bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cysteine scanning mutagenesis of putative transmembrane helices IX and X in the lactose permease of Escherichia coli
Authors: Sahin‐Tóth Miklós , Kaback H. Ronald 
Published: February 2009

AbstractUsing a functional lactose permease mutant devoid of Cys residues (C‐less permease), each amino‐acid residue in putative transmembrane helices IX and X and the short intervening loop was systematically replaced with Cys (from Asn‐290 to Lys‐335). Thirty‐four of 46 mutants accumulate lactose to high levels (70‐100% or more of C‐less), and an additional 7 mutants exhibit lower but highly significant lactose accumulation. As expected (see Kaback, H.R., 1992, Int. Rev. Cytol. 137A, 97‐125), Cys substitution for Arg‐302, His‐322, or Glu‐325 results in inactive permease molecules. Although Cys replacement for Lys‐319 or Phe‐334 also inactivates lactose accumulation, Lys‐319 is not essential for active lactose transport (Sahin‐Tóth, M., Dunten, R.L., Gonzalez, A., & Kaback, H.R., 1992, Proc. Natl. Acad. Sci. USA 89, 10547‐10551), and replacement of Phe‐334 with leucine yields permease with considerable activity. All single‐Cys mutants except Gly‐296 → Cys are present in the membrane in amounts comparable to C‐less permease, as judged by immunological techniques. In contrast, mutant Gly‐296 → Cys is hardly detectable when expressed at a relatively low rate from the lac promoter/operator but present in the membrane in stable form when expressed at a high rate from the T7 promoter. Finally, studies with N‐ethylmaleimide (NEM) show that only a few mutants are inactivated significantly. Remarkably, the rate of inactivation of Val‐315 → Cys permease is enhanced at least 10‐fold in the presence of β‐galactopyranosyl 1‐thio‐β,d‐galactopyranoside (TDG) or an H+ electrochemical gradient (Δ). The results demonstrate that only three residues in this region of the permease–Arg‐302, His‐322, and Glu‐325–are essential for active lactose transport. Furthermore, the enhanced reactivity of the Val‐315 → Cys mutant toward NEM in the presence of TDG or Δ probably reflects a conformational alteration induced by either substrate binding or Δ.
In situ conversion of coproporphyrinogen to heme by murine mitochondria: Terminal steps of the heme biosynthetic pathway
Authors: Proulx Karen L. , Woodard Steven I. , Dailey Harry A. 
Published: February 2009

AbstractCoproporphyrinogen oxidase (EC 1.3.3.3), protoporphyrinogen oxidase (EC 1.3.3.4), and ferrochelatase (EC 4.99.1.1) catalyze the terminal three steps of the heme biosynthetic pathway. All three are either bound to or associated with the inner mitochondrial membrane in higher eukaryotic cells. A current model proposes that these three enzymes may participate in some form of multienzyme complex with attendant substrate channeling (Grandchamp, B., Phung, N., &Nordmann, Y., 1978, Biochem. J. 176, 97–102; Ferreira, G.C., et al., 1988, J. Biol. Chem. 263, 3835–3839). In the present study we have examined this question in isolated mouse mitochondria using two experimental approaches: one that samples substrate and product levels during a timed incubation, and a second that follows dilution of radiolabeled substrate by pathway intermediates. When isolated mouse mitochondria are incubated with coproporphyrinogen alone there is an accumulation of free protoporphyrin. When Zn is added as a substrate for the terminal enzyme, ferrochelatase, along with coproporphyrinogen, there is formation of Zn protoporphyrin with little accumulation of free protoporphyrin. When EDTA is added to this incubation mixture with Zn, Zn protoporphyrin formation is eliminated and protoporphyrin is formed. We have examined the fate of radiolabeled substrates in vitro to determine if exogenously supplied pathway intermediates can compete with the endogenously produced compounds. The data demonstrate that while coproporphyrinogen is efficiently converted to heme in vitro when the pathway is operating below maximal capacity, exogenous protoporphyrinogen can compete with endogenously formed protoporphyrinogen in heme production. Likewise, exogenously supplied protoporphyrin efficiently competes with protoporphyrinogen in the formation of heme. These two lines of experiments strongly suggest that an obligatory channeling complex does not exist for the terminal three enzymes of the heme biosynthetic pathway, but that the enzymes function in a manner such that significant accumulation of intermediates does not normally occur.
Effects of alanine substitutions in α‐helices of sperm whale myoglobin on protein stability
Authors: Pinker Rachel J. , Lin Laura , Kallenbach Neville R. , Rose George D. 
Published: February 2009

