bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynamics of a truncated prion protein, PrP(113–231), from 15N NMR relaxation: Order parameters calculated and slow conformational fluctuations localized to a distinct region
Authors: O'Sullivan Denis B. D. , Jones Christopher E. , Abdelraheim Salama R. , Brazier Marcus W. , Toms Harold , Brown David R. , Viles John H.
Published: February 2009

Prion diseases are associated with the misfolding of the prion protein (PrPC) from a largely α‐helical isoform to a β‐sheet rich oligomer (PrPSc). Flexibility of the polypeptide could contribute to the ability of PrPC to undergo the conformational rearrangement during PrPC–PrPSc interactions, which then leads to the misfolded isoform. We have therefore examined the molecular motions of mouse PrPC, residues 113–231, in solution, using 15N NMR relaxation measurements. A truncated fragment has been used to eliminate the effect of the 90‐residue unstructured tail of PrPC so the dynamics of the structured domain can be studied in isolation. 15N longitudinal (T1) and transverse relaxation (T2) times as well as the proton‐nitrogen nuclear Overhauser effects have been used to calculate the spectral density at three frequencies, 0, ωN, and 0.87ωH. Spectral densities at each residue indicate various time‐scale motions of the main‐chain. Even within the structured domain of PrPC, a diverse range of motions are observed. We find that removal of the tail increases T2 relaxation times significantly indicating that the tail is responsible for shortening of T2 times in full‐length PrPC. The truncated fragment of PrP has facilitated the determination of meaningful order parameters (S2) from the relaxation data and shows for the first time that all three helices in PrPC have similar rigidity. Slow conformational fluctuations of mouse PrPC are localized to a distinct region that involves residues 171 and 172. Interestingly, residues 170–175 have been identified as a segment within PrP that will form a steric zipper, believed to be the fundamental amyloid unit. The flexibility within these residues could facilitate the PrPC–PrPSc recognition process during fibril elongation.
Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn
Authors: Pan Hai , Chen Kenneth , Chu Liping , Kinderman Francis , Apostol Izydor , Huang Gang
Published: February 2009

Susceptibility of methionine residues to oxidation is a significant issue of protein therapeutics. Methionine oxidation may limit the product's clinical efficacy or stability. We have studied kinetics of methionine oxidation in the Fc portion of the human IgG2 and its impact on the interaction with FcRn and Protein A. Our results confirm previously published observations for IgG1 that two analogous solvent‐exposed methionine residues in IgG2, Met 252 and Met 428, oxidize more readily than the other methionine residue, Met 358, which is buried inside the Fc. Met 397, which is not present in IgG1 but in IgG2, oxidizes at similar rate as Met 358. Oxidation of two labile methionines, Met 252 and Met 428, weakens the binding of the intact antibody with Protein A and FcRn, two natural protein binding partners. Both of these binding partners share the same binding site on the Fc. Additionally, our results shows that Protein A may serve as a convenient and inexpensive surrogate for FcRn binding measurements.
Structural plasticity of the phage P22 tail needle gp26 probed with xenon gas
Authors: Olia Adam S. , Casjens Sherwood , Cingolani Gino
Published: February 2009

The tail needle, gp26, is a highly stable homo‐trimeric fiber found in the tail apparatus of bacteriophage P22. In the mature virion, gp26 is responsible for plugging the DNA exit channel, and likely plays an important role in penetrating the host cell envelope. In this article, we have determined the 1.98 Å resolution crystal structure of gp26 bound to xenon gas. The structure led us to identify a calcium and a chloride ion intimately bound at the interior of α‐helical core, as well as seven small cavities occupied by xenon atoms. The two ions engage in buried polar interactions with gp26 side chains that provide specificity and register to gp26 helical core, thus enhancing its stability. Conversely, the distribution of xenon accessible cavities correlates well with the flexibility of the fiber observed in solution and in the crystal structure. We suggest that small internal cavities in gp26 between the helical core and the C‐terminal tip allow for flexible swinging of the latter, without affecting the overall stability of the protein. The C‐terminal tip may be important in scanning the bacterial surface in search of a cell‐envelope penetration site, or for recognition of a yet unidentified receptor on the surface of the host.
Conformational stability of porcine serum transferrin
Authors: Shen Zhi Min , Yang Jen Tsi , Feng You‐Min , Wu Chuen‐Shang C.
Published: February 2009

