracellular matrix fibrils, including human fibrillin‐1, the defective protein in the Marfan syndrome. Analysis of the nuclear magnetic resonance (NMR) data for the sixth TB module from human fibrillin‐1 has revealed the existence of two stable conformers that differ in the isomerization states of two proline residues. Unusually, the two isoforms do not readily interconvert and are stable on the time scale of milliseconds. We have computed independent structures of the major and minor conformers of TB6 to assess how the domain fold adjusts to incorporate alternatively cis‐ or trans‐ prolines. Based on previous observations, it has been suggested that multiple conformers can only be accommodated in flexible regions of protein structure. In contrast, P22, which exists in trans in the major form and cis in the minor form of TB6, is in a rigid region of the domain, which is confirmed by backbone dynamics measurements. Overall, the structures of the major and minor conformers are similar. However, the secondary structure topologies of the two forms differ as a direct consequence of the changes in proline conformation.
Characterization of a folding intermediate from HIV‐1 ribonuclease H
Authors: Kern Gunther , Handel Tracy , Marqusee Susan
Published: February 2009

The RNase H domain from HIV‐1 (HIV RNase H) encodes an essential retroviral activity. Refolding of the isolated HIV RNase H domain shows a kinetic intermediate detectable by stopped‐flow far UV circular dichroism and pulse‐labeling H/D exchange. In this intermediate, strands 1, 4, and 5 as well as helices A and D appear to be structured. Compared to its homolog fromEscherichia coli, the rate limiting step in refolding of HIV RNase H appears closer to the native state. We have modeled this kinetic intermediate using a C‐terminal deletion fragment lacking helix E. Like the kinetic intermediate, this variant folds rapidly and shows a decrease in stability. We propose that inhibition of the docking of helix E to this folding intermediate may present a novel strategy for anti HIV‐1 therapy.
Aggregation states of mitochondrial malate dehydrogenase
Authors: Sánchez Susana A. , Brunet Juan E. , Hazlett Theodore L. , Jameson David M.
Published: February 2009

The oligomeric state of fluorescein‐labeled mitochondrial malate dehydrogenase (L‐malate NAD+ oxidoreductase; mMDH; EC 1.1.1.37), as a function of protein concentration, has been examined using steady‐state and dynamic polarization methodologies. A “global” rotational relaxation time of 103 ± 7 ns was found for micromolar concentrations of mMDH‐fluorescein, which is consistent with the reported size and shape of mMDH. Dilution of the mMDH‐fluorescein conjugates, prepared using a phosphate buffer protocol, to nanomolar concentrations had no significant effect on the rotational relaxation time of the adduct, indicating that the dimer‐monomer dissociation constant for mMDH is below 10−9 M. In contrast to reports in the literature suggesting a pH‐dependent dissociation of mMDH, the oligomeric state of this mMDH‐fluorescein preparation remained unchanged between pH 5.0 and 8.0. Application of hydrostatic pressure up to 2.5 kilobars was ineffective in dissociating the mMDH dimer. However, the mMDH dimer was completely dissociated in 1.5 M guanidinium hydrochloride. Dilution of a mMDH‐fluorescein conjugate, prepared using a Tris buffer protocol, did show dissociation, which can be attributed to aggregates present in these preparations. These results are considered in light of the disparities in the literature concerning the properties of the mMDH dimer‐monomer equilibrium.
Analysis of the interactions between streptokinase domains and human plasminogen
Authors: Conejero‐Lara Francisco , Parrad Juan , Azuaga Ana I. , Dobson Christopher M. , Ponting Chris P.
Published: February 2009

