bluuebunny/crossref_metadata_2025_split huggingface dataset:
Why is Leu55→Pro55 transthyretin variant the most amyloidogenic: Insights from molecular dynamics simulations of transthyretin monomers
Authors: Yang Mingfeng , Lei Ming , Huo Shuanghong 
Published: May 2003

AbstractTransthyretin (TTR) is one of the known human amyloidogenic proteins. Its native state is a homotetramer with each monomer having a β‐sandwich structure. Strong experimental evidence suggests that TTR dissociates into monomeric intermediates and that the monomers subsequently self‐assemble to form amyloid deposits and insoluble fibrils. However, details on the early steps along the pathway of TTR amyloid formation are unclear, although various experimental approaches with resolutions at the molecular or residue level have provided some clues. It is highly likely that the stability and flexibility of monomeric TTR play crucial roles in the early steps of amyloid formation; thereby, it is essential to characterize initial conformational changes of TTR monomers. In this article we probe the possibility that the differences in the monomeric forms of wild‐type (WT) TTR and its variants are responsible for differential amyloidogenesis. We begin with the simulations of WT, Val30→Met (V30M), and Leu55→Pro (L55P) TTR monomers. Nanosecond time scale molecular dynamics simulations at 300 K were performed using AMBER. The results indicate that the L55P‐TTR monomer undergoes substantial structural changes relative to fluctuations observed in the WT and V30M TTR monomers. The observation supports earlier speculation that the L55P mutation may lead to disruption of the β‐sheet structure through the disorder of the “edge strands” that might facilitate amyloidogenesis.
The role of α‐, 310‐, and π‐helix in helix→coil transitions
Authors: Armen Roger , Alonso Darwin O.V. , Daggett Valerie 
Published: May 2003

AbstractThe conformational equilibrium between 310‐ and α‐helical structure has been studied via high‐resolution NMR spectroscopy by Millhauser and coworkers using the MW peptide Ac‐AMAAKAWAAKA AAARA‐NH2. Their 750‐MHz nuclear Overhauser effect spectroscopy (NOESY) spectra were interpreted to reflect appreciable populations of 310‐helix throughout the peptide, with the greatest contribution at the N and C termini. The presence of simultaneous αN(i,i + 2) and αN(i,i + 4) NOE cross‐peaks was proposed to represent conformational averaging between 310‐ and α‐helical structures. In this study, we describe 25‐nsec molecular dynamics simulations of the MW peptide at 298 K, using both an 8 Å and a 10 Å force‐shifted nonbonded cutoff. The ensemble averages of both simulations are in reasonable agreement with the experimental helical content from circular dichroism (CD), the 3JHNα coupling constants, and the 57 observed NOEs. Analysis of the structures from both simulations revealed very little formation of contiguous i → i + 3 hydrogen bonds (310‐helix); however, there was a large population of bifurcated i → i + 3 and i → i + 4 α‐helical hydrogen bonds. In addition, both simulations contained considerable populations of π‐helix (i → i + 5 hydrogen bonds). Individual turns formed over residues 1–9, which we predict contribute to the intensities of the experimentally observed αN(i,i + 2) NOEs. Here we show how sampling of both folded and unfolded structures can provide a structural framework for deconvolution of the conformational contributions to experimental ensemble averages.
A wheat germ cell‐free system is a novel way to screen protein folding and function
Authors: Morita Eugene Hayato , Sawasaki Tatsuya , Tanaka Rikou , Endo Yaeta , Kohno Toshiyuki 
Published: May 2003