The conformation of porcine serum ferric transferrin (Tf) and its stability against denaturation were studied by circular dichroism. Tf was estimated to have 19–24% α‐helix and 50–55% β‐sheet based on the methods of Chang et al. (Chang, C.T., Wu, C.‐S.C., & Yang, J.T., 1978, Anal. Biochem. 91, 13–31) and Provencher and Glöckner (Provencher, S.W. & Glöckner, J., 1981, Biochemistry 20, 33–37). Removal of the bound ferric ions (apo‐Tf) did not alter the overall conformation, but there were subtle changes in local conformation based on its near‐UV CD spectrum. The Tfs were stable between pH 3.5 and 11. Denaturation by guanidine hydrochloride (Gu‐HCl) showed two transitions at 1.6 and 3.4 M denaturant. The process of denaturation by acid and base was reversible, whereas that by Gu‐HCl was partially reversible. The irreversible thermal unfolding of Tfs began at temperatures above 60 °C and was not complete even at 80 °C. The bound irons (based on absorbance at 460 nm) were completely released at pH <4 or in Gu‐HCl solution above 1.7 M, when the protein began to unfold, but they remained intact in neutral solution even at 85 °C. The NH2‐ and COOH‐terminal halves of the Tf molecule obtained by limited trypsin digestion had CD spectra similar to the spectrum of native Tf, and the COOH‐terminal fragment had more stable secondary structure than the NH2‐terminal fragment.
Kinetic studies of the refolding of yeast phosphoglycerate kinase: Comparison with the isolated engineered domains
Authors: Missiakas Dominique , Betton Jean‐Michel , Chaffotte Alain , Minard Philippe , Yon Jeannine M.
Published: February 2009

Unfolding and refolding kinetics of yeast phosphoglycerate kinase were studied by following the time‐dependent changes of two signals: the ellipticity at 218 nm and 222 nm, and the fluorescence emission at 330 nm (following excitation at 295 nm). The protein is composed of two similar‐sized structural domains. Each domain has been produced by recombinant DNA techniques. It has been previously demonstrated that the engineered isolated domains are able to fold into a quasinative structure (Minard, P., et al., 1989b, Protein Eng. 3, 55–60; Missiakas, D., Betton, J.M., Minard, P., & Yon, J.M., 1990, Biochemistry 29, 8683–8689). The behavior of the isolated domains was studied using the same two conformational probes as for the whole enzyme. We found that the refolding kinetics of each domain are multiphasic. In the whole protein, domain folding and pairing appeared to be simultaneous events. However, it was found that some refolding steps occurring during the refolding of the isolated C‐domain are masked during the refolding of yeast phosphoglycerate kinase. The N‐domain was also found to refold faster when it was isolated than when integrated.
Comparison of the X‐ray structure of native rubredoxin from pyrococcus furiosus with the NMR structure of the zinc‐substituted protein
Authors: Blake Paul R. , Day Michael W. , Hsu Barbara T. , Joshua‐Tor Leemor , Park Jae‐Bum , Hare Dennis R. , Adams Michael W. W. , Rees Douglas C. , Summers Michael F.
Published: February 2009

The three‐dimensional X‐ray structures of the oxidized and reduced forms of rubredoxin from Pyrococcus furiosus, determined at ‐161°C, and the NMR structure of the zinc‐substituted protein, determined in solution at 45°C, are compared. The NMR and X‐ray structures, which were determined independently, are very similar and lead to similar conclusions regarding the interactions that confer hyperthermostability.
The curious case of protein splicing: Mechanistic insights suggested by protein semisynthesis
Authors: Wallace CARMICHAEL J.A.
Published: February 2009

