bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural studies of the streptavidin binding loop
Authors: Stenkamp Ronald E. , Trong Isolde Le , Klumb Lisa , Stayton Patrick S. , Freitag Stefanie 
Published: February 2009

AbstractThe streptavidin‐biotin complex provides the basis for many important biotechnological applications and is an interesting model system for studying high‐affinity protein‐ligand interactions. We report here crystallographic studies elucidating the conformation of the flexible binding loop of streptavidin (residues 45 to 52) in the unbound and bound forms. The crystal structures of unbound streptavidin have been determined in two monoclinic crystal forms. The binding loop generally adopts an open conformation in the unbound species. In one subunit of one crystal form, the flexible loop adopts the closed conformation and an analysis of packing interactions suggests that protein‐protein contacts stabilize the closed loop conformation. In the other crystal form all loops adopt an open conformation. Co‐crystallization of streptavidin and biotin resulted in two additional, different crystal forms, with ligand bound in all four binding sites of the first crystal form and biotin bound in only two subunits in a second. The major change associated with binding of biotin is the closure of the surface loop incorporating residues 45 to 52. Residues 49 to 52 display a 310 helical conformation in unbound subunits of our structures as opposed to the disordered loops observed in other structure determinations of streptavidin. In addition, the open conformation is stabilized by a β‐sheet hydrogen bond between residues 45 and 52, which cannot occur in the closed conformation. The 310 helix is observed in nearly all unbound subunits of both the co‐crystallized and ligand‐free structures. An analysis of the temperature factors of the binding loop regions suggests that the mobility of the closed loops in the complexed structures is lower than in the open loops of the ligand‐free structures. The two biotin bound subunits in the tetramer found in the MONO‐b1 crystal form are those that contribute Trp 120 across their respective binding pockets, suggesting a structural link between these binding sites in the tetramer. However, there are no obvious signatures of binding site communication observed upon ligand binding, such as quaternary structure changes or shifts in the region of Trp 120. These studies demonstrate that while crystallographic packing interactions can stabilize both the open and closed forms of the flexible loop, in their absence the loop is open in the unbound state and closed in the presence of biotin. If present in solution, the helical structure in the open loop conformation could moderate the entropic penalty associated with biotin binding by contributing an order‐to‐disorder component to the loop closure.
De novo design of the hydrophobic core of ubiquitin
Authors: Lazar Greg A. , Desjarlais John R. , Handel Tracy M. 
Published: February 2009

AbstractWe have previously reported the development and evaluation of a computational program to assist in the design of hydrophobic cores of proteins. In an effort to investigate the role of core packing in protein structure, we have used this program, referred to as Repacking of Cores (ROC), to design several variants of the protein ubiquitin. Nine ubiquitin variants containing from three to eight hydrophobic core mutations were constructed, purified, and characterized in terms of their stability and their ability to adopt a uniquely folded native‐like conformation. In general, designed ubiquitin variants are more stable than control variants in which the hydrophobic core was chosen randomly. However, in contrast to previous results with 434 cro, all designs are destabilized relative to the wild‐type (WT) protein. This raises the possibility that β‐sheet structures have more stringent packing requirements than α‐helical proteins. A more striking observation is that all variants, including random controls, adopt fairly well‐defined conformations, regardless of their stability. This result supports conclusions from the cro studies that non‐core residues contribute significantly to the conformational uniqueness of these proteins while core packing largely affects protein stability and has less impact on the nature or uniqueness of the fold.Concurrent with the above work, we used stability data on the nine ubiquitin variants to evaluate and improve the predictive ability of our core packing algorithm. Additional versions of the program were generated that differ in potential function parameters and sampling of side chain conformers. Reasonable correlations between experimental and predicted stabilities suggest the program will be useful in future studies to design variants with stabilities closer to that of the native protein. Taken together, the present study provides further clarification of the role of specific packing interactions in protein structure and stability, and demonstrates the benefit of using systematic computational methods to predict core packing arrangements for the design of proteins.
Identification of compact, hydrophobically stabilized domains and modules containing multiple peptide chains
Authors: Zehfus Micheal H. 
Published: February 2009