AbstractThe peptide backbones in folded native proteins contain distinctive secondary structures, α‐helices, β‐sheets, and turns, with significant frequency. One question that arises in folding is how the stability of this secondary structure relates to that of the protein as a whole. To address this question, we substituted the α‐helix‐stabilizing alanine side chain at 16 selected sites in the sequence of sperm whale myoglobin, 12 at helical sites on the surface of the protein, and 4 at obviously internal sites. Substitution of alanine for bulky side chains at internal sites destabilizes the protein, as expected if packing interactions are disrupted. Alanine substitutions do not uniformly stabilize the protein, either in capping positions near the ends of helices or at mid‐helical sites near the surface of myoglobin. When corrected for the extent of exposure of each side chain replaced by alanine at a mid‐helix position, alanine replacement still has no clear effect in stabilizing the native structure. Thus linkage between the stabilization of secondary structure and tertiary structure in myoglobin cannot be demonstrated, probably because of the relatively small free energy differences between side chains in stabilizing isolated helix. By contrast, about 80% of the variance in free energy observed can be accounted for by the loss in buried surface area of the native residue substituted by alanine. The differential free energy of helix stabilization does not account for any additional variation.
Crystal structure to 2.45 Å resolution of a monoclonal Fab specific for the Brucella A cell wall polysaccharide antigen
Authors: Rose D. R. , Przybylska M. , To R. J. , Kayden C. S. , Vorberg E. , Young N. M. , Bundle D. R. , Oomen R. P. 
Published: February 2009

AbstractThe atomic structure of an antibody antigen‐binding fragment (Fab) at 2.45Åresolution shows that polysaccharide antigen conformation and Fab structure dictated by combinatorial diversity and domain association are responsible for the fine specificity of the Brucella‐specific antibody, YsT9.1. It discriminates the Brucella abortus A antigen from the nearly identical Brucella melitensis M antigen by forming a groove‐type binding site, lined with tyrosine residues, that accommodates the rodlike A antigen but excludes the kinked structure of the M antigen, as envisioned by a model of the antigen built into the combining site. The variable‐heavy (VH) and variable‐light (VL) domains are derived from genes closely related to two used in previously solved structures, M603 and R19.9, respectively. These genes combine in YsT9.1 to form an antibody of totally different specificity. Comparison of this X‐ray structure with a previously built model of the YsT9.1 combining site based on these homologies highlights the importance of VL:VH association as a determinant of specificity and suggests that small changes at the VL:VH interface, unanticipated in modeling, may cause significant modulation of binding‐site properties.
Binding of ferredoxin to ferredoxin: NADP+ oxidoreductase: The role of carboxyl groups, electrostatic surface potential, and molecular dipole moment
Authors: De Pascalis Antonio R. , Jelesarov Ilian , Ackermann Friederike , Bosshard Hans Rudolf , Koppenol Willem H. , Hirasawa Masakazu , Knaff David B. 
Published: February 2009

AbstractThe small, soluble, (2Fe‐2S)‐containing protein ferredoxin (Fd) mediates electron transfer from the chloroplast photosystem I to ferredoxin:NADP+ oxidoreductase (FNR), a flavoenzyme located on the stromal side of the thylakoid membrane. Ferredoxin and FNR form a 1:1 complex, which is stabilized by electrostatic interactions between acidic residues of Fd and basic residues of FNR. We have used differential chemical modification of Fd to locate aspartic and glutamic acid residues at the intermolecular interface of the Fd:FNR complex (both proteins from spinach). Carboxyl groups of free and FNR‐bound Fd were amidated with carbodiimide/2‐aminoethane sulfonic acid (taurine). The differential reactivity of carboxyl groups was assessed by double isotope labeling. Residues protected in the Fd:FNR complex were D‐26, E‐29, E‐30, D‐34, D‐65, and D‐66. The protected residues belong to two domains of negative electrostatic surface potential on either side of the iron‐sulfur cluster. The negative end of the molecular dipole moment vector of Fd (377 Debye) is close to the iron‐sulfur cluster, in the center of the area demarcated by the protected carboxyl groups. The molecular dipole moment and the asymmetric surface potential may help to orient Fd in the reaction with FNR. In support, we find complementary domains of positive electrostatic potential on either side of the FAD redox center of FNR. The results allow a binding model for the Fd:FNR complex to be constructed.
Crystal structure of activated tobacco rubisco complexed with the reaction‐intermediate analogue 2‐carboxy‐arabinitol 1, 5‐bisphosphate
Authors: Schreuder Herman A. , Curmi Paul M. G. , Cascio Duilio , Eisenberg David , Andersson Inger , Knight Stefan , Brändén Carl‐Ivar , Sweet Robert M. 
Published: February 2009