The gradual accumulation of examples of protein splicing, in which a nested intervening sequence is spliced out of the interior of a polyprotein precursor, suggests that this curious phenomenon might prove to have universal phylogenetic distribution and biological significance. The known examples are reviewed, with the aim of establishing underlying patterns, and a generalized mechanism of autocatalytic protein splicing is proposed. The testable consequences of such a proposal and the possible evolutionary origins of the phenomenon are discussed.
Covalent binding properties of the human complement protein C4 and hydrolysis rate of the internal thioester upon activation
Authors: Sepp Armin , Dodds Alister W. , Anderson Michael J. , Duncan Campbell R. , Willis Antony C. , Alex Law S.K.
Published: February 2009

The complement proteins C3 and C4 have an internal thioester. Upon activation on the surface of a target cell, the thioester becomes exposed and reactive to surface‐bound amino and hydroxyl groups, thus allowing covalent deposition of C3 and C4 on these targets. The two human C4 isotypes, C4A and C4B, which differ by only four amino acids, have different binding specificities. C4A binds more efficiently than C4B to amino groups, and C4B is more effective than C4A in binding to hydroxyl groups. By site‐directed mutagenesis, the four residues in a cDNA clone of C4B were modified. The variants were expressed and their binding properties studied. Variants with a histidine residue at position 1106 showed C4B‐like binding properties, and those with aspartic acid, alanine, or asparagine at the same position were C4A‐like. These results suggest that the histidine is important in catalyzing the reaction of the thioester with water and other hydroxyl group‐containing compounds. When substituted with other amino acids, this reaction is not catalyzed and the thioester becomes apparently more reactive with amino groups. This interpretation also predicts that the stability of the thioester in C4A and C4B, upon activation, will be different. We measured the time course of activation and binding of glycine to C4A and C4B. The lag in the binding curve behind the activation curve for C4A is significantly greater than that for C4B. The hydrolysis rates (k0) of the thioester in the activated proteins were estimated to be 0.068 s−1 (t1/2 of 10.3 s) for C4A and 1.08 s−1 (t1/2 of 0.64 s) for C4B. These results indicate that the difference in hydrolysis rate of the thioester accounts, at least in part, for the difference in the binding properties of C4A and C4B.
Chymotrypsin inhibitory activity of normal C1‐inhibitor and a P1 Arg to His mutant: Evidence for the presence of overlapping reactive centers
Authors: Aulak K.S. , Davis A.E. , Donaldson V.H. , Harrison R.A.
Published: February 2009

C1‐inhibitor is a serine proteinase inhibitor that is active against C1s, C1r, kallikrein, and factor XII. Recently, it has been shown that it also has inhibitory activity against chymotrypsin. We have investigated this activity of normal human C1‐inhibitor, normal rabbit C1‐inhibitor, and P1 Arg to His mutant human C1‐inhibitors and find that all are able to inhibit chymotrypsin and form stable sodium dodecyl sulfate‐resistant complexes. The Kass values show that the P1 His mutant is a slightly better inhibitor of chymotrypsin than normal human C1‐inhibitor (3.4 × 104 compared with 7.3 × 103). The carboxy‐terminal peptide of normal human C1‐inhibitor, derived from the dissociated protease‐inhibitor complex, shows cleavage between the P2 and P1 residues. Therefore, as with α2‐antiplasmin, C1‐inhibitor possesses two overlapping P1 residues, one for chymotrypsin and the other for Arg‐specific proteinases. In contrast, with the P1 His mutant, the peptide generated from the dissociation of its complex with chymotrypsin demonstrated cleavage between the P1 and P'1 residues. Therefore, unlike α2‐antiplasmin, chymotrypsin utilizes the P2 residue as its reactive site in normal C1‐inhibitor but utilizes the P1 residue as its reactive site in the P1 His mutant protein. This suggests that the reactive center loop allows a degree of induced fit and therefore must be relatively flexible.
Crystal structure analysis of amicyanin and apoamicyanin from paracoccus denitrificans at 2.0 Å and 1.8 Å resolution
Authors: Durley Rosemary , Chen Longyin , Scott Mathews F. , Lim Louis W. , Davidson Victor L.
Published: February 2009

The crystal structure of amicyanin, a cupredoxin isolated from Paracoccus denitrificans, has been determined by molecular replacement. The structure has been refined at 2.0 Å resolution using energy‐restrained least‐squares procedures to a crystallographic residual of 15.7%. The copper‐free protein, apoamicyanin, has also been refined to 1.8 Å resolution with residual 15.5%.