AbstractFor high‐throughput protein structural analysis, it is indispensable to develop a reliable protein overexpression system. Although many protein overexpression systems, such as that involving Escherichia coli cells, have been developed, the number of overexpressed proteins showing the same biological activities as those of the native proteins is limited. A novel wheat germ cell‐free protein synthesis system was developed recently, and most of the proteins functioning in solution were synthesized as soluble forms. This suggests the applicability of this protein synthesis method to determination of the solution structures of functional proteins. To examine this possibility, we have synthesized two 15N‐labeled proteins and obtained 1H‐15N HSQC spectra for them. The structural analysis of these proteins has already progressed with an E. coli overexpression system, and 1H‐15N HSQC spectra for biologically active proteins have already been obtained. Comparing the spectra, we have shown that proteins synthesized with a wheat germ cell‐free system have the proper protein folding and enough biological activity. This is the first experimental evidence of the applicability of the wheat germ cell‐free protein synthesis system to high‐throughput protein structural analysis.
An electronic effect on protein structure
Authors: Hinderaker Matthew P. , Raines Ronald T. 
Published: May 2003

AbstractThe well‐known preference of the peptide bond for the trans conformation has been attributed to steric effects. Here, we show that a proline residue with an N‐formyl group (Hi−1−C′i−1=Oi−1), in which Hi−1 presents less steric hindrance than does Oi−1, likewise prefers a trans conformation. Thus, the preference of the peptide bond for the trans conformation cannot be explained by steric effects alone. Rather, an n → π* interaction between the oxygen of the peptide bond (Oi−1), and the subsequent carbonyl carbon in the polypeptide chain (C′i) also contributes to this preference. The Oi−1 and C′i distance and Oi−1···C′i=Oi angle are especially favorable for such an n → π* interaction in a polyproline II helix. We propose that this electronic effect provides substantial stabilization to this and other elements of protein structure.
Effects of sucrose on conformational equilibria and fluctuations within the native‐state ensemble of proteins
Authors: Kim Yong‐Sung , Jones Latoya S. , Dong Aichun , Kendrick Brent S. , Chang Byeong S. , Manning Mark C. , Randolph Theodore W. , Carpenter John F. 
Published: May 2003

AbstractOsmolytes increase the thermodynamic conformational stability of proteins, shifting the equilibrium between native and denatured states to favor the native state. However, their effects on conformational equilibria within native‐state ensembles of proteins remain controversial. We investigated the effects of sucrose, a model osmolyte, on conformational equilibria and fluctuations within the native‐state ensembles of bovine pancreatic ribonuclease A and S and horse heart cytochrome c. In the presence of sucrose, the far‐ and near‐UV circular dichroism spectra of all three native proteins were slightly altered and indicated that the sugar shifted the native‐state ensemble toward species with more ordered, compact conformations, without detectable changes in secondary structural contents. Thermodynamic stability of the proteins, as measured by guanidine HCl‐induced unfolding, increased in proportion to sucrose concentration. Native‐state hydrogen exchange (HX) studies monitored by infrared spectroscopy showed that addition of 1 M sucrose reduced average HX rate constants at all degrees of exchange of the proteins, for which comparison could be made in the presence and absence of sucrose. Sucrose also increased the exchange‐resistant core regions of the proteins. A coupling factor analysis relating the free energy of HX to the free energy of unfolding showed that sucrose had greater effects on large‐scale than on small‐scale fluctuations. These results indicate that the presence of sucrose shifts the conformational equilibria toward the most compact protein species within native‐state ensembles, which can be explained by preferential exclusion of sucrose from the protein surface.
Structure of two fungal β‐1,4‐galactanases: Searching for the basis for temperature and pH optimum
Authors: Le Nours Jérôme , Ryttersgaard Carsten , Lo Leggio Leila , Østergaard Peter Rahbek , Borchert Torben Vedel , Christensen Lars Lehmann Hylling , Larsen Sine 
Published: May 2003