AbstractThe crystal structure of activated tobacco rubisco, complexed with the reaction‐intermediate analogue 2‐carboxy‐arabinitol 1, 5‐bisphosphate (CABP) has been determined by molecular replacement, using the structure of activated spinach rubisco (Knight, S., Andersson, I., &Brändén, C.‐I., 1990, J. Mol. Biol. 215, 113‐160) as a model. The R‐factor after refinement is 21.0% for 57, 855 reflections between 9.0 and 2.7Åresolution.The local fourfold axis of the rubisco hexadecamer coincides with a crystallographic twofold axis. The result is that the asymmetric unit of the crystals contains half of the L8S8 complex (molecular mass 280 kDa in the asymmetric unit). The activated form of tobacco rubisco is very similar to the activated form of spinach rubisco. The root mean square difference is 0.4Åfor 587 equivalent Cα atoms. Analysis of mutations between tobacco and spinach rubisco revealed that the vast majority of mutations concerned exposed residues. Only 7 buried residues were found to be mutated versus 54 residues at or near the surface of the protein.The crystal structure suggests that the Cys 247‐Cys 247 and Cys 449‐Cys 459 pairs are linked via disulfide bridges. This pattern of disulfide links differs from the pattern of disulfide links observed in crystals of unactivated tobacco rubisco (Curmi, P.M.G., et al., 1992, J. Biol. Chem. 267, 16980‐16989) and is similar to the pattern observed for activated spinach tobacco.
Leucine 332 influences the CO2/O2 specificity factor of ribulose‐1, 5‐bisphosphate carboxylase/oxygenase from Anacystis nidulans
Authors: Lee Garrett J. , McDonald Kelly A. , McFadden Bruce A. 
Published: February 2009

AbstractThe role of Leu 332 in ribulose‐1, 5‐bisphosphate carboxylase/oxygenase from the cyanobacterium Anacystis nidulans was investigated by site‐directed mutagenesis. Substitutions of this residue with Met, Ile, Val, Thr, or Ala decreased the CO2/O2 specificity factor by as much as 67% and 96% for the Ile mutant in the presence of Mg2+ and Mn2+, respectively. For the Met, Ile, and Ala mutants in the presence of Mg2+, no loss of oxygenase activity was observed despite the loss of greater than 65% of the carboxylase activity relative to the wild‐type enzyme. In the presence of Mn2+, carboxylase activities for mutant enzymes were reduced to approximately the same degree as was observed in the presence of Mg2+, although oxygenase activities were also reduced to similar extents as carboxylase activities. Only minor changes in Km(RuBp) were observed for all mutants in the presence of Mg2+ relative to the wild‐type enzyme, indicating that Leu 332 does not function in RuBP binding. These results suggest that in the presence of Mg2+, Leu 332 contributes to the stabilization of the transition state for the carboxylase reaction, and demonstrate that it is possible to affect only one of the activities of this bifunctional enzyme.
Characterization of the stable, acid‐induced, molten globule‐like state of staphylococcal nuclease
Authors: Fink Anthony L. , Calciano Linda J. , Goto Yuji , Nishimura Midori , Swedberg Sally A. 
Published: February 2009

AbstractTitration of a salt‐free solution of native staphylococcal nuclease by HC1 leads to an unfolding transition in the vicinity of pH 4, as determined by near‐ and far‐UV circular dichroism. At pH 2‐3, the protein is substantially unfolded. The addition of further HC1 results in a second transition, this one to a more structured species (the A state) with the properties of an expanded molten globule, namely substantial secondary structure, little or no tertiary structure, relatively compact size as determined by hydrodynamic radius, and the ability to bind the hydrophobic dye 1‐anilino‐8‐naphthalene sulfonic acid. The addition of anions, in the form of neutral salts, to the acid‐unfolded state at pH 2 also causes a transition leading to the A state. Fourier transform infrared analysis of the amide I band was used to compare the amount and type of secondary structure in the native and A states. A significant decrease in α‐helix structure, with a corresponding increase in β or extended structure, was observed in the A state, compared to the native state. A model to account for such compact denatured states is proposed.
Refolding and proton pumping activity of a polyethylene glycol‐bacteriorhodopsin water‐soluble conjugate
Authors: Sirokmán Géza , Fasman Gerald D. 
Published: February 2009