The protein is found to have a β‐sandwich topology with nine β‐strands forming two mixed β‐sheets. The secondary structure is very similar to that observed in the other classes of cupredoxins, such as plastocyanin and azurin. Amicyanin has approximately 20 residues at the N‐terminus that have no equivalents in the other proteins; a portion of these residues forms the first β‐strand of the structure.

The copper atom is located in a pocket between the β‐sheets and is found to have four coordinating ligands: two histidine nitrogens, one cysteine sulfur, and, at a longer distance, one methionine sulfur. The geometry of the copper coordination is very similar to that in the plant plastocyanins. Three of the four copper ligands are located in the loop between β‐strands eight and nine. This loop is shorter than that in the other cupredoxins, having only two residues each between the cysteine and histidine and the histidine and methionine ligands. The amicyanin and apoamicyanin structures are very similar; in particular, there is little difference in the positions of the coordinating ligands with or without copper.

One of the copper ligands, a histidine, lies close to the protein surface and is surrounded on that surface by seven hydrophobic residues. This hydrophobic patch is thought to be important as an electron transfer site.
Binding of amino acid side chains to preformed cavities: Interaction of serine proteinases with turkey ovomucoid third domains with coded and noncoded P1 residues
Authors: Bigler Theresa L. , Lu Wuyuan , Park Soon Jae , Tashiro Misao , Wieczorek Maciej , Wynn Richard , Laskowski Michael
Published: February 2009

In the association of serine proteinases with their cognate substrates and inhibitors an important interaction is the fitting of the P1 side chain of the substrate or inhibitor into a preformed cavity of the enzyme called the S1 pocket. In turkey ovomucoid third domain, which is a canonical protein proteinase inhibitor, the P1 residue is Leu18. Here we report the values of equilibrium constants, Ka, for turkey ovomucoid third domain and 13 additional Leu18X variants with six serine proteinases: bovine α chymotrypsin A, porcine pancreatic elastase, subtilisin Carlsberg, Streptomyces griseus proteinases A and B, and human leukocyte elastase. Eight of the Xs are coded amino acids: Ala, Ser, Val, Met, Gln, Glu, Lys, and Phe, and five are noncoded: Abu, Ape, Ahx, Ahp, and Hse. They were chosen to simplify the interamino acid comparisons. In the homologous series of straight‐chain side chains Ala, Abu, Ape, Ahx, Ahp, free energy of binding decreases monotonically with the side‐chain length for chymotrypsin with large binding pocket, but even for this enzyme shows curvature. For the two S. griseus enzymes a minimum appears to be reached at Ahp. A minimum is clearly evident for the two elastases, where increasing the side‐chain length from Ahx to Ahp greatly weakens binding, but much more so for the apparently more rigid pancreatic enzyme than for the more flexible leukocyte enzyme. β‐Branching (Ape/Val) is very deleterious for five of the six enzymes; it is only slightly deleterious for the more flexible human leukocyte elastase. The effect of γ‐branching (Ahx/Leu), of introduction of heteroatoms (Abu/Ser), (Ape/Hse), and (Ahx/Met), and of introduction of charge (Gln/Glu) and (Ahp/Lys) are tabulated and discussed. An important component of the free energy of interaction is the distortion of the binding pocket by bulky or branched side chains.

