bluuebunny/crossref_metadata_2025_split huggingface dataset:
Amino acid repeat patterns in protein sequences: Their diversity and structural‐functional implications
Authors: Katti Mukund V. , Sami‐Subbu R. , Ranjekar Prabhakar K. , Gupta Vidya S. 
Published: February 2009

AbstractAll the protein sequences from SWISS‐PROT database were analyzed for occurrence of single amino acid repeats, tandem oligo‐peptide repeats, and periodically conserved amino acids. Single amino acid repeats of glutamine, serine, glutamic acid, glycine, and alanine seem to be tolerated to a considerable extent in many proteins. Tandem oligo‐peptide repeats of different types with varying levels of conservation were detected in several proteins and found to be conspicuous, particularly in structural and cell surface proteins. It appears that repeated sequence patterns may be a mechanism that provides regular arrays of spatial and functional groups, useful for structural packing or for one to one interactions with target molecules. To facilitate further explorations, a database of Tandem Repeats in Protein Sequences (TRIPS) has been developed and is available at URL: http://www.ncl‐india.org/trips.
Molecular simulation study of cooperativity in hydrophobic association
Authors: Czaplewski Cezary , Rodziewicz‐Motowidlo Sylwia , Liwo Adam , Ripoll Daniel R. , Wawak Ryszard J. , Scheraga Harold A. 
Published: February 2009

AbstractTo investigate the cooperativity of hydrophobic interactions, the potential of mean force of two‐ and three‐molecule methane clusters in water was determined by molecular dynamics simulations using two methods: umbrella‐sampling with the weighted histogram analysis method and thermodynamic integration. Two water models, TIP3P and TIP4P, were used, while each methane molecule was modeled as a united atom. It was found that the three‐body potential of mean force is not additive, i.e., it cannot be calculated as a sum of two‐body contributions, but requires an additional three‐body cooperative term. The cooperative term, which amounts to only about 10% of the total hydrophobic association free energy, was found to increase the strength of hydrophobic association; this finding differs from the results of earlier Monte Carlo studies with the free energy perturbation method of Rank and Baker (1997). As in the work of Rank and Baker, the solvent contribution to the potential of mean force was found to be well approximated by the molecular surface of two methane molecules. Moreover, we also found that the cooperative term is well represented by the difference between the molecular surface of the three‐methane cluster and those of all three pairs of methane molecules. In addition, it was found that, while there is a cooperative contribution to the hydrophobic association free energy albeit a small one, the errors associated with the use of pairwise potentials are comparable to or larger than this contribution.
Assisted folding of D‐glyceraldehyde‐3‐phosphate dehydrogenase by trigger factor
Authors: Huang Guo‐Chang , Li Zhen‐Yu , Zhou Jun‐Mei , Fischer Gunter 
Published: February 2009

AbstractThe Escherichia coli trigger factor is a peptidyl‐prolyl cis‐trans isomerase that catalyzes proline‐limited protein folding extremely well. Here, refolding of D‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) in the presence of trigger factor was investigated. The regain of activity of GAPDH was markedly increased by trigger factor after either long‐or short‐term denaturation, and detectable aggregation of GAPDH intermediates was prevented. In both cases, time courses of refolding of GAPDH were decelerated by trigger factor. The reactivation yield of GAPDH showed a slow down‐turn when molar ratios of trigger factor to GAPDH were above 5, due to tight binding between trigger factor and GAPDH intermediates. Such inactive bound GAPDH could be partially rescued from trigger factor by addition of reduced αLA as competitor, by further diluting the refolding mixture, or by disrupting hydrophobic interactions in the complexes. A model for trigger factor assisted refolding of GAPDH is proposed. We also suggest that assisted refolding of GAPDH is due mainly to the chaperone function of trigger factor.
Topography of a 2.0 Å structure of α1‐antitrypsin reveals targets for rational drug design to prevent conformational disease
Authors: Elliott Peter R. , Pei Xue Y. , Dafforn Timothy R. , Lomas David A. 
Published: February 2009