The contrasting roles of streptokinase (SK) domains in binding human Glu l‐plasminogen (Plg) have been studied using a set of proteolytic fragments, each of which encompasses one or more of SK's three structural domains (A, B, C). Direct binding experiments have been performed using gel filtration chromatography and surface plasmon resonance. The latter technique has allowed estimation of association and dissociation rate constants for interactions between Plg and intact SK or SK fragments. Each of the SK fragments that contains domain B (fragments A2‐B‐C, A2‐B, B‐C, and B) binds Plg with similar affinity, at a level approximately 100‐ to 1,000‐fold lower than intact SK. Experiments using 10 mM 6‐aminohexanoic acid or 50 mM benzamidine demonstrate that either of these two lysine analogues abolishes interaction of domain B with Plg. Isolated domain C does not show detectable binding to Plg. Moreover, the additional presence of domain C within other SK fragments (B‐C and A2‐B‐C) does not alter significantly their affinities for Plg. In addition, Plg‐binding by a noncovalent complex of two SK fragments that contains domains A and B is similar to that of domain B. By contrast, species containing domain B and both domains A and C (intact SK and the two‐chain complex A1·A2‐B‐C) show a significantly higher affinity for Plg, which could not be completely inhibited by saturating amounts of 6‐AHA. These results show that SK domain B interacts with Plg in a lysine‐dependent manner and that although domains A and C do not appear independently to possess affinity for Plg, they function cooperatively to establish the additional interactions with Plg to form an efficient native‐like Plg activator complex.
Delineation of an evolutionary salvage pathway by compensatory mutations of a defective lysozyme
Authors: Jucovic Milan , Poteete Anthony R.
Published: February 2009

Model‐free approaches (random mutagenesis, DNA shuffling) in combination with more “rational,” three‐dimensional information‐guided randomization have been used for directed evolution of lysozyme activity in a defective T4 lysozyme mutant. A specialized lysozyme cloning vector phage, derived from phage λ, depends upon T4 lysozyme function for its ability to form plaques. The substitution W138P in T4 lysozyme totally abolishes its plaque‐forming ability. Compensating mutations in W138PT4 lysozyme after sequential random mutagenesis of the whole gene as well as after targeted randomization of residues in the vicinity of Trp138 were selected. In a second stage, these mutations were randomly recombined by the recombinatorial PCR method of DNA shuffling. Shuffled and selected W138P T4 lysozyme variants provide the hybrid λ phage with sufficient lysozyme activity to produce normal‐size plaques, even at elevated temperature (42 °C). The individual mutations with the highest compensatory information for W138P repair are the substitutions A146F and A146M, selected after targeted randomization of three residues in the neighborhood of Trp138 by combinatorial mutagenesis. The best evolved W138P T4 lysozymes, however, accumulated mutations originating from both randomly mutagenized as well as target‐randomized variants.
Probing the contribution of internal cavities to the volume change of protein unfolding under pressure
Authors: Frye Kelly J. , Royer Catherine A.
Published: February 2009

The structural origin of the decrease in system volume upon protein denaturation by pressure has remained a puzzle for decades. This negative volume change upon unfolding is assumed to arise globally from more intimate interactions between the polypeptide chain and water, including electrostriction of buried charges that become exposed upon unfolding, hydration of the polypeptide backbone and amino acid side chains and elimination of packing defects and internal void volumes upon unfolding of the chain. However, the relative signs and magnitudes of each of these contributing factors have not been experimentally determined. Our laboratory has probed the fundamental basis for the volume change upon unfolding of staphylococcal nuclease (Snase) using variable solution conditions and point mutants of Snase (Royer CA et al., 1993, Biochemistry 32:5222‐5232; Frye KJ et al., 1996, Biochemistry 35:10234‐10239). Our prior results indicate that for Snase, neither electrostriction nor polar or nonpolar hydration contributes significantly to the value of the volume change of unfolding. In the present work, we investigate the pressure induced unfolding of three point mutants of Snase in which internal cavity size is altered. The experimentally determined volume changes of unfolding for the mutants suggest that loss of internal void volume upon unfolding represents the major contributing factor to the value of the volume change of Snase unfolding.
Phage P22 tailspike protein: Removal of head‐binding domain unmasks effects of folding mutations on native‐state thermal stability
Authors: Miller Stefan , Schuler Benjamin , Seckler Robert
Published: February 2009