Most of the variants studied were obtained by enzymatic semisynthesis. X18 variants of the 6–18 peptide GlyNH2 were synthesized and combined with natural reduced peptide 19–56. Disulfide bridges were formed. The GlyNH2 was removed and the reactive‐site peptide bond X18‐Glu19 was synthesized by complex formation with proteinase K. The resultant complexes were dissociated by sudden pH drop. This kinetically controlled dissociation afforded virgin, reactive‐site‐intact inhibitor variants.
Comparison of the crystal structures of genetically engineered human manganese superoxide dismutase and manganese superoxide dismutase from thermus thermophilus: Differences in dimer–dimer interaction
Authors: Wagner Ulrike G. , Frolow Felix , Sussman Joel L. , Pattridge Katherine A. , Ludwig Martha L. , Stallings William C. , Werber Moshe M. , Oefner Christian
Published: February 2009

The three‐dimensional X‐ray structure of a recombinant human mitochondrial manganese superoxide dismutase (MnSOD) (chain length 198 residues) was determined by the method of molecular replacement using the related structure of MnSOD from Thermus thermophilus as a search model. This tetrameric human MnSOD crystallizes in space group P21212 with a dimer in the asymmetric unit (Wagner, U.G., Werber, M.M., Beck, Y., Hartman, J.R., Frolow, F., & Sussman, J.L., 1989, J. Mol. Biol. 206, 787–788). Refinement of the protein structure (3, 148 atoms with Mn and no solvents), with restraints maintaining noncrystallographic symmetry, converged at an R‐factor of 0.207 using all data from 8.0 to 3.2 Å resolution and group thermal parameters. The monomer–monomer interactions typical of bacterial Fe‐ and Mn‐containing SODs are retained in the human enzyme, but the dimer–dimer interactions that form the tetramer are very different from those found in the structure of MnSOD from T. thermophilus. In human MnSOD one of the dimers is rotated by 84° relative to its equivalent in the thermophile enzyme. As a result the monomers are arranged in an approximately tetrahedral array, the dimer–dimer packing is more intimate than observed in the bacterial MnSOD from T. thermophilus, and the dimers interdigitate. The metal—ligand interactions, determined by refinement and verified by computation of omit maps, are identical to those observed in T. thermophilus MnSOD.
Stress and strain in staphylococcal nuclease
Authors: Hodel Alec , Kautz Roger A. , Jacobs Marc D. , Fox Robert O.
Published: February 2009

Protein molecules generally adopt a tertiary structure in which all backbone and side chain conformations are arranged in local energy minima; however, in several well‐refined protein structures examples of locally strained geometries, such as cis peptide bonds, have been observed. Staphylococcal nuclease A contains a single cis peptide bond between residues Lys 116 and Pro 117 within a type VIaβ‐turn. Alternative native folded forms of nuclease A have been detected by NMR spectroscopy and attributed to a mixture of cis and trans isomers at the Lys 116‐Pro 117 peptide bond. Analyses of nuclease variants K116G and K116A by NMR spectroscopy and X‐ray crystallography are reported herein. The structure of K116A is indistinguishable from that of nuclease A, including a cis 116–117 peptide bond (92% populated in solution). The overall fold of K116G is also indistinguishable from nuclease A except in the region of the substitution (residues 112–117), which contains a predominantly trans Gly 116–Pro 117 peptide bond (80% populated in solution). Both Lys and Ala would be prohibited from adopting the backbone conformation of Gly 116 due to steric clashes between the β‐carbon and the surrounding residues. One explanation for these results is that the position of the ends of the residue 112–117 loop only allow trans conformations where the local backbone interactions associated with the ϕ and Ψ torsion angles are strained. When the 116–117 peptide bond is cis, less strained backbone conformations are available. Thus the relaxation of the backbone strain intrinsic to the trans conformation compensates for the energetically unfavorable cis X‐Pro peptide bond. With the removal of the side chain from residue 116 (K116G), the backbone strain of the trans conformation is reduced to the point that the conformation associated with the cis peptide bond is no longer favorable.
The molten globule intermediate of apomyoglobin and the process of protein folding
Authors: Barrick Doug , Baldwin Robert L.
Published: February 2009