Abstractβ‐1,4‐Galactanases hydrolyze the galactan side chains that are part of the complex carbohydrate structure of the pectin. They are assigned to family 53 of the glycoside hydrolases and display significant variations in their pH and temperature optimum and stability. Two fungal β‐1,4‐galactanases from Myceliophthora thermophila and Humicola insolens have been cloned and heterologously expressed, and the crystal structures of the gene products were determined. The structures are compared to the previously only known family 53 structure of the galactanase from Aspergillus aculeatus (AAGAL) showing ∼56% identity. The M. thermophila and H. insolens galactanases are thermophilic enzymes and are most active at neutral to basic pH, whereas AAGAL is mesophilic and most active at acidic pH. The structure of the M. thermophila galactanase (MTGAL) was determined from crystals obtained with HEPES and TRIS buffers to 1.88 Å and 2.14 Å resolution, respectively. The structure of the H. insolens galactanase (HIGAL) was determined to 2.55 Å resolution. The thermostability of MTGAL and HIGAL correlates with increase in the protein rigidity and electrostatic interactions, stabilization of the α‐helices, and a tighter packing. An inspection of the active sites in the three enzymes identifies several amino acid substitutions that could explain the variation in pH optimum. Examination of the activity as a function of pH for the D182N mutant of AAGAL and the A90S/ H91D mutant of MTGAL showed that the difference in pH optimum between AAGAL and MTGAL is at least partially associated with differences in the nature of residues at positions 182, 90, and/or 91.
Automated protein fold determination using a minimal NMR constraint strategy
Authors: Zheng Deyou , Huang Yuanpeng J. , Moseley Hunter N.B. , Xiao Rong , Aramini James , Swapna G.V.T. , Montelione Gaetano T. 
Published: May 2003

AbstractDetermination of precise and accurate protein structures by NMR generally requires weeks or even months to acquire and interpret all the necessary NMR data. However, even medium‐accuracy fold information can often provide key clues about protein evolution and biochemical function(s). In this article we describe a largely automatic strategy for rapid determination of medium‐accuracy protein backbone structures. Our strategy derives from ideas originally introduced by other groups for determining medium‐accuracy NMR structures of large proteins using deuterated, 13C‐, 15N‐enriched protein samples with selective protonation of side‐chain methyl groups (13CH3). Data collection includes acquiring NMR spectra for automatically determining assignments of backbone and side‐chain 15N, HN resonances, and side‐chain 13CH3 methyl resonances. These assignments are determined automatically by the program AutoAssign using backbone triple resonance NMR data, together with Spin System Type Assignment Constraints (STACs) derived from side‐chain triple‐resonance experiments. The program AutoStructure then derives conformational constraints using these chemical shifts, amide 1H/2H exchange, nuclear Overhauser effect spectroscopy (NOESY), and residual dipolar coupling data. The total time required for collecting such NMR data can potentially be as short as a few days. Here we demonstrate an integrated set of NMR software which can process these NMR spectra, carry out resonance assignments, interpret NOESY data, and generate medium‐accuracy structures within a few days. The feasibility of this combined data collection and analysis strategy starting from raw NMR time domain data was illustrated by automatic analysis of a medium accuracy structure of the Z domain of Staphylococcal protein A.
Insights into dimerization and four‐helix bundle formation found by dissection of the dimer interface of the GrpE protein from Escherichia coli
Authors: Mehl Andrew F. , Heskett Luke D. , Jain Sumesh S. , Demeler Borries 
Published: May 2003