A shortened, recombinant protein comprising residues 109‐666 of the tailspike endorhamnosidase of Salmonella phage P22 was purified fromEscherichia coliand crystallized. Like the full‐length tailspike, the protein lacking the amino‐terminal head‐binding domain is an SDS‐resistant, thermostable trimer. Its fluorescence and circular dichroism spectra indicate native structure. Oligosaccharide binding and endoglycosidase activities of both proteins are identical. A number of tailspike folding mutants have been obtained previously in a genetic approach to protein folding. Two temperature‐sensitive‐folding (tsf) mutations and the four known global second‐site suppressor (su) mutations were introduced into the shortened protein and found to reduce or increase folding yields at high temperature. The mutational effects on folding yields and subunit folding kinetics parallel those observed with the full‐length protein. They mirror the in vivo phenotypes and are consistent with the substitutions altering the stability of thermolabile folding intermediates. Because full‐length and shortened tailspikes aggregate upon thermal denaturation, and their denaturant‐induced unfolding displays hysteresis, kinetics of thermal unfolding were measured to assess the stability of the native proteins. Unfolding of the shortened wild‐type protein in the presence of 2% SDS at 71 °C occurs at a rate of 9.2 × 10−4s−1. It reflects the second kinetic phase of unfolding of the full‐length protein. All six mutations were found to affect the thermal stability of the native protein. Both tsf mutations accelerate thermal unfolding about 10‐fold. Two of the su mutations retard thermal unfolding up to 5‐fold, while the remaining two mutations accelerate unfolding up to 5‐fold. The mutational effects can be rationalized on the background of the recently determined crystal structure of the protein.
A single amino acid substitution makes ERK2 susceptible to pyridinyl imidazole inhibitors of p38 MAP kinase
Authors: Fox Ted , Coll Joyce T. , Xie Xiaoling , Ford Pamella J. , Germann Ursula A. , Porter Margaret D. , Pazhanisamy S. , Fleming Mark A. , Galullo Vincent , Su Michael S. S. , Wilson Keith P.
Published: February 2009

Mitogen‐activated protein (MAP) kinases are serine/threonine kinases that mediate intracellular signal transduction pathways.Pyridinyl imidazole compounds block pro‐inflammatory cytokine production and are specific p38 kinase inhibitors. ERK2 is related to p38 in sequence and structure, but is not inhibited by pyridinyl imidazole inhibitors. Crystal structures of two pyridinyl imidazoles complexed with p38 revealed these compounds bind in the ATP site. Mutagenesis data suggested a single residue difference at threonine 106 between p38 and other MAP kinases is sufficient to confer selectivity of pyridinyl imidazoles. We have changed the equivalent residue in human ERK2, Q105, into threonine and alanine, and substituted four additional ATP binding site residues. The single residue change Q105A in ERK2 enhances the binding of SB202190 at least 25,000‐fold compared to wild‐type ERK2. We report enzymatic analyses of wild‐type ERK2 and the mutant proteins, and the crystal structure of a pyridinyl imidazole, SB203580, bound to an ERK2 pentamutant, I103L, Q105T, D106H, E109G, T110A. These ATP binding site substitutions induce low nanomolar sensitivity to pyridinyl imidazoles. Furthermore, we identified 5‐iodotubercidin as a potent ERK2 inhibitor, which may help reveal the role of ERK2 in cell proliferation.
Solution structure and backbone dynamics of Mason‐Pfizer monkey virus (MPMV) nucleocapsid protein
Authors: Gao Yun , Kaluarachchi Kumaralal , Giedroc David P.
Published: February 2009