AbstractCompactness has been used to locate discontinuous structural units containing one or more polypeptide chains in proteins of known structure. Rather than exhaustively calculating the compactness of all possible units, our procedure uses a screening algorithm to find discontinuous regions that are potentially compact. Precise calculations of compactness are restricted only to units in these regions. With our procedure, compactness can be used to discover discontinuous domains with virtually any number of disjoint peptides. Small, single‐domain proteins may contain several compact regions: thus, compact regions do not always correspond to folding domains. Because a domain is an independent folding unit and should contain a hydrophobic core, compact units were further examined for the presence of hydro‐phobic clusters (Zehfus MH, 1995, Protein Sci 41188‐1202). This added constraint limits the number of acceptable units and helps greatly in the location of the true structural domains. The larger hydrophobically stabilized compact units correspond to domains, while the smaller units may correspond to folding intermediates.
Refined solution structure and backbone dynamics of HIV‐1 Nef
Authors: Grzesiek Stephan , Bax Ad , Hu Jin‐Shan , Tjandra Nico , Kaufman Joshua , Palmer Ira , Stahl Stephen J. , Wingfield Paul T. 
Published: February 2009

AbstractThe tendency of HIV‐1 Nef to form aggregates in solution, particularly at pH values below 8, together with its large fraction of highly mobile residues seriously complicated determination of its three‐dimensional structure, both for heteronuclear solution NMR (Grzesiek et al., 1996a, Nat Struct Biol 3:340‐345) and for X‐ray crystallography (Lee et al., 1996, Cell 85:931‐942). Methods used to determine the Nef structure by NMR at pH 8 and 0.6 mM concentration are presented, together with a detailed description of Nef's secondary and tertiary structure. The described techniques have general applicability for the NMR structure determination of proteins that are aggregating and/or have limited stability at low pH values. Extensive chemical shift assignments are reported for backbone and side chain 1H, 13C, and 15N resonances of the HIV‐1 Nef deletion mutants NEFδ2‐39, NEFδ2‐39,δ159‐173, and of NEFδ2‐39,δ159‐173 in complex with the SH3 domain of the Hck tyrosine protein kinase. Besides a type II polyproline helix, Nef's structure consists of three α‐helices, a 310 helix, and a five‐stranded anti‐parallel β‐sheet. The analysis of 15N relaxation parameters of the backbone amide sites reveals that all the secondary structure elements are non‐mobile on the picosecond to nanosecond and on the millisecond time scale. A large number of slowly exchanging amide protons provides evidence for the stability of the Nef core even on the time scale of hours. Significant internal motions on the ps to ns time scale are detected for residues 60 to 71 and for residues 149 to 180, which form solvent‐exposed loops. The residues of the HIV‐1 protease cleavage site (W57/L58) do not exhibit large amplitude motions on the sub‐nanosecond time scale, and their side chains insert themselves into a hydrophobic crevice formed between the C‐terminus of helix 1 and the N‐terminus of helix 2. A refined structure has been determined based on additional constraints for side‐chain and backbone dihedral angles derived from a large number of three‐bond J‐couplings and ROE data.
The role of context on α‐helix stabilization: Host‐guest analysis in a mixed background peptide model
Authors: Yang Jianxin , Spek Erik J. , Gong Youxiang , Zhou Hongxing , Kallenbach Neville R. 
Published: February 2009

AbstractThe helix content of a series of peptides containing single substitutions of the 20 natural amino acids in a new designed host sequence, succinyl‐YSEEEEKAKKAXAEEAEKKKK‐NH2, has been determined using CD spectroscopy. This host is related to one previously studied, in which triple amino acid substitutions were introduced into a background of Glu‐Lys blocks completely lacking alanine. The resulting free energies show that only Ala and Glu− prove to be helix stabilizing, while all other side chains are neutral or destabilizing. This agrees with results from studies of alanine‐rich peptide models, but not the previous Glu‐Lys block oligomers in which Leu and Met also stabilize helix. The helix propensity scale derived from the previous block oligomers correlated well with the frequencies of occurrence of different side chains in helical sequences of proteins, whereas the values from the present series do not. The role of context in determining scales of helix propensity values is discussed, and the ability of algorithms designed to predict helix structure from sequence is compared.
Hydrophobicity regained
Authors: Andrew Karplus P. 
Published: February 2009

AbstractA widespread practice is to use free energies of transfer between organic solvents and water (ΔGtransfer°) to define hydrophobicity scales for the amino acid side chains. A comparison of four ΔGtransfer° scales reveals that the values for hydrogen‐bonding side chains are highly dependent on the non‐aqueous environment. This property of polar side chains violates the assumptions underlying the paradigm of equating ΔGtransfer° with hydrophobicity or even with a generic solvation energy that is directly relevant to protein stability and ligand binding energetics. This simple regaining of the original concept of hydrophobicity reveals a flaw in approaches that use ΔGtransfer° values to derive generic estimates of the energetics of the burial of polar groups, and allows the introduction of a “pure” hydrophobicity scale for the amino acid residues.
Determination of the disulfide bond arrangement of human respiratory syncytial virus attachment (G) protein by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry
Authors: Gorman Jeffrey J. , Ferguson Bettina L. , Speelman Dan , Mills John 
Published: February 2009