AbstractMembers of the serpin family of serine proteinase inhibitors play important roles in the inflammatory, coagulation, fibrinolytic, and complement cascades. An inherent part of their function is the ability to undergo a structural rearrangement, the stressed (S) to relaxed (R) transition, in which an extra strand is inserted into the central A β‐sheet. In order for this transition to take place, the A sheet has to be unusually flexible. Malfunctions in this flexibility can lead to aberrant protein linkage, serpin inactivation, and diseases as diverse as cirrhosis, thrombosis, angioedema, emphysema, and dementia. The development of agents that control this conformational rearrangement requires a high resolution structure of an active serpin. We present here the topology of the archetypal serpin α1‐antitrypsin to 2 Å resolution. This structure allows us to define five cavities that are potential targets for rational drug design to develop agents that will prevent conformational transitions and ameliorate the associated disease.
NMR structure of a concatemer of the first and second ligand‐binding modules of the human low‐density lipoprotein receptor
Authors: Kurniawan Nyoman D. , Atkins Annette R. , Bieri Stephan , Brown Catherine J. , Kroon Paulus A. , Smith Ross , Brereton Ian M. 
Published: February 2009

AbstractThe ligand‐binding domain of the human low‐density lipoprotein receptor consists of seven modules, each of 40–45 residues. In the presence of calcium, these modules adopt a common polypeptide fold with three conserved disulfide bonds. A concatemer of the first and second modules (LB1‐2) folds efficiently in the presence of calcium ions, forming the same disulfide connectivities as in the isolated modules. The three‐dimensional structure of LB1‐2 has now been solved using two‐dimensional 1H NMR spectroscopy and restrained molecular dynamics calculations. No intermodule nuclear Overhauser effects were observed, indicating the absence of persistent interaction between them. The near random‐coil NH and Hα chemical shifts and the low ϕ and ψ angle order parameters of the four‐residue linker suggest that it has considerable flexibility. The family of LB1‐2 structures superimposed well over LB1 or LB2, but not over both modules simultaneously. LB1 and LB2 have a similar pattern of calcium ligands, but the orientations of the indole rings of the tryptophan residues W23 and W66 differ, with the latter limiting solvent access to the calcium ion. From these studies, it appears that although most of the modules in the ligand‐binding region of the receptor are joined by short segments, these linkers may impart considerable flexibility on this region.
Soft metal ions, Cd(II) and Hg (II), induce triple‐stranded α‐helical assembly and folding of a de novo designed peptide in their trigonal geometries
Authors: Li Xiangqun , Suzuki Kazuo , Kashiwada Ayumi , Hiroaki Hidekazu , Kohda Daisuke , Tanaka Toshiki 
Published: February 2009

AbstractWe previously reported the de novo design of an amphiphilic peptide [YGG(IEKKIEA)4] that forms a native‐like, parallel triple‐stranded coiled coil. Starting from this peptide, we sought to regulate the assembly of the peptide by a metal ion. The replacement of the Ile 18 and Ile22 residues with Ala and Cys residues, respectively, in the hydrophobic positions disrupted of the triple‐stranded α‐helix structure. The addition of Cd(II), however, resulted in the reconstitution of the triple‐stranded α‐helix bundle, as revealed by circular dichroism (CD) spectroscopy and sedimentation equilibrium analysis. By titration with metal ions and monitoring the change in the intensity of the CD spectra at 222 nm, the dissociation constant Kd was determined to be 1.5 ± 0.8 μ for Cd(II). The triple‐stranded complex formed by the 113Cd(II) ion showed a single 113Cd NMR resonance at 572 ppm whose chemical shift was not affected by the presence of Cl− ions. The 113Cd NMR resonance was connected with the βH protons of the cysteine residue by 1H‐113Cd heteronuclear multiple quantum correlation spectroscopy. These NMR results indicate that the three cysteine residues are coordinated to the cadmium ion in a trigonal‐planar complex. Hg(II) also induced the assembly of the peptide into a triple‐stranded α‐helical bundle below the Hg (II)/peptide ratio of 1/3. With excess Hg(II), however, the α‐helicity of the peptide was decreased, with the change of the Hg(II) coordination state from three to two. Combining this construct with other functional domains should facilitate the production of artificial proteins with functions controlled by metal ions.
Bovine β‐lactoglobulin: Interaction studies with palmitic acid
Authors: Ragona Laura , Zetta Lucia , Fogolari Federico , Molinari Henriette , Pérez Dolores M. , Puyol Pilar , Kruif Kees De , Löhr Frank , Rüterjans Heinz 
Published: February 2009