Retroviral nucleocapsid proteins (NCPs) are CCHC‐type zinc finger proteins that mediate virion RNA binding activities associated with retrovirus assembly and genomic RNA encapsidation. Mason‐Pfizer monkey virus (MPMV), a type D retrovirus, encodes a 96‐amino acid nucleocapsid protein, which contains two Cys‐X2‐Cys‐X4‐His‐X4‐Cys (CCHC) zinc fingers connected by an unusually long 15‐amino acid linker. Homonuclear, two‐dimensional sensitivity‐enhanced 15N‐H, three‐dimensional 15N‐H, and triple resonance NMR spectroscopy have been used to determine the solution structure and residue‐specific backbone dynamics of the structured core domain of MPMV NCP containing residues 21–80. Structure calculations and spectral density mapping of N‐H bond vector mobility reveal that MPMV NCP 21–80 is best described as two independently folded, rotationally uncorrelated globular domains connected by a seven‐residue flexible linker consisting of residues 42–48. The N‐terminal CCHC zinc finger domain (residues 24–37) appears to adopt a fold like that described previously for HIV‐1 NCP; however, residues within this domain and the immediately adjacent linker region (residues 38–41) are characterized by extensive conformational averaging on the μs‐ms time scale at 25C. In contrast to other NCPs, residues 49–77, which includes the C‐terminal CCHC zinc‐finger (residues 53–66), comprise a well‐folded globular domain with the Val49‐Pro‐Gly‐Leu52 sequence and C‐terminal tail residues 67–77 characterized by amide proton exchange properties and 15N R1, R2, and {H‐15N} NOE values indistinguishable to residues in the core C‐terminal finger. Twelve refined structural models of MPMV NCP residues 49–80 (pairwise backbone RMSD of 0. 77 Å) reveal that the side chains of the conserved Pro50 and Trp62 are in van der Waals contact with one another. Residues 70–73 in the C‐terminal tail adopt a reverse turn‐like structure. Ile77 is involved in extensive van der Waals contact with the core finger domain, while the side chains of Ser68 and Asn75 appear to form hydrogen bonds that stabilize the overall fold of this domain. These residues outside of the core finger structure are conserved in D‐type and related retroviral NCPs, e. g., MMTV NCP, suggesting that the structure of MPMV NCP may be representative of this subclass of retroviral NCPs.
Determinants of strand register in antiparallel β‐sheets of proteins
Authors: Hutchinson E. Gail , Sessions Richard B. , Thornton Janet M. , Woolfson Derek N.
Published: February 2009

Antiparallel β‐sheets present two distinct environments to inter‐strand residue pairs: βA, HB sites have two backbone hydrogen bonds; whereas at βA, NHB positions backbone hydrogen bonding is precluded. We used statistical methods to compare the frequencies of amino acid pairs at each site. Only ˜ 10% of the 210 possible pairs showed occupancies that differed significantly between the two sites. Trends were clear in the preferred pairs, and these could be explained using stereochemical arguments. Cys‐Cys, Aromatic‐Pro, Thr‐Thr, and Val‐Val pairs all preferred the ßA, NHB site. In each case, the residues usually adopted sterically favored X1 conformations, which facilitated intra‐pair interactions: Cys‐Cys pairs formed disulfide bonds; Thr‐Thr pairs made hydrogen bonds; Aromatic‐Pro and Val‐Val pairs formed close van der Waals contacts. In contrast, to make intimate interactions at a ßA. HB site, one or both residues had to adopt less favored X1 geometries. Nonetheless, pairs containing glycine and/or aromatic residues were favored at this site. Where glycine and aromatic side chains combined, the aromatic residue usually adopted the gauche conformation, which promoted novel aromatic ring‐peptide interactions. This work provides rules that link protein sequence and tertiary structure, which will be useful in protein modeling, redesign, and de novo design. Our findings are discussed in light of previous analyses and experimental studies.
Stabilizing the subtilisin BPN' pro‐domain by phage display selection: How restrictive is the amino acid code for maximum protein stability?
Authors: Ruan Biao , Hoskins Joel , Wang Lan , Bryan Philip N.
Published: February 2009

We have devised a procedure using monovalent phage display to select for stable mutants in the pro‐domain of the serine protease, subtilisin BPN'. In complex with subtilisin, the pro‐domain assumes a compact structure with a four‐stranded antiparallel β‐sheet and two three‐turn α‐helices. When isolated, however, the pro‐domain is 97% unfolded. These experiments use combinatorial mutagenesis to select for stabilizing amino acid combinations at a particular structural locus and determine how many combinations are close to the maximum protein stability. The selection for stability is based on the fact that the independent stability of the pro‐domain is very low and that binding to subtilisin is thermodynamically linked to folding. Two libraries of mutant pro‐domains were constructed and analyzed to determine how many combinations of amino acids at a particular structural locus result in the maximum stability. A library comprises all combinations of four amino acids at a structural locus. Previous studies using combinatorial genetics have shown that many different combinations of amino acids can be accommodated in a selected locus without destroying function. The present results indicate that the number of sequence combinations at a structural locus, which are close to the maximum stability, is small. The most striking example is a selection at an interior locus of the pro‐domain. After two rounds of phagemid selection, one amino acid combination is found in 40% of sequenced mutants. The most frequently selected mutant has a δGunfolding = 4 kcal/mol at 25°C, an increase of 6 kcal/mol relative to the naturally occurring sequence. Some implications of these results on the amount of sequence information needed to specify a unique tertiary fold are discussed. Apart from possible implications on the folding code, the phage display selection described here should be useful in optimizing the stability of other proteins, which can be displayed on the phage surface.
The interaction between the chaperone SecB and its ligands: Evidence for multiple subsites for binding
Authors: Randall Linda L. , Hardy Simon J. S. , Topping Traci B. , Smith Virginia F. , Bruce James E. , Smith Richard D.
Published: February 2009