AbstractThe attachment protein or G protein of the A2 strain of human respiratory syncytial virus (RSV) was digested with trypsin and the resultant peptides separated by reverse‐phase high‐performance liquid chromatography (HPLC). One tryptic peptide produced a mass by matrix‐assisted laser desorption/ionization (MALDI) time‐of‐flight (TOF) mass spectrometry (MS) corresponding to residues 152‐187 with the four Cys residues of the ectodomain (residues 173, 176, 182, and 186) in disulfide linkage and absence of glycosylation. Sub‐digestion of this tryptic peptide with pepsin and thermolysin produced peptides consistent with disulfide bonds between Cys 173 and Cys 186 and between Cys 176 and Cys 182. Analysis of ions produced by post‐source decay of a peptic peptide during MALDI‐TOF‐MS revealed fragmentation of peptide bonds with minimal fission of an inter‐chain disulfide bond. Ions produced by this unprecedented MALDI‐induced post‐source fragmentation corroborated the existence of the disulfide arrangement deduced from mass analysis of proteolysis products. These findings indicate that the ectodomain of the G protein has a non‐glycosylated subdomain containing a “cystine noose.”
Automated design of the surface positions of protein helices
Authors: Dahiyat Bassil I. , Benjamin Gordon D. , Mayo Stephen L. 
Published: February 2009

AbstractUsing a protein design algorithm that quantitatively considers side‐chain interactions, the design of surface residues of α helices was examined. Three scoring functions were tested: a hydrogen‐bond potential, a hydrogen‐bond potential in conjunction with a penalty for uncompensated burial of polar hydrogens, and a hydrogen‐bond potential in combination with helix propensity. The solvent exposed residues of a homodimeric coiled coil based on GCN4‐p1 were designed by using the Dead‐End Elimination Theorem to find the optimal amino acid sequence for each scoring function. The corresponding peptides were synthesized and characterized by circular dichroism spectroscopy and size exclusion chromatography. The designed peptides were dimeric and nearly 100% helical at 1°C, with melting temperatures from 69‐72°C, over 12°C higher than GCN4‐p1, whereas a random hydrophilic sequence at the surface positions produced a peptide that melted at 15°C. Analysis of the designed sequences suggests that helix propensity is the key factor in sequence design for surface helical positions.
Crystallographic analysis of the pH‐dependent binding of iminobiotin by streptavidin
Authors: Athappilly Francis K. , Hendrickson Wayne A. 
Published: February 2009

AbstractStreptavidin binds 2′‐iminobiotin in a pH‐dependent fashion—affinity decreases as the pH is lowered. This property makes the purification of compounds conjugated to streptavidin or iminobiotin possible under mild conditions by affinity chromatography. In order to understand the molecular details of this pH‐dependent binding, we analyzed the crystal structures of the complex of core streptavidin with 2′‐iminobiotin at pH values 4.0 and 7.5. The two structures are very similar to each other even at their binding sites. Although the relative abundance of the protonated species of the ligand is increased more than 3,000‐fold on going from pH 7.5 to pH 4.0, both structures contain only the nonprotonated form of the ligand. Streptavidin selects the nonprotonated form, which, at pH 4.0, is one part in 7.9 × 107.
Characterization and crystallization of human uroporphyrinogen decarboxylase
Authors: Phillips John D. , Kushner James P. , Whitby Frank G. , Hill Christopher P. 
Published: February 2009

AbstractThe cytosolic enzyme uroporphyrinogen decarboxylase (URO‐D) catalyzes the fifth step in the heme biosynthetic pathway, converting uroporphyrinogen to coproporphyrinogen by decarboxylating the four acetate side chains of the substrate. Recombinant human URO‐D has been expressed in Escherichia coli with a histidine tag and has been purified to homogeneity. Purified protein was determined to be a monodisperse dimer by dynamic light scattering. Equilibrium sedimentation analysis confirmed that the protein is dimeric, with a dissociation constant of 0.1 μM. URO‐D containing an amino‐terminal histidine tag was crystallized in space group P3121 or its enantiomer P3221 with unit cell dimensions a = b = 103.6 Å, c = 75.2 Å. There is one molecule in the asymmetric unit, consistent with generation of the dimer by the twofold axis of this crystallographic operator. Native data have been collected to 3.0 Å resolution.
Nuclear magnetic resonance assignment and secondary structure of an ankyrin‐like repeat‐bearing protein: Myotrophin
Authors: