bluuebunny/crossref_metadata_2025_split huggingface dataset:
Delineation of an evolutionary salvage pathway by compensatory mutations of a defective lysozyme
Authors: Jucovic Milan , Poteete Anthony R. 
Published: February 2009

AbstractModel‐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

AbstractThe 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

AbstractA 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

AbstractMitogen‐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

AbstractRetroviral 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

AbstractAntiparallel 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

AbstractWe 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

AbstractThe 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

AbstractThe 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

AbstractThe 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

AbstractX‐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

AbstractA 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

AbstractStringent 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.