The chaperone protein SecB is dedicated to the facilitation of export of proteins from the cytoplasm to the periplasm and outer membrane of Escherichia coli. It functions to bind and deliver precursors of exported proteins to the membrane‐associated translocation apparatus before the precursors fold into their native stable structures. The binding to SecB is characterized by a high selectivity for ligands having nonnative structure but a low specificity for consensus in sequence among the ligands. A model previously presented (Randall LL, Hardy SJS, 1995, Trends Biochem Sci 20: 65–69) to rationalize the ability of SecB to distinguish between the native and nonnative states of a polypeptide proposes that the SecB tetramer contains two types of subsites for ligand binding: one kind that would interact with extended flexible stretches of polypeptides and the other with hydrophobic regions. Here we have used titration calorimetry, analytical ultracentrifugation, and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry to obtain evidence that such distinguishable subsites exist.
The kinetic cycle of cardiac troponin C: Calcium binding and dissociation at site II trigger slow conformational rearrangements
Authors: Hazard Andrea L. , Kohout Susy C. , Stricker Nicole L. , Putkey John A. , Falke Joseph J.
Published: February 2009

The goal of this study is to characterize the kinetic mechanism of Ca2+ activation and inactivation of cardiac troponin C (cTnC), the Ca2+ signaling protein which triggers heart muscle contraction. Previous studies have shown that IAANS covalently coupled to Cys84 of wild‐type cTnC is sensitive to conformational change caused by Ca2+ binding to the regulatory site II; the present study also utilizes the C35S mutant, in which Cys84 is the lone cysteine, to ensure the specificity of IAANS labeling. Site II Ca2+ affinities for cTnC‐wt, cTnC‐C35S, cTnC‐wt‐IAANS2, and cTnC‐C35S‐IAANS were similar (KD = 2–5 μM at 25°C; KD = 2–8 μM at 4°C), indicating that neither the IAANS label nor the C35S mutation strongly perturbs site II Ca2+ affinity. To directly determine the rate of Ca2+ dissociation from site II, the Ca2+‐loaded protein was rapidly mixed with a spectroscopically sensitive chelator in a stopped flow spectrometer. The resulting site II Ca2+ off‐rates were kon = 700–800 s−1 (4°C) for both cTnC‐wt and cTnC‐C35S, yielding calculated macroscopic site II Ca2+ on‐rates of kon = koff/KD = 2–4 × 108 M−1 s−1 (4°C). As observed for Ca2+ affinities, neither the C35S mutation nor IAANS labeling significantly altered the Ca2+ on‐ and off‐rates. Using IAANS fluorescence as a monitor of the protein conformational state, the intramolecular conformational changes (Δ) induced by Ca2+ binding and release at site II were found to be significantly slower than the Ca2+ on‐ and off‐rates. The conformational rate constants measured for cTnC‐wt‐IAANS2 and cTnC‐C35S‐IAANS were kΔon = 120–210 s−1 and kΔoff = 90–260 s−1 (4°C). Both conformational events were slowed in cTnC‐wt‐IAANS2 relative to cTnC‐C35S‐IAANS, presumably due to the bulky IAANS probe coupled to Cys35. Together, the results provide a nearly complete kinetic description of the Ca2+ activation cycle of isolated cTnC, revealing rapid Ca2+ binding and release at site II accompanied by slow conformational steps that are likely to be retained by the full troponin complex during heart muscle contraction and relaxation.
The identification of metal‐binding ligand residues in metalloproteins using nuclear magnetic resonance spectroscopy
Authors: Scrofani Sergiod. B. , Wright Peter E. , Dyson H. Jane
Published: February 2009