AbstractBovine β‐lactoglobulin (BLG) in vivo has been found complexed with fatty acids, especially palmitic and oleic acid. To elucidate the still unknown structure‐function relationship in this protein, the interactions between 13C enriched palmitic acid (PA) and BLG were investigated by means of one‐, two‐, and three‐dimensional NMR spectroscopy in the pH range 8.4‐2.1. The NMR spectra revealed that at neutral pH the ligand is bound within the central cavity of BLG, with the methyl end deeply buried within the protein. The analysis of 13C spectra of the holo protein revealed the presence of conformational variability of bound PA carboxyl end in the pH range 8.4–5.9, related to the Tanford transition. The release of PA starts at pH lower than 6.0, and it is nearly complete at acidic pH. This finding is relevant in relation to the widely reported hypothesis that this protein can act as a transporter through the acidic gastric tract. Ligand binding and release is shown to be completely reversible over the entire pH range examined, differently from other fatty acid binding proteins whose behavior is analyzed throughout the paper. The mode of interaction of BLG is compatible with the proposed function of facilitating the digestion of milk fat during the neonatal period of calves.
The disulfide bond pattern of catrocollastatin C, a disintegrin‐like/cysteine‐rich protein isolated from Crotalus atrox venom
Authors: Calvete Juan J. , Moreno‐Murciano M. Paz , Sanz Libia , Jurgens Michael , Schrader Michael , Raida Manfred , Benjamin David C. , Fox Jay W. 
Published: February 2009

AbstractThe disulfide bond pattern of catrocollastatin‐C was determined by N‐terminal sequencing and mass spectrometry. The N‐terminal disintegrin‐like domain is a compact structure including eight disulfide bonds, seven of them in the same pattern as the disintegrin bitistatin. The protein has two extra cysteine residues (XIII and XVI) that form an additional disulfide bond that is characteristically found in the disintegrin‐like domains of cellular metalloproteinases (ADAMs) and PIII snake venom Zn‐metalloproteinases (SVMPs). The C‐terminal cysteine‐rich domain of catrocollastatin‐C contains five disulfide bonds between nearest‐neighbor cysteines and a long range disulfide bridge between CysV and CysX. These results provide structural evidence for a redefinition of the disintegrin‐like and cysteine‐rich domain boundaries. An evolutionary pathway for ADAMs, PIII, and PII SVMPs based on disulfide bond engineering is also proposed.
Structure of a protein G helix variant suggests the importance of helix propensity and helix dipole interactions in protein design
Authors: Strop Pavel , Marinescu Andrei M. , Mayo Stephen L. 
Published: February 2009

AbstractSix helix surface positions of protein G (Gβ1) were redesigned using a computational protein design algorithm, resulting in the five fold mutant Gβ1m2. Gβ1m2 is well folded with a circular dichroism spectrum nearly identical to that of Gβ1, and a melting temperature of 91 °C, ∼6°C higher than that of Gβ1. The crystal structure of Gβ1m2 was solved to 2.0 Å resolution by molecular replacement. The absence of hydrogen bond or salt bridge interactions between the designed residues in Gβ1m2 suggests that the increased stability of Gβ1m2 is due to increased helix propensity and more favorable helix dipole interactions.
Charge–charge interactions influence the denatured state ensemble and contribute to protein stability
Authors: Nick Pace C. , Alston Roy W. , Shaw Kevin L. 
Published: February 2009

AbstractSeveral recent studies have shown that it is possible to increase protein stability by improving electrostatic interactions among charged groups on the surface of the folded protein. How–ever, the stability increases are considerably smaller than predicted by a simple Coulomb's law calculation, and in some cases, a charge reversal on the surface leads to a decrease in stability when an increase was predicted. These results suggest that favorable charge–charge interactions are important in determining the dena–tured state ensemble, and that the free energy of the denatured state may be decreased more than that of the native state by reversing the charge of a side chain. We suggest that when the hydrophobic and hydrogen bonding interactions that stabilize the folded state are disrupted, the unfolded polypeptide chain rearranges to com–pact conformations with favorable long–range electrostatic inter–actions. These charge–charge interactions in the denatured state will reduce the net contribution of electrostatic interactions to protein stability and will help determine the denatured state ensemble. To support this idea, we show that the denatured state ensemble of ribonuclease Sa is considerably more compact at pH 7 where fa–vorable charge–charge interactions are possible than at pH 3, where unfavorable electrostatic repulsion among the positive charges causes an expansion of the denatured state ensemble. Further support is provided by studies of the ionic strength dependence of the sta–bility of charge–reversal mutants of ribonuclease Sa. These results may have important implications for the mechanism of protein folding.
Mesoscopic surfactant organization and membrane protein crystallization
Authors: Wiener Michael C. , Verkman Alan S. , Stroud Robert M. , Van Hoek Alfred N. 
Published: February 2009