AbstractBacteriorhodopsin (BR), from the purple membrane (PM) of Halobacterium halobium, was chemically modified with methoxypolyethylene glycol (m‐PEG; molecular weight = 5, 000 Da) succinimidyl carbonate. The polyethylene glycol‐bacteriorhodopsin (m‐PEG‐SC‐BR33) conjugate, containing one polyethylene glycol chain, was water soluble. The secondary structure of the conjugate in water appeared partially denatured, but was shown to contain α‐helical segments by circular dichroism spectroscopy. The isolated bacteriorhodopsin conjugate, with added retinal, was refolded in a mixed detergent‐lipid micelle and had an absorption maximum at 555 nm. The refolded conjugate was transferred into vesicles that pumped protons, upon illumination, as efficiently as did native BR. Modification of the PM with m‐PEG did not alter the native structure or inhibit proton pumping, and therefore it is suggested that the glycol polymer is present as a moiety covalently linked to residues unnecessary for proton pumping and proper folding. The site of attachment of m‐PEG was determined to be at either Lys 129 or Lys 159, with position Lys 129 the most probable site of attachment. The m‐PEG‐SC‐BR33 could be stepwise refolded to the native conformation by the addition of trifluoroethanol to lower the dielectric constant, simulating the insertion of the BR into the phospholipid bilayer.
Cross‐validation of protein structural class prediction using statistical clustering and neural networks
Authors: Metfessel Brent A. , Connelly Donald P. , Rich Stephen S. , Saurugger Peter N. 
Published: February 2009

AbstractWe present an approach to predicting protein structural class that uses amino acid composition and hydrophobic pattern frequency information as input to two types of neural networks: (1) a three‐layer back‐propagation network and (2) a learning vector quantization network. The results of these methods are compared to those obtained from a modified Euclidean statistical clustering algorithm. The protein sequence data used to drive these algorithms consist of the normalized frequency of up to 20 amino acid types and six hydrophobic amino acid patterns. From these frequency values the structural class predictions for each protein (all‐alpha, all‐beta, or alpha‐beta classes) are derived. Examples consisting of 64 previously classified proteins were randomly divided into multiple training (56 proteins) and test (8 proteins) sets. The best performing algorithm on the test sets was the learning vector quantization network using 17 inputs, obtaining a prediction accuracy of 80.2%. The Matthews correlation coefficients are statistically significant for all algorithms and all structural classes. The differences between algorithms are in general not statistically significant. These results show that information exists in protein primary sequences that is easily obtainable and useful for the prediction of protein structural class by neural networks as well as by standard statistical clustering algorithms.
Synthetic chimeras of mouse growth factor‐associated glandular kallikreins. I. Kinetic properties
Authors: Blaber Michael , Isackson Paul J. , Bradshaw Ralph A. , Burnier John P. , Marsters James C. 
Published: February 2009

AbstractA series of six chimeric proteins, composed of fragments corresponding to either one or the other of the growth factor‐associated mouse glandular kallikreins–epidermal growth factor binding protein (EGF‐BP) and the γ‐subunit of nerve growth factor (γ‐NGF)–were expressed in Escherichia coli and isolated, and their kinetic properties were characterized. The assembly of these synthetic proteases involved the substitution of regions of the proteins containing four specific surface loops that have been postulated to influence both kinetic specificity and the formation of growth factor complexes. The substrates utilized in the kinetic characterization of these chimeric kallikreins were tripeptide nitroanilides representing carboxyl termini of both the EGF and β‐NGF mature hormones, putative processing sites for these kallikreins in the precursors. Characterization of these hybrid enzymes demonstrates that Km and kcat kinetic constants may be independently affected by the regions utilized in construction of these chimeric kallikreins. Specifically, loop 1, located in the amino terminal region (Bode, W., et al., J. Mol. Biol. 164, 237–282, 1983), in γ‐NGF enhanced the kcat for substrates containing threonine in the P2 position, as is the case during the processing of the carboxy terminus of the β‐NGF precursor. Also, the central regions of the kallikreins containing loop 2 and the kallikrein loop dictated the generally inverted Km and kcat kinetic constants observed between EGF‐BP and γ‐NGF. Finally, in γ‐NGF the autolysis loop, found in the carboxyl terminal region, functions to lower the Km kinetic constant for a variety of substrates. The results allow previously characterized kinetic differences between EGF‐BP and γ‐NGF to be interpreted in terms of specific regions of the proteins and identify a subset of amino acid positions responsible for these functional characteristics.
Synthetic chimeras of mouse growth factor‐associated glandular kallikreins. II. Growth factor binding properties
Authors: Blaber Michael , Isackson Paul J. , Bradshaw Ralph A. , Holden Hazel M. 
Published: February 2009