The identification of metal‐binding ligands in metalloproteins is an important step in gaining detailed information regarding the environment of the active site. Traditionally, techniques such as 113Cd‐substitution for the active metal followed by isotope‐filtered NMR techniques have been used to this end. However, for medium to high molecular weight proteins (>20 kDa), these experiments may not be beneficial due to extensive H spectral overlap. Here, we describe an alternative approach, where metal‐binding ligands such as histidine and cysteine are specifically 15N backbone labeled, excess EDTA is added and changes to {H‐15N} HSQC spectra are followed. Under these conditions, the amide groups of all 15N labeled histidine and cysteine residues, which were either ligands or residues close to the active site, were identified unambiguously for metallo‐ß‐lactamse from Bacteroides fragilis.
Structural analysis of inhibitor binding to human carbonic anhydrase II
Authors: Boriack‐Sjodin P. Ann , Zeitlin Samantha , Christianson David W. , Chen Huang‐Hsing , Crenshaw Lori , Gross Sharon , Dantanarayana Anura , Delgado Pete , May Jesse A. , Dean Tom
Published: February 2009

X‐ray crystal structures of carbonic anhydrase II (CAII) complexed with sulfonamide inhibitors illuminate the structural determinants of high affinity binding in the nanomolar regime. The primary binding interaction is the coordination of a primary sulfonamide group to the active site zinc ion. Secondary interactions fine‐tune tight binding in regions of the active site cavity >5 Å away from zinc, and this work highlights three such features: (1) advantageous conformational restraints of a bicyclic thienothiazene‐6‐sulfonamide‐1,1‐dioxide inhibitor skeleton in comparison with a monocyclic 2,5‐thiophenedisulfonamide skeleton; (2) optimal substituents attached to a secondary sulfonamide group targeted to interact with hydrophobic patches defined by Phe131, Leu198, and Pro202; and (3) optimal stereochemistry and configuration at the C‐4 position of bicyclic thienothiazene‐6‐sulfonamides; the C‐4 substituent can interact with His64, the catalytic proton shuttle. Structure‐activity relationships rationalize affinity trends observed during the development of brinzolamide (AzoptTM), the newest carbonic anhydrase inhibitor approved for the treatment of glaucoma.
Identification of kinetically hot residues in proteins
Authors: Demirel Melik C. , Atilgan Ali Rana , Bahar Ivet , Jernigan Robert L. , Erman Burak
Published: February 2009

A number of recent studies called attention to the presence of kinetically important residues underlying the formation and stabilization of folding nuclei in proteins, and to the possible existence of a correlation between conserved residues and those participating in the folding nuclei. Here, we use the Gaussian network model (GNM), which recently proved useful in describing the dynamic characteristics of proteins for identifying the kinetically hot residues in folded structures. These are the residues involved in the highest frequency fluctuations near the native state coordinates. Their high frequency is a manifestation of the steepness of the energy landscape near their native state positions. The theory is applied to a series of proteins whose kinetically important residues have been extensively explored: chymotrypsin inhibitor 2, cytochrome c, and related C2 proteins. Most of the residues previously pointed out to underlie the folding process of these proteins, and to be critically important for the stabilization of the tertiary fold, are correctly identified, indicating a correlation between the kinetic hot spots and the early forming structural elements in proteins. Additionally, a strong correlation between kinetically hot residues and loci of conserved residues is observed. Finally, residues that may be important for the stability of the tertiary structure of CheY are proposed.
Dominant role of local dipolar interactions in phosphate binding to a receptor cleft with an electronegative charge surface: Equilibrium, kinetic, and crystallographic studies
Authors: Ledvina Polly S. , Koehl Edward , Tsai Ah‐Lim , Wang Zhongmin , Quiocho Florante A.
Published: February 2009

