bluuebunny/crossref_metadata_2025_split huggingface dataset:
An Insight into the Molecular Basis of Salt Tolerance of l-myo-Inositol 1-P Synthase (PcINO1) from Porteresia coarctata (Roxb.) Tateoka, a Halophytic Wild Rice
Authors: Ghosh Dastidar Krishnarup , Maitra Susmita , Goswami Lily , Roy Debjani , Das Kali Pada , Majumder Arun Lahiri 
Published: February 2006

Abstract
The molecular basis of salt tolerance of l-myo-inositol 1-P synthase (MIPS; EC 5.5.1.4) from Porteresia coarctata (Roxb.) Tateoka (PcINO1, AF412340) earlier reported from this laboratory, has been analyzed by in vitro mutant and hybrid generation and subsequent biochemical and biophysical studies of the recombinant proteins. A 37-amino acid stretch between Trp-174 and Ser-210 has been confirmed as the salt-tolerance determinant domain in PcINO1 both by loss or gain of salt tolerance by either deletion or by addition to salt-sensitive MIPS(s) of Oryza (OsINO1) and Brassica juncea (BjINO1). This was further verified by growth analysis under salt environment of Schizosaccharomyces pombe transformed with the various gene constructs and studies on the differential behavior of mutant and wild proteins by Trp fluorescence, aggregation, and circular dichroism spectra in the presence of salt. 4,4′-Dianilino-1,1′-binaphthyl-5,5-disulfonic acid binding experiments revealed a lower hydrophobic surface on PcINO1 than OsINO1, contributed by this 37-amino acid stretch explaining the differential behavior of OsINO1 and PcINO1 both with respect to their enzymatic functions and thermodynamic stability in high salt environment. Detailed amino acid sequence comparison and modeling studies revealed the interposition of polar and charged residues and a well-connected hydrogen-bonding network formed by Ser and Thr in this stretch of PcINO1. On the contrary, hydrophobic residues clustered in two continuous stretches in the corresponding region of OsINO1 form a strong hydrophobic patch on the surface. It is conceivable that salt-tolerant MIPS proteins may be designed out of the salt-sensitive plant MIPS proteins by replacement of the corresponding amino acid stretch by the designated 37-amino acid stretch of PcINO1.
ArabidopsisSHORT HYPOCOTYL UNDER BLUE1 Contains SPX and EXS Domains and Acts in Cryptochrome Signaling
Authors: Kang Xiaojun , Ni Min 
Published: February 2006

AbstractPhotomorphogenesis is regulated by red/far-red light–absorbing phytochromes and blue/UV-A light–absorbing cryptochromes. We isolated an Arabidopsis thaliana blue light mutant, short hypocotyl under blue1 (shb1), a knockout allele. However, shb1-D, a dominant allele, exhibited a long-hypocotyl phenotype under red, far-red, and blue light. The phenotype conferred by shb1-D was caused by overaccumulation of SHB1 transcript and recapitulated by overexpression of SHB1 in Arabidopsis. Therefore, SHB1 acts in cryptochrome signaling but overexpression may expand its signaling activity to red and far-red light. Consistent with this, overexpression of SHB1 enhanced the expression of PHYTOCHROME-INTERACTING FACTOR4 (PIF4) under red light. PIF4 appears to specifically mediate SHB1 regulation of hypocotyl elongation and CHLOROPHYLL a/b BINDING PROTEIN3 or CHALCONE SYNTHASE expression under red light. Overexpression of SHB1 also promoted proteasome-mediated degradation of phytochrome A and hypocotyl elongation under far-red light. Under blue light, shb1 suppressed LONG HYPOCOTYL IN FAR-RED LIGHT1 (HFR1) expression and showed several deetiolation phenotypes similar to hfr1-201. However, the hypocotyl and cotyledon-opening phenotypes of shb1 were opposite to those of hfr1-201, and HFR1 acts downstream of SHB1. SHB1 encodes a nuclear and cytosolic protein that has motifs homologous with SYG1 protein family members. Therefore, our studies reveal a signaling step in regulating cryptochrome- and possibly phytochrome-mediated light responses.
“Zero‐length” cross‐linking in solid state as an approach for analysis of protein–protein interactions
Authors: El‐Shafey Ahmed , Tolic Nikola , Young Malin M. , Sale Kenneth , Smith Richard D. , Kery Vladimir 
Published: February 2006