AbstractThe paucity of detailed X–ray crystallographic struc–tures of integral membrane proteins arises from substantive technical obstacles in the overexpression of multimilligram quantities of protein, and in the crystallization of purified protein–detergent complexes (PDCs). With rare exception, crystal contacts within the lattice are mediated by protein‐protein interaction, and the detergent surrounding the protein behaves as a disordered solvent. The addition and use of surfactants that display mesoscopic self–assembly behavior in membrane protein crystallization experiments presents a novel alternative strategy. Well–ordered crystals of the water channel human aquaporin–1 (hAQP1) that diffract to 4 Å resolution have been obtained with this approach.
Recombinant decorsin: Dynamics of the RGD recognition site
Authors: Krezel Andrzej M. , Ulmer Jana S. , Wagner Gerhard , Lazarus Robert A. 
Published: February 2009

AbstractDecorsin is an antagonist of integrin αIIbβ3 and a potent platelet aggregation inhibitor. A synthetic gene encoding decorsin, originally isolated from the leech Macrobdella decora, was designed, constructed, and expressed in Escherichia coli. The synthetic gene was fused to the stII signal sequence and expressed under the transcriptional control of the E. coli alkaline phosphatase promoter. The protein was purified by size‐exclusion filtration of the periplasmic contents followed by reversed‐phase high‐performance liquid chromatography. Purified recombinant decorsin was found to be indistinguishable from leech‐derived decorsin based on amino acid composition, mass spectral analysis, and biological activity assays. Complete sequential assignments of 1H and proton bound 13C resonances were established. Stereospecific assignments of 21 of 25 nondegenerate β‐methylene groups were determined. The RGD adhesion site recognized by integrin receptors was found at the apex of a most exposed hairpin loop. The dynamic behavior of decorsin was analyzed using several independent NMR parameters. Although the loop containing the RGD sequence is the most flexible one in decorsin, the conformation of the RGD site itself is more restricted than in other proteins with similar activities.
Solution structure of a zinc substituted eukaryotic rubredoxin from the cryptomonad alga Guillardia theta
Authors: Schweimer Kristian , Hoffmann Silke , Rösch Paul , Sticht Heinrich , Wastl Jürgen , Maier Uwe G. 
Published: February 2009

AbstractThe rubredoxin from the cryptomonad Guillardia theta is one of the first examples of a rubredoxin encoded in a eukaryotic organism. The structure of a soluble zinc‐substituted 70‐residue G. theta rubredoxin lacking the membrane anchor and the thylakoid targeting sequence was determined by multidimensional heteronuclear NMR, representing the first three‐dimensional (3D) structure of a eukaryotic rubredoxin. For the structure calculation a strategy was applied in which information about hydrogen bonds was directly inferred from a long‐range HNCO experiment, and the dynamics of the protein was deduced from heteronuclear nuclear Overhauser effect data and exchange rates of the amide protons. The structure is well defined, exhibiting average root‐mean‐square deviations of 0.21 Å for the backbone heavy atoms and 0.67 Å for all heavy atoms of residues 7–56, and an increased flexibility toward the termini. The structure of this core fold is almost identical to that of prokaryotic rubredoxins. There are, however, significant differences with respect to the charge distribution at the protein surface, suggesting that G. theta rubredoxin exerts a different physiological function compared to the structurally characterized prokaryotic rubredoxins. The amino‐terminal residues containing the putative signal peptidase recognition/cleavage site show an increased flexibility compared to the core fold, but still adopt a defined 3D orientation, which is mainly stabilized by nonlocal interactions to residues of the carboxy‐terminal region. This orientation might reflect the structural elements and charge pattern necessary for correct signal peptidase recognition of the G. theta rubredoxin precursor.
Bypassing the kinetic trap of serpin protein folding by loop extension
Authors: Im Hana , Ahn Hee‐Young , Yu Myeong‐Hee 
Published: February 2009