Stringent specificity and complementarity between the receptor, a periplasmic phosphate‐binding protein (PBP) with a two‐domain structure, and the completely buried and dehydrated phosphate are achieved by hydrogen bonding or dipolar interactions. We recently found that the surface charge potential of the cleft between the two domains that contains the anion binding site is intensely electronegative. This novel finding prompted the study reported here of the effect of ionic strength on the equilibrium and rapid kinetics of phosphate binding. To facilitate this study, Alal97, located on the edge of the cleft, was replaced by a Trp residue (A197W PBP) to generate a fluorescence reporter group. The A197W PBP‐phosphate complex retains wild‐type Kd and X‐ray structure beyond the replacement residue. The Kd (0.18 μM) at no salt is increased by 20‐fold at greater than 0.30 M NaCl. Stopped‐flow fluorescence kinetic studies indicate a two‐step binding process: (1) The phosphate (L) binds, at near diffusion‐controlled rate, to the open cleft form (Po) of PBP to produce an intermediate, PoL. This rate decreases with increasing ionic strength. (2) The intermediate isomerizes to the closed‐conformation form, PcL. The results indicate that the high specificity, affinity, and rate of phosphate binding are not influenced by the noncomplementary electronegative surface potential of the cleft. That binding depends almost entirely on local dipolar interactions with the receptor has important ramification in electrostatic interactions in protein structures and in ligand recognition.
Interaction of wheat α‐thionin with large unilamellar vesicles
Authors: Caaveiro José Manuel M. , Goñi Félix M. , González‐Mañas Juan Manuel , Molina Antonio , Rodriguez‐Palenzuela Pablo
Published: February 2009

The interaction of the wheat antibacterial peptide α‐thionin with large unilamellar vesicles has been investigated by means of fluorescence spectroscopy. Binding of the peptide to the vesicles is followed by the release of vesicle contents, vesicle aggregation, and lipid mixing. Vesicle fusion, i.e., mixing of the aqueous contents, was not observed. Peptide binding is governed by electrostatic interactions and shows no cooperativity. The amphipatic nature of wheat α‐fhionin seems to destabilize the membrane bilayer and trigger the aggregation of the vesicles and lipid mixing. The presence of distearoylphosphatidylethanolamine‐poly(ethylene glycol 2000) (PEG‐PE) within the membrane provides a steric barrier that inhibits vesicle aggregation and lipid mixing but does not prevent leakage. Vesicle leakage through discrete membrane channels is unlikely, because the release of encapsulated large fluorescent dextrans is very similar to that of 8‐aminonaphthalene‐1,3,6, trisulfonic acid (ANTS). A minimum number of 700 peptide molecules must bind to each vesicle to produce complete leakage, which suggests a mechanism in which the overall destabilization of the membrane is due to the formation of transient pores rather than discrete channels.
Empirical solvent‐mediated potentials hold for both intra‐molecular and inter‐molecular inter‐residue interactions
Authors: Keskin O. , Bahar I. , Jernigan R. L. , Badretdinov A. Y. , Ptitsyn O. B.
Published: February 2009

Whether knowledge‐based intra‐molecular inter‐residue potentials are valid to represent inter‐molecular interactions taking place at protein‐protein interfaces has been questioned in several studies. Differences in the chain connectivity effect and in residue packing geometry between interfaces and single chain monomers have been pointed out as possible sources of distinct energetics for the two cases. In the present study, the interfacial regions of protein‐protein complexes are examined to extract inter‐molecular inter‐residue potentials, using the same statistical methods as those previously adopted for intra‐molecular residue pairs. Two sets of energy parameters are derived, corresponding to solvent‐mediation and “average residue” mediation. The former set is shown to be highly correlated (correlation coefficient 0.89) with that previously obtained for inter‐residue interactions within single chain monomers, while the latter exhibits a weaker correlation (0.69) with its intra‐molecular counterpart. In addition to the close similarity of intra‐ and inter‐molecular solvent‐mediated potentials, they are shown to be significantly more residue‐specific and thereby discriminative compared to the residue‐mediated ones, indicating that solvent‐mediation plays a major role in controlling the effective inter‐residue interactions, either at interfaces, or within single monomers. Based on this observation, a reduced set of energy parameters comprising 20 one‐body and 3 two‐body terms is proposed (as opposed to the 20 × 20 tables of inter‐residue potentials), which reproduces the conventional 20 × 20 tables with a correlation coefficient of 0.99.
Domain structure analysis of elongation factor‐3 from saccharomyces cerevisiae by limited proteolysis and differential scanning calorimetry
Authors: Ladror Uri S. , Egan David A. , Snyder Seth W. , Capobianco John O. , Goldman Robert C. , Dorwin Sarah A. , Johnson Robert W. , Edalji Rohinton , Sarthy Aparna V. , Mcgonigal Tom , Walter Karl A. , Holzman Thomas F.
Published: February 2009