AbstractWe have developed a new approach for the analysis of interacting interfaces in protein complexes and protein quaternary structure based on cross‐linking in the solid state. Protein complexes are freeze‐dried under vacuum, and cross‐links are introduced in the solid phase by dehydrating the protein in a nonaqueous solvent creating peptide bonds between amino and carboxyl groups of the interacting peptides. Cross‐linked proteins are digested into peptides with trypsin in both H216O and H218O and then readily distinguished in mass spectra by characteristic 8 atomic mass unit (amu) shifts reflecting incorporation of two 18O atoms into each C terminus of proteolytic peptides. Computer analysis of mass spectrometry (MS) and MS/MS data is used to identify the cross‐linked peptides. We demonstrated specificity and reproducibility of our method by cross‐linking homo‐oligomeric protein complexes of glutathione‐S‐transferase (GST) from Schistosoma japonicum alone or in a mixture of many other proteins. Identified cross‐links were predominantly of amide origin, but six esters and thioesters were also found. The cross‐linked peptides were validated against the GST monomer and dimer X‐ray structures and by experimental (MS/MS) analyses. Some of the identified cross‐links matched interacting peptides in the native 3D structure of GST, indicating that the structure of GST and its oligomeric complex remained primarily intact after freeze‐drying. The pattern of oligomeric GST obtained in solid state was the same as that obtained in solution by Ru (II) Bpy32+ catalyzed, oxidative “zero‐length” cross‐linking, confirming that it is feasible to use our strategy for analyzing the molecular interfaces of interacting proteins or peptides.
Physical characterization of MxiH and PrgI, the needle component of the type III secretion apparatus from Shigella and Salmonella
Authors: Darboe Numukunda , Kenjale Roma , Picking Wendy L. , Picking William D. , Middaugh C. Russell 
Published: February 2006

AbstractShigella and Salmonella use similar type III secretion systems for delivering effector proteins into host cells. This secretion system consists of a base anchored in both bacterial membranes and an extracellular “needle” that forms a rod‐like structure exposed on the pathogen surface. The needle is composed of multiple subunits of a single protein and makes direct contact with host cells to facilitate protein delivery. The proteins that make up the needle of Shigella and Salmonella are MxiH and PrgI, respectively. These proteins are attractive vaccine candidates because of their essential role in virulence and surface exposure. We therefore isolated, purified, and characterized the monomeric forms of MxiH and PrgI. Their far‐UV circular dichroism spectra show structural similarities with hints of subtle differences in their secondary structure. Both proteins are highly helical and thermally unstable, with PrgI having a midpoint of thermal unfolding (Tm) near 37°C and MxiH having a value near 42°C. The two proteins also have comparable intrinsic stabilities as measured by chemically induced (urea) unfolding. MxiH, however, with a free energy of unfolding (ΔG°0,un) of 1.6 kcal/mol, is slightly more stable than PrgI (1.2 kcal/mol). The relatively low m‐values obtained for the urea‐induced unfolding of the proteins suggest that they undergo only a small change in solvent‐accessible surface area. This argues that when MxiH and PrgI are incorporated into the needle complex, they obtain a more stable structural state through the introduction of protein–protein interactions.
Role of unfolded state heterogeneity and en‐route ruggedness in protein folding kinetics
Authors: Ellison Paul A. , Cavagnero Silvia 
Published: February 2006