The molten globule model for the beginning of the folding process, which originated with Kuwajima's studies of α‐lactalbumin (Kuwajima, K., 1989, Proteins Struct. Funct. Genet. 6, 87–103, and references therein), states that, for those proteins that exhibit equilibrium molten globule intermediates, the molten globule is a major kinetic intermediate near the start of the folding pathway. Pulsed hydrogen‐deuterium exchange measurements confirm this model for apomyoglobin (Jennings, P.A. & Wright, P.E., in prep.). The energetics of the acid‐induced unfolding transition, which have been determined by fitting a minimal three‐state model (N ↔ I ↔ U; N = native, I = molten globule intermediate, U = unfolded) show that I is more stable than U at neutral pH (Barrick, D. & Baldwin, R.L., 1993, Biochemistry 32, in press), which provides an explanation for why I is formed from U at the start of folding. Hydrogen exchange rates measured by two‐dimensional NMR for individual peptide NH protons, taken together with the CD spectrum of I, indicate that moderately stable helices are present in I at the locations of the A, G, and H helices of native myoglobin (Hughson, F.M., Wright, P.E., & Baldwin, R.L., 1990, Science 249, 1544–1548). Directed mutagenesis experiments indicate that the interactions between the A, G, and H helices in I are loose (Hughson, F.M., Barrick, D., & Baldwin, R.L., 1991, Biochemistry 30, 4113–4118), which can explain why I is formed rapidly from U at the start of folding. These experiments are consistent with the explanation proposed earlier (Baldwin, R.L., 1989, Trends Biochem. Sci. 14, 291–294) for the stabilization of native secondary structures in molten globule intermediates, namely that each unit of native secondary structure has a hydrophobic face, and the hydrophobic surfaces of two units of secondary structure can interact loosely to provide mutual stabilization. These topics are discussed here in the light of some additional results.
Families and the structural relatedness among globular proteins
Authors: Yee David P. , Dill Ken A.
Published: February 2009

Protein structures come in families. Are families “closely knit” or “loosely knit” entities? We describe a measure of relatedness among polymer conformations. Based on weighted distance maps, this measure differs from existing measures mainly in two respects: (1) it is computationally fast, and (2) it can compare any two proteins, regardless of their relative chain lengths or degree of similarity. It does not require finding relative alignments. The measure is used here to determine the dissimilarities between all 12, 403 possible pairs of 158 diverse protein structures from the Brookhaven Protein Data Bank (PDB). Combined with minimal spanning trees and hierarchical clustering methods, this measure is used to define structural families. It is also useful for rapidly searching a dataset of protein structures for specific substructural motifs. By using an analogy to distributions of Euclidean distances, we find that protein families are not tightly knit entities.
Structure of synthetic peptide analogues of an eggshell protein of Schistosoma mansoni
Authors: Middaugh C. Russell , Ryan James A. , Burke Carl J. , Mach Henryk , Naylor Adel M. , Bogusky Michael J. , Pitzenberger Steven M. , Ji Hanlee , Cordingley John S. , Thomson John A.
Published: February 2009

The peptide (Gly‐l‐Tyr‐l‐Asp‐l‐Lys‐l‐Tyr)6, referred to as F4–6, was synthesized as a model for a schistosome eggshell protein in which the Gly‐Tyr‐Asp‐Lys‐Tyr consensus sequence is repeated over 40 times. Analysis by CD, Fourier transform infrared spectroscopy, potentiometric and spectrophotometric titrations, NMR, and molecular modeling suggests that F4–6 forms some type of left‐handed structure. A likely possibility appears to be a left‐handed α‐helix stabilized by Lysi‐Aspi+4 salt bridges and possibly Aspi‐Tyri+4 hydrogen bonding and Tyr‐Tyr interactions. Spectroscopic studies of a number of F4–6 analogues support this conclusion. For example, substitution of d‐Ala for Gly produces a peptide with enhanced left‐handed helical spectral characteristics, whereas an l‐Ala substitution results in a peptide with minimal structure. These studies suggest that the F4 protein from Schistosoma mansoni may be the first example of a naturally occurring protein devoid of proline and carbohydrate that forms a left‐handed helix composed of l‐amino acids, although alternative forms of other left‐handed structures have yet to be rigorously excluded.
Brownian dynamics simulations of molecular recognition in an antibody‐antigen system
Authors: Kozack Richard E. , Subramaniam Shankar
Published: February 2009