AbstractThe native form of some proteins such as strained plasma serpins (serine protease inhibitors) and the spring‐loaded viral membrane fusion proteins are in a metastable state. The metastable native form is thought to be a folding intermediate in which conversion into the most stable state is blocked by a very high kinetic barrier. In an effort to understand how the spontaneous conversion of the metastable native form into the most stable state is prevented, we designed mutations of α1‐antitrypsin, a prototype serpin, which can bypass the folding barrier. Extending the reactive center loop of α1‐antitrypsin converts the molecule into a more stable state. Remarkably, a 30‐residue loop extension allows conversion into an extremely stable state, which is comparable to the relaxed cleaved form. Biochemical data strongly suggest that the strain release is due to the insertion of the reactive center loop into the major β‐sheet, A sheet, as in the known stable conformations of serpins. Our results clearly show that extending the reactive center loop is sufficient to bypass the folding barrier of α1‐antitrypsin and suggest that the constrain held by polypeptide connection prevents the conversion of the native form into the lowest energy state.
Chemical cross‐linking with thiol‐cleavable reagents combined with differential mass spectrometric peptide mapping—A novel approach to assess intermolecular protein contacts
Authors: Bennett Keiryn L. , Kussmann Martin , Mikkelsen Marie , Roepstorff Peter , Björk Per , Godzwon Magdalena , Sörensen Poul 
Published: February 2009

AbstractThe intermolecular contact regions between monomers of the homodimeric DNA binding protein ParR and the interaction between the glycoproteins CD28 and CD80 were investigated using a strategy that combined chemical cross‐linking with differential MALDI‐MS analyses. ParR dimers were modified in vitro with the thiol‐cleavable cross‐linker 3,3′‐dithio‐bis(succinimidylproprionate) (DTSSP), proteolytically digested with trypsin and analyzed by MALDI‐MS peptide mapping. Comparison of the peptide maps obtained from digested cross‐linked ParR dimers in the presence and absence of a thiol reagent strongly supported a “head‐to‐tail” arrangement of the monomers in the dimeric complex. Glycoprotein fusion constructs CD28‐IgG and CD80‐Fab were cross‐linked in vitro by DTSSP, characterized by non‐reducing SDS‐PAGE, digested in situ with trypsin and analyzed by MALDI‐MS peptide mapping (± thiol reagent). The data revealed the presence of an intermolecular cross‐link between the receptor regions of the glycoprotein constructs, as well as a number of unexpected but nonetheless specific interactions between the fusion domains of CD28‐IgG and the receptor domain of CD80‐Fab. The strategy of chemical cross‐linking combined with differential MALDI‐MS peptide mapping ( thiol reagent) enabled localization of the interface region(s) of the complexes studied and clearly demonstrates the utility of such an approach to obtain structural information on interacting noncovalent complexes.
Ligand binding and thermodynamic stability of a multidomain protein, calmodulin
Authors: Masino Laura , Martin Stephen R. , Bayley Peter M. 
Published: February 2009

AbstractChemical and thermal denaturation of calmodulin has been monitored spectroscopically to determine the stability for the intact protein and its two isolated domains as a function of binding of Ca2+ or Mg2+. The reversible urea unfolding of either isolated apo‐domain follows a two‐state mechanism with relatively low δG°20 values of ˜2.7 (N‐domain) and ˜1.9 kcal/mol (C‐domain). The apo‐C‐domain is significantly unfolded at normal temperatures (20‐25 °C!. The greater affinity of the C‐domain for Ca2+ causes it to be more stable than the N‐domain at [Ca2+] ≤0.3 mM. By contrast, Mg2+ causes a greater stabilization of the N‐ rather than the C‐domain, consistent with measured Mg2+ affinities.For the intact protein (+Ca2+), the bimodal denaturation profiles can be analyzed to give two δG°20 values, which differ significantly from those of the isolated domains, with one domain being less stable and one domain more stable. The observed stability of the domains is strongly dependent on solution conditions such as ionic strength, as well as specific effects due to metal ion binding. In the intact protein, different folding intermediates are observed, depending on the ionic composition. The results illustrate that a protein of low intrinsic stability is liable to major perturbation of its unfolding properties by environmental conditions and liganding processes and, by extension, mutation. Hence, the observed stability of an isolated domain may differ significantly from the stability of the same structure in a multidomain protein. These results address questions involved in manipulating the stability of a protein or its domains by site directed mutagenesis and protein engineering.