AbstractSix chimeric constructs of the sequentially similar growth factor‐associated kallikreins–epidermal growth factor binding protein (EGF‐BP) and the γ‐subunit of nerve growth factor (γ‐NGF)–have been expressed, and their ability to generate complexes with epidermal growth factor (EGF) and β‐NGF, analogous to the high molecular weight forms (7S NGF and HMW‐EGF) found in the mouse submaxillary gland, evaluated. The chimeras are distinguished by the interchange of three regions composing the amino, middle, and carboxyl terminal regions that encompass four surface loops possibly involved in specific growth factor interactions. Native β‐NGF (along with native α‐NGF) formed complexes indistinguishable from naturally occurring 7S NGF, characterized by an α2βγ2 structure (where β‐NGF is itself a dimer), with recombinant (r) γ‐NGF and with a chimera in which the amino terminal region from EGF‐BP was substituted. Two other chimeras containing either the middle or carboxyl terminal regions of γ‐NGF showed weaker ability to form 7S complexes. Thus, all chimeras containing two segments from γ‐NGF retained at least some ability to form the 7S complex. rEGF‐BP reacted weakly with EGF, but the chimera composed of the amino and middle segments of EGF‐BP and the carboxyl terminal segment of γ‐NGF formed a nativelike HMW‐EGF complex. None of the other chimeras appeared to bind EGF. These results identify amino acid positions within each kallikrein that participate in strong growth factor interactions and demonstrate that, outside of active site contacts, different regions of the kallikreins are involved in the binding of EGF and β‐NGF, respectively.
Design of synthetic gene libraries encoding random sequence proteins with desired ensemble characteristics
Authors: Labean Thomas H. , Kauffman Stuart A. 
Published: February 2009

AbstractLibraries of random sequence polypeptides are useful as sources of unevolved proteins, novel ligands, and potential lead compounds for the development of vaccines and therapeutics. The expression of small random peptides has been achieved previously using DNA synthesized with equimolar mixtures of nucleotides. For many potential uses of random polypeptide libraries, concerns such as avoiding termination codons and matching target amino acid compositions make more complex designs necessary. In this study, three mixtures of nucleotides, corresponding to the three positions in the codon, were designed such that semirandom DNA synthesized by repeated cycles of the three mixtures created an open reading frame encoding random sequence polypeptides with desired ensemble characteristics. Two methods were used to design the nucleotide mixtures: the manual use of a spreadsheet and a refining grid search algorithm. Using design targets of less than or equal to 1% stop codons and an amino acid composition based on the average ratios observed in natural, globular proteins, the search methods yielded similar nucleotide ratios. Semirandom DNA, synthesized with a designed, three‐residue repeat pattern, can encode libraries of very high diversity and represents an important tool for the construction of random polypeptide libraries.
Independence of metal binding between tandem Cys2His2 zinc finger domains
Authors: Krizek Beth Allyn , Berg Jeremy M. , Zawadzke Laura E. 
Published: February 2009

AbstractMost Cys2His2 zinc finger proteins contain tandem arrays of metal binding domains. The tandem nature of these arrays suggests that metal binding by these domains may not be independent but rather that metal binding may occur in a cooperative manner. This is especially true in light of the crystal structure of a three zinc finger array bound to DNA that revealed several types of interactions between domains. To address this question, peptides containing two tandem domains have been prepared. While metal binding studies do show that the two finger peptide has a metal ion affinity about threefold higher than that for a single domain peptide with the same sequence, additional studies reveal that this behavior is due to increased single site affinities in the context of the two domain peptide rather than to cooperativity. These studies indicate that domains of this type are independent of one another with regard to metal binding, at least in the absence of DNA. This observation has implications with regard to the question of whether the activities of proteins of this class might be modulated by available zinc concentrations.