AbstractIn order to improve our understanding of the physical bases of protein folding, there is a compelling need for better connections between experimental and computational approaches. This work addresses the role of unfolded state conformational heterogeneity and en‐route intermediates, as an aid for planning and interpreting protein folding experiments. The expected kinetics were modeled for different types of energy landscapes, including multiple parallel folding routes, preferential paths dominated by one primary folding route, and distributed paths with a wide spectrum of microscopic folding rate constants. In the presence of one or more preferential routes, conformational exchange among unfolded state populations slows down the observed rates for native protein formation. We find this to be a general phenomenon, taking place even when unfolded conformations interconvert much faster than the “escape” rate constants to folding. Dramatic kinetic deceleration is expected in the presence of an increasing number of folding‐incompetent unfolded conformations. This argues for the existence of parallel folding paths involving several folding‐competent unfolded conformations, during the early stages of protein folding. Deviations from single‐exponential behavior are observed for unfolded conformations exchanging at comparable rates or more slowly than folding events. Analysis of the effect of en‐route (on‐path) intermediate formation and landscape ruggedness on folding kinetics leads to the following unexpected conclusions: (1) intermediates, which often retard native state formation, may in some cases accelerate folding, and (2) rugged landscapes, usually associated with stretched exponentials, display single‐exponential behavior in the presence of late high‐friction paths.
Amyloid β‐protein monomer structure: A computational and experimental study
Authors: Baumketner Andrij , Bernstein Summer L. , Wyttenbach Thomas , Bitan Gal , Teplow David B. , Bowers Michael T. , Shea Joan‐Emma 
Published: February 2006

AbstractThe structural properties of the Aβ42 peptide, a main constituent of the amyloid plaques formed in Alzheimer's disease, were investigated through a combination of ion‐mobility mass spectrometry and theoretical modeling. Replica exchange molecular dynamics simulations using a fully atomic description of the peptide and implicit water solvent were performed on the −3 charge state of the peptide, its preferred state under experimental conditions. Equilibrated structures at 300 K were clustered into three distinct families with similar structural features within a family and with significant root mean square deviations between families. An analysis of secondary structure indicates the Aβ42 peptide conformations are dominated by loops and turns but show some helical structure in the C‐terminal hydrophobic tail. A second calculation on Aβ42 in a solvent‐free environment yields compact structures turned “inside out” from the solution structures (hydrophobic parts on the outside, polar parts on the inside). Ion mobility experiments on the Aβ42 −3 charge state electrosprayed from solution yield a bimodal arrival time distribution. This distribution can be quantitatively fit using cross‐sections from dehydrated forms of the three families of calculated solution structures and the calculated solvent‐free family of structures. Implications of the calculations on the early stages of aggregation of Aβ42 are discussed.
Automated extraction of backbone deuteration levels from amide H/2H mass spectrometry experiments
Authors: Hotchko Matthew , Anand Ganesh S. , Komives Elizabeth A. , Ten Eyck Lynn F. 
Published: February 2006

AbstractA Fourier deconvolution method has been developed to explicitly determine the amount of backbone amide deuterium incorporated into protein regions or segments by hydrogen/deuterium (H/D) exchange with high‐resolution mass spectrometry. Determination and analysis of the level and number of backbone amide exchanging in solution provide more information about the solvent accessibility of the protein than do previous centroid methods, which only calculate the average deuterons exchanged. After exchange, a protein is digested into peptides as a way of determining the exchange within a local area of the protein. The mass of a peptide upon deuteration is a sum of the natural isotope abundance, fast exchanging side‐chain hydrogens (present in MALDI‐TOF H/2H data) and backbone amide exchange. Removal of the components of the isotopic distribution due to the natural isotope abundances and the fast exchanging side‐chains allows for a precise quantification of the levels of backbone amide exchange, as is shown by an example from protein kinase A. The deconvoluted results are affected by overlapping peptides or inconsistent mass envelopes, and evaluation procedures for these cases are discussed. Finally, a method for determining the back exchange corrected populations is presented, and its effect on the data is discussed under various circumstances.
On the precision of experimentally determined protein folding rates and ϕ‐values
Authors: De Los Rios Miguel A. , Muralidhara B.K. , Wildes David , Sosnick Tobin R. , Marqusee Susan , Wittung‐Stafshede Pernilla , Plaxco Kevin W. , Ruczinski Ingo 
Published: February 2006