Elongation‐factor‐3 (EF‐3) is an essential factor of the fungal protein synthesis machinery. In this communication the structure of EF‐3 from Saccharomyces cerevisiae is characterized by differential scanning calorimetry (DSC), ultra‐centrifugation, and limited tryptic digestion. DSC shows a major transition at a relatively low temperature of 39 °C, and a minor transition at 58 °C. Ultracentrifugation shows that EF‐3 is a monomer; thus, these transitions could not reflect the unfolding or dissociation of a multimeric structure. EF‐3 forms small aggregates, however, when incubated at room temperature for an extended period of time. Limited proteolysis of EF‐3 with trypsin produced the first cleavage at the N‐side of Gln775, generating a 90‐kDa N‐terminal fragment and a 33‐kDa C‐terminal fragment. The N‐terminal fragment slowly undergoes further digestion generating two major bands, one at ∼75 kDa and the other at ∼55 kDa. The latter was unusually resistant to further tryptic digestion. The 33‐kDa C‐terminal fragment was highly sensitive to tryptic digestion. A 30‐min tryptic digest showed that the N‐terminal 60% of EF‐3 was relatively inaccessible to trypsin, whereas the C‐terminal 40% was readily digested. These results suggest a tight structure of the N‐terminus, which may give rise to the 58 °C transition, and a loose structure of the C‐terminus, giving rise to the 39 °C transition. Three potentially functional domains of the protein were relatively resistant to proteolysis: the supposed S5‐homologous domain (Lys102‐Ile368), the N‐terminal ATP‐binding cassette (Gly463‐Lys622), and the aminoacyl‐tRNA‐synthase homologous domain (Glu820‐Gly865). Both the basal and ribosome‐stimulated ATPase activities were inactivated by trypsin, but the ribosome‐stimulated activity was inactivated faster.
Localization of basic residues required for receptor binding to the single α‐helix of the receptor binding domain of human α2‐macroglobulin
Authors: Huang Wen , Dolmer Klavs , Liao Xiubei , Gettins Peter G. W.
Published: February 2009

To better understand the structural basis for the binding of proteinase‐transformed human α2‐macroglobulin (α2M) to its receptor, we have used three‐dimensional multinuclear NMR spectroscopy to determine the secondary structure of the receptor binding domain (RBD) of human α2M. Assignment of the backbone NMR resonances of RBD was made using 13C/15N and 15N‐enriched RBD expressed in Escherichia coli. The secondary structure of RBD was determined using 1H and 13C chemical shift indices and inter‐ and intrachain nuclear Overhauser enhancements. The secondary structure consists of eight strands in β‐conformation and one α‐helix, which together comprise 44% of the protein. The β‐strands form three regions of antiparallel β‐sheet. The two lysines previously identified as being critical for receptor binding are located in (Lys1374), and immediately adjacent to (Lys1370) the α‐helix, which also contains an (Arg1378). Secondary structure predictions of other α‐macroglobulins show the conservation of this α‐helix and suggest an important role for this helix and for basic residues within it for receptor binding.
Self‐organizing tree‐growing network for the classification of protein sequences
Authors: Fraga Luis Gerardo De La , Carazo Jose Maria , Wang Huai‐Chun , Dopazo Joaquin , Zhu Yun‐Ping
Published: February 2009

The self‐organizing tree algorithm (SOTA) was recently introduced to construct phylogenetic trees from biological sequences, based on the principles of Kohonen's self‐organizing maps and on Fritzke's growing cell structures. SOTA is designed in such a way that the generation of new nodes can be stopped when the sequences assigned to a node are already above a certain similarity threshold. In this way a phylogenetic tree resolved at a high taxonomic level can be obtained. This capability is especially useful to classify sets of diversified sequences. SOTA was originally designed to analyze pre‐aligned sequences. It is now adapted to be able to analyze patterns associated to the frequency of residues along a sequence, such as protein dipeptide composition and other n‐gram compositions. In this work we show that the algorithm applied to these data is able to not only successfully construct phylogenetic trees of protein families, such as cytochrome c, triosephophate isomerase, and hemoglobin alpha chains, but also classify very diversified sequence data sets, such as a mixture of interleukins and their receptors.