AbstractThe GrpE heat shock protein from Escherichia coli has a homodimeric structure. The dimer interface encompasses two long α‐helices at the NH2‐terminal end from each monomer (forming a “tail”), which lead into a small four‐helix bundle from which each monomer contributes two short sequential α‐helices in an antiparallel topological arrangement. We have created a number of different deletion mutants of GrpE that have portions of the dimer interface to investigate requirements for dimerization and to study four‐helix bundle formation. Using chemical crosslinking and analytical ultracentrifugation techniques to probe for multimeric states, we find that a mutant containing only the long α‐helical tail portion (GrpE1–88) is unable to form a dimer, most likely due to a decrease in α‐helical content as determined by circular dichroism spectroscopy, thus one reason for a dimeric structure for the GrpE protein is to support the tail region. Mutants containing both of the short α‐helices (GrpE1–138 and GrpE88–197) are able to form a dimer and presumably the four‐helix bundle at the dimer interface. These two mutants have equilibrium constants for the monomer–dimer equilibrium that are very similar to the full‐length protein suggesting that the tail region does not contribute significantly to the stability of the dimer. Interestingly, one mutant that contains just one of the short α‐helices (GrpE1–112) exists as a tetrameric species, which presumably is forming a four‐helix bundle structure. A proposed model is discussed for this mutant and its relevance for factors influencing four‐helix bundle formation.
The long‐range electrostatic interactions control tRNA–aminoacyl‐tRNA synthetase complex formation
Authors: Tworowski Dmitry , Safro Mark 
Published: May 2003

AbstractIn most cases aminoacyl‐tRNA synthetases (aaRSs) are negatively charged, as are the tRNA substrates. It is apparent that there are driving forces that provide a long‐range attraction between like charge aaRS and tRNA, and ensure formation of “close encounters.” Based on numerical solutions to the nonlinear Poisson‐Boltzmann equation, we evaluated the electrostatic potential generated by different aaRSs. The 3D‐isopotential surfaces calculated for different aaRSs at 0.01 kT/e contour level reveal the presence of large positive patches—one patch for each tRNA molecule. This is true for classes I and II monomers, dimers, and heterotetramers. The potential maps keep their characteristic features over a wide range of contour levels. The results suggest that nonspecific electrostatic interactions are the driving forces of primary stickiness of aaRSs–tRNA complexes. The long‐range attraction in aaRS–tRNA systems is explained by capture of negatively charged tRNA into “blue space area” of the positive potential generated by aaRSs. Localization of tRNA in this area is a prerequisite for overcoming the barrier of Brownian motion.
Importance of α–helix N–capping motif in stabilization of ββα fold
Authors: Koscielska‐Kasprzak Katarzyna , Cierpicki Tomasz , Otlewski Jacek 
Published: May 2003

AbstractFSD‐1 (full sequence design 1) is a protein folded in a ββα motif, designed on the basis of the second zinc finger domain of Zif268 by a substitution of its metal coordination site with a hydrophobic core. In this work, we analyzed the possibility of introducing the DNA recognition motif of the template zinc finger (S13RSDH17) into FSD‐1 sequence in order to obtain a small DNA‐binding module devoid of cross‐link(s) or metal cofactors. The hybrid protein was unfolded, as judged by CD and NMR criteria. To reveal the role of each of the five amino acids, which form the N‐capping motif of the α‐helix, we analyzed conformational and stability properties of eight FSD‐1 mutants. We used a shielded methyl group of Leu 18 and a CD signal at 215 nm as a convenient measure of the folded state. Glu 17→His substitution at the N3 in S13NEKE17 variant decreased the folded structure content from 90% to 25% (equivalent to 1.8 kcal • mole−1 destabilization) by disruption of N‐capping interactions, and had the most significant effect among single mutants studied here. The Ncap Asn 14 substitution with Arg considerably decreased stability, reducing structure content from 90% to 40% (1.4 kcal • mole−1 destabilization) by disruption of a helix‐capping hydrogen bond and destabilization of a helix macrodipole. The N1 Glu 15→Ser mutation also produced a considerable effect (1.0 kcal • mole−1 destabilization), again emphasizing the significance of electrostatic interactions in α‐helix stabilization.
Structural perturbation and enhancement of the chaperone‐like activity of α‐crystallin by arginine hydrochloride
Authors: Srinivas Volety , Raman Bakthisaran , Rao Kunchala Sridhar , Ramakrishna Tangirala , Rao Ch Mohan 
Published: May 2003

AbstractStructural perturbation of α‐crystallin is shown to enhance its molecular chaperone‐like activity in preventing aggregation of target proteins. We demonstrate that arginine, a biologically compatible molecule that is known to bind to the peptide backbone and negatively charged side‐chains, increases the chaperone‐like activity of calf eye lens α‐crystallin as well as recombinant human αA‐ and αB‐crystallins. Arginine‐induced increase in the chaperone activity is more pronounced for αB‐crystallin than for αA‐crystallin. Other guanidinium compounds such as aminoguanidine hydrochloride and guanidine hydrochloride also show a similar effect, but to different extents. A point mutation, R120G, in αB‐crystallin that is associated with desmin‐related myopathy, results in a significant loss of chaperone‐like activity. Arginine restores the activity of mutant protein to a considerable extent. We have investigated the effect of arginine on the structural changes of α‐crystallin by circular dichroism, fluorescence, and glycerol gradient sedimentation. Far‐UV CD spectra show no significant changes in secondary structure, whereas near‐UV CD spectra show subtle changes in the presence of arginine. Glycerol gradient sedimentation shows a significant decrease in the size of α‐crystallin oligomer in the presence of arginine. Increased exposure of hydrophobic surfaces of α‐crystallin, as monitored by pyrene‐solubilization and ANS‐fluorescence, is observed in the presence of arginine. These results show that arginine brings about subtle changes in the tertiary structure and significant changes in the quaternary structure of α‐crystallin and enhances its chaperone‐like activity significantly. This study should prove useful in designing strategies to improve chaperone function for therapeutic applications.
Noncharged amino acid residues at the solvent‐exposed positions in the middle and at the C terminus of the α‐helix have the same helical propensity
Authors: Ermolenko Dmitri N. , Richardson John M. , Makhatadze George I. 
Published: May 2003

AbstractIt was established previously that helical propensities of different amino acid residues in the middle of α‐helix in peptides and in proteins are very similar. The statistical analysis of the protein helices from the known three‐dimensional structures shows no difference in the frequency of noncharged residues in the middle and at the C terminus. Yet, experimental studies show distinctive differences for the helical propensities of noncharged residues in the middle and in the C terminus in model peptides. Is this a general effect, and is it applicable to protein helices or is it specific to the model alanine‐based peptides? To answer this question, the effects of substitutions at positions 28 (middle residue) and 32 (C2 position at the C terminus) of the α‐helix of ubiquitin on the stability of this protein are measured by using differential scanning calorimetry. The two data sets produce similar values for intrinsic helix propensity, leading to a conclusion that noncharged amino acid residues at the solvent‐exposed positions in the middle and at the C terminus of the α‐helix have the same helical propensity. This conclusion is further supported with an excellent correlation between the helix propensity scale obtained for the two positions in ubiquitin with the experimental helix propensity scale established previously and with the statistical distribution of the residues in protein helices.
Propensities, probabilities, and the Boltzmann hypothesis
Authors: Shortle David 
Published: May 2003

AbstractThe relative strengths of interactions involving polypeptide chains can be estimated with reasonable accuracy with statistical potentials, free‐energy functions derived from the frequency of occurrence of structural arrangements of residues or atoms in collections of protein structures. Recent published work has shown that the energetics of side‐chain/backbone interactions can be modeled by the ϕ/Ψ propensities of the 20 amino acids. In this report, the more commonly used ϕ/Ψ probabilities are demonstrated to fail in evaluating the free energies of protein conformations because of an overriding preference for all helical structures. Comparison of the hypothetical reactions implied by these two different statistics—propensities versus probabilities—leads to the conclusion that the Boltzmann hypothesis may only be applicable for the calculation of statistical potentials after the starting conformation has been specified. This conclusion supports a simple conjecture: The surprising success of the Boltzmann hypothesis in explaining the energetics of protein structures is a direct consequence of a real equilibrium, one extending over evolutionary time that has maintained the stability of each protein within a narrow range of values.