Abstractϕ‐Values, a relatively direct probe of transition‐state structure, are an important benchmark in both experimental and theoretical studies of protein folding. Recently, however, significant controversy has emerged regarding the reliability with which ϕ‐values can be determined experimentally: Because ϕ is a ratio of differences between experimental observables it is extremely sensitive to errors in those observations when the differences are small. Here we address this issue directly by performing blind, replicate measurements in three laboratories. By monitoring within‐ and between‐laboratory variability, we have determined the precision with which folding rates and ϕ‐values are measured using generally accepted laboratory practices and under conditions typical of our laboratories. We find that, unless the change in free energy associated with the probing mutation is quite large, the precision of ϕ‐values is relatively poor when determined using rates extrapolated to the absence of denaturant. In contrast, when we employ rates estimated at nonzero denaturant concentrations or assume that the slopes of the chevron arms (mf and mu) are invariant upon mutation, the precision of our estimates of ϕ is significantly improved. Nevertheless, the reproducibility we thus obtain still compares poorly with the confidence intervals typically reported in the literature. This discrepancy appears to arise due to differences in how precision is calculated, the dependence of precision on the number of data points employed in defining a chevron, and interlaboratory sources of variability that may have been largely ignored in the prior literature.
Solution structure and backbone dynamics of an N‐terminal ubiquitin‐like domain in the GLUT4‐regulating protein, TUG
Authors: Tettamanzi M. Cristina , Yu Chenfei , Bogan Jonathan S. , Hodsdon Michael E. 
Published: February 2006

AbstractThe GLUT4‐regulating protein, TUG, functions to retain GLUT4‐containing membrane vesicles intracellularly and, in response to insulin stimulation, releases these vesicles to the cellular exocytic machinery for translocation to the plasma membrane. As part of our on going effort to describe the molecular basis for TUG function, we have determined the tertiary structure and characterized the backbone dynamics for an N‐terminal ubiquitin‐like domain (TUG–UBL1) using NMR spectroscopy. A well‐ordered conformation is observed for residues 10–83 of full‐length TUG, and confirms a β‐grasp or ubiquitin‐like topology. Although not required for in vitro association with GLUT4, the functional role of the TUG–UBL1 domain has not yet been described. We undertook a limited literature review of similar N‐terminal UBL domains and noted that a majority participate in protein–protein interactions, generally functioning as adaptor modules to physically associate the over all activity of the protein with a specific cellular process, such as the ubiquitin–proteasome pathway. In consistent fashion, TUG–UBL1 is not expected to participate in a covalent protein modification reaction as it lacks the characteristic C‐terminal diglycine (“GG”) motif required for conjugation to an acceptor lysine, and also lacks the three most common acceptor lysine residues involved in polyubiquitination. Additionally, analysis of the TUG–UBL1 molecular surface reveals a lack of conservation of the “Ile‐44 hydrophobic face” typically involved in ubiquitin recognition. Instead, we speculate on the possible significance of a concentrated area of negative electrostatic potential with increased backbone mobility, both of which are features suggestive of a potential protein–protein interaction site.
Substitutions of prolines examine their role in kinetic trap formation of the caspase recruitment domain (CARD) of RICK
Authors: Chen Yun‐Ru , Clark A. Clay 
Published: February 2006

AbstractCaspase recruitment domains (CARDs) are small helical protein domains that adopt the Greek key fold. For the two CARDs studied to date, RICK‐CARD and caspase‐1‐CARD (CP1‐CARD), the proteins unfold by an apparent two‐state process at equilibrium. However, the folding kinetics are complex for both proteins and may contain kinetically trapped species on the folding pathway. In the case of RICK‐CARD, the time constants of the slow refolding phases are consistent with proline isomerism. RICK‐CARD contains three prolines, P47 in turn 3, and P85 and P87. The latter two prolines constitute a nonconserved PxP motif in helix 6. To examine the role of the prolines in the complex folding kinetics of RICK‐CARD, we generated seven proline‐to‐alanine mutants, including three single mutants, three double mutants, and one triple mutant. We examined the spectroscopic properties, equilibrium folding, binding to CP1‐CARD, and folding kinetics. The results show that P85 is critical for maintaining the function of the protein and that all mutations decrease the stability. Results from single mixing and sequential mixing stopped‐flow studies strongly suggest the presence of parallel folding pathways consisting of at least two unfolded populations. The mutations affect the distribution of the two unfolded species, thereby affecting the population that folds through each channel. The two conformations also are present in the triple mutant, demonstrating that interconversion between them is not due to prolyl isomerism. Overall, the data show that the complex folding pathway, especially formation of kinetically trapped species, is not due to prolyl isomerism.
The early history of the ubiquitin field
Authors: Varshavsky Alexander 
Published: February 2006

AbstractThis is a personal account of the early history of ubiquitin research, by one of its protagonists. The field of ubiquitin and regulated protein degradation was created in the 1980s, largely through the complementary discoveries by the laboratory of A. Hershko (Technion, Haifa, Israel) and by my laboratory, then at MIT (Cambridge, MA). I describe the elegant insights by Hershko and his colleagues that yielded the initial understanding of ubiquitin conjugation and ubiquitin‐mediated proteolysis in cell extracts, including the identification of E1, E2, and E3 enzymes. These advances were followed by a set of interconnected discoveries in my laboratory that revealed the biology of the ubiquitin system, i.e., its necessity for the protein degradation in vivo, its specific physiological functions (in the cell cycle, DNA repair, protein synthesis, transcriptional regulation, and stress responses), the source of its selectivity (specific degradation signals in short‐lived proteins), and its key mechanistic attributes, such as the polyubiquitin chain and the subunit selectivity of protein degradation. The above biological (function‐based) insights produced the main discovery of the physiological regulation by intracellular protein degradation. These advances caused the enormous expansion of the ubiquitin field in the 1990s. Together with the initial discovery of ubiquitin‐mediated proteolysis by Hershko and coworkers, our biological discoveries in the 1980s led to a radically changed understanding of the logic of intracellular circuits, as it became clear that the control through regulated protein degradation rivals, and often surpasses in significance, the classical regulation through transcription and translation.
Adult‐onset Still's disease with prurigo pigmentosa‐like skin eruption
Authors: TOMARU Kahori , NAGAI Yayoi , OHYAMA Naoko , HASEGAWA Michiko , ENDO Yukie , TAMURA Atsushi , ISHIKAWA Osamu 
Published: March 2006

ABSTRACTA 34‐year‐old woman with adult‐onset Still's disease (AOSD) developed prurigo pigmentosa‐like lesions on her chest and upper back in addition to the typical rash of AOSD. A biopsy specimen taken from the upper back showed characteristic features of prurigo pigmentosa. The eruption and fever subsided immediately after the administration of 40 mg/day prednisolone, but arthralgia persisted even after intravenous pulse methylprednisolone therapy in combination with immunosuppressive drugs. Various atypical skin rashes, including prurigo pigmentosa‐like lesions, have been reported in association with AOSD. Therefore, one should carefully follow the clinical course of a patient in order not to overlook these atypical cutaneous manifestations of AOSD.
Fusion of ETV6 with an intronic sequence of the BAZ2A gene in a paediatric pre‐B acute lymphoblastic leukaemia with a cryptic chromosome 12 rearrangement
Authors: Panagopoulos Ioannis , Strömbeck Bodil , Isaksson Margareth , Heldrup Jesper , Olofsson Tor , Johansson Bertil 
Published: March 2006

Summary ETV6 at 12p13 is rearranged in a variety of haematological malignancies and solid tumours, with more than 20 different partners having been reported. These fusions result in either chimeric proteins or activation of the partner gene. However, there are a few examples of abnormalities resulting in truncated and, most likely, unproductive ETV6 proteins, suggesting that haploinsufficiency of ETV6 and/or the partner is leukaemogenic. We present a novel ETV6 rearrangement, identified in a paediatric pre‐B acute lymphoblastic leukaemia. Fluorescence in situ hybridisation (FISH) and molecular genetic analyses revealed a fusion of ETV6 and BAZ2A (at 12q13), generated through a cryptic rearrangement between 12p13 and 12q13, consisting of exons 1 and 2 of ETV6 and a sequence from intron 1 of BAZ2A. This transcript is not expected to produce any chimeric protein, but may encode a truncated form of ETV6, containing the first 54 amino acids (aa), followed by 16 aa from the 3′ fusion sequence, reminiscent of ETV6 fusions with MDS2, LOC115548, PER1, and STL. The rearrangement might also modify the regulation of BAZ2A by either activating a cryptic promoter or by coming under the control of the ETV6 promoter. The present case emphasises that ‘unproductive’ETV6 rearrangements may play an important pathogenetic role in leukaemia.