The crystal structure for an antibody‐antigen system, that of the anti‐hen egg lysozyme monoclonal antibody HyHEL‐5 complexed to lysozyme, is used as the starting point for computer simulations of diffusional encounters between the two proteins. The investigation consists of two parts: first, the linearized Poisson‐Boltzmann equation is solved to determine the long‐range electrostatic forces between antibody and antigen, and then, the relative motion as influenced by these forces is modeled within Brownian motion theory. The effects of various point mutations on the calculated reaction rate are considered. It is found that charged residues close to the binding site exert the greatest influence in steering the proteins into a configuration favorable for their binding, while more distant mutations are qualitatively described by the Smoluchowski model for the mutual diffusion of two uniformly charged spheres. The antibody residues involved in forming salt links with the lysozyme, Glu‐H35 and Glu‐H50, appear to be particularly important in electrostatic steering, as neutralization of both of them yields reaction rates that are two to three orders of magnitude below those of wild‐type rates. The relative rates obtained from the simulations can be tested through kinetic measurements on mutant protein complexes. Kinetically efficient partners can also be designed and constructed through directed mutagenesis.
Molecular dynamics simulations and rigid body (TLS) analysis of aspartate carbamoyltransferase: Evidence for an uncoupled R state
Authors: Tanner John J. , Smith Paul E. , Krause Kurt L.
Published: February 2009

In the R form of ATCase complexed with the bisubstrate analogue, N‐(phosphonacetyl)‐l‐aspartate, large temperature factors are reported for the allosteric domains of the regulatory chains. We studied the conformational flexibility of the holoenzyme with molecular dynamics simulations and rigid body (TLS) analysis. The results of the molecular dynamics simulations suggest that, although local atomic fluctuations account for the temperature factors of the catalytic and zinc domains, they do not account for the large temperature factors of the allosteric regions. However, the temperature factors of the allosteric domains can be satisfactorily analyzed using a rigid body model. The simulations and rigid body analysis support the idea that the allosteric regions are mechanically uncoupled from the rest of the enzyme in the PALA structure. Implications of this uncoupling for allosteric regulation are discussed.
Structures of DNA‐binding mutant zinc finger domains: Implications for DNA binding
Authors: Hoffman Ross C. , Klevit Rachel E. , Horvath Suzanna J.
Published: February 2009

Studies of Cys2‐His2 zinc finger domains have revealed that the structures of individual finger domains in solution determined by NMR spectroscopy are strikingly similar to the structure of fingers bound to DNA determined by X‐ray diffraction. Therefore, detailed structural analyses of single finger domains that contain amino acid substitutions known to affect DNA binding in the whole protein can yield information concerning the structural ramifications of such mutations. We have used this approach to study two mutants in the N‐terminal finger domain of ADR1, a yeast transcription factor that contains two Cys2‐His2 zinc finger sequences spanning residues 102‐159. Two point mutants at position 118 in the N‐terminal zinc finger (ADR1b: 102‐130) that adversely affect the DNA‐binding activity of ADR1 have previously been identified: H118A and H118Y. The structures of wild‐type ADR1b and the two mutant zinc finger domains were determined using two‐dimensional nuclear magnetic resonance spectroscopy and distance geometry and were refined using a complete relaxation matrix method approach (REPENT) to improve agreement between the models and the nuclear Overhauser effect spectroscopy data from which they were generated. The molecular architecture of the refined wild‐type ADR1b domain is presented in detail. Comparisons of wild‐type ADR1b and the two mutants revealed that neither mutation causes a significant structural perturbation. The structures indicate that the DNA binding properties of the His 118 mutants are dependent on the identity of the side chain at position 118, which has been postulated to make a direct DNA contact in the wild‐type ADR1 protein. The results suggest that the identity of the side chain at the middle DNA contact position in Cys2‐His2 zinc fingers may be changed with impunity regarding the domain structure and can affect the affinity of the protein‐DNA interaction.
Disulfide bonds in homo‐ and heterodimers of EF‐hand subdomains of calbindin D9k: Stability, calcium binding, and NMR studies
Authors: Linse Sara , Thulin Eva , Sellers Peter
Published: February 2009

The effect of decreased protein flexibility on the stability and calcium binding properties of calbindin D9k has been addressed in studies of a disulfide bridged calbindin D9k mutant, denoted (L39C + P43M + I73C), with substitutions Leu 39 → Cys, Ile 73 → Cys, and Pro 43 → Met. Backbone 1H NMR assignments show that the disulfide bond, which forms spontaneously under air oxidation, is well accommodated. The disulfide is inserted on the opposite end of the protein molecule with respect to the calcium sites, to avoid direct interference with these sites, as confirmed by 113Cd NMR. The effect of the disulfide bond on calcium binding was assessed by titrations in the presence of a chromophoric chelator. A small but significant effect on the cooperativity was found, as well as a very modest reduction in calcium affinity. The disulfide bond increases Tm, the transition midpoint of thermal denaturation, of calcium free calbindin D9k from 85 to 95 °C and Cm, the urea concentration of half denaturation, from 5.3 to 8.0 M. Calbindins with one covalent bond linking the two EF‐hand subdomains are equally stable regardless if the covalent link is the 43‐44 peptide bond or the disulfide bond.

Kinetic remixing experiments show that separated CNBr fragments of (L39C + P43M + I73C), each comprising one EF‐hand, form disulfide linked homodimers. Each homodimer binds two calcium ions with positive cooperativity, and an average affinity of 106 M−1. Disulfide linkage dramatically increases the stability of each homodimer. For the homodimer of the C‐terminal fragment Tm increases from 59 + 2 without covalent linkage to 91 + 2 °C with disulfide, and Cm from = 1.5 to 7.5 M. The overall topology of this homodimer is derived from 1H NMR assignments and a few key NOEs.
In vivo formation of active aspartate transcarbamoylase from complementing fragments of the catalytic polypeptide chains
Authors: Yang Ying R. , Schachman H. K.
Published: February 2009

Despite the complexity of Escherichia coli aspartate transcarbamoylase (ATCase), composed of 12 polypeptide chains organized as two catalytic (C) trimers and three regulatory (R) dimers, it is possible to form active stable enzyme in vivo even with fragmented catalytic (c) chains. Based on the observation that chymotryptic digestion of the C trimers yields an active protein that can be dissociated into fragmented chains and then reconstituted in high yield, genetically engineered plasmids carrying the genes encoding each of the fragments were constructed. When the N‐terminal peptide (residues 1‐242) and the C‐terminal peptide (residues 235‐310) were expressed separately, each incomplete polypeptide chain was found in the insoluble fraction of the individual cell extracts. Mixing the two insoluble pellets in 6.5 M urea, followed by a 10‐fold dilution in buffer, led to the formation of active C trimers composed of incomplete polypeptide chains with an 8‐amino acid redundancy. When the two partial genes were linked into a single transcriptional unit separated by a 15‐nucleotide untranslated region containing a sequence for ribosome binding, the cells produced high yields of active C trimers composed of the incomplete, partially overlapping chains. The resulting protein, purified as C trimers or as holoenzyme formed by the addition of R subunits, has a specific activity (Vmax) only slightly less than that of the wild‐type C trimer and ATCase. However, Km for aspartate exhibited by the C trimer composed of fragmented chains is more than 10‐fold larger than that of the wild‐type trimer. The holoenzyme formed from the C trimer containing the coexpressed peptides is devoid of cooperativity with a Hill coefficient of 1.0, as contrasted to wild‐type ATCase for which the Hill coefficient is 1.7. Km for aspartate as well as Kd for the binding of the bisubstrate analog N‐(phosphonacetyl)‐l‐aspartate are significantly higher than the analogous values for wild‐type ATCase. Sedimentation velocity experiments indicate that the holoenzyme containing the incomplete chains has a conformation analogous to that of the R state of wild‐type ATCase.
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

Using 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

Coproporphyrinogen 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

The 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

The 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

The 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

The 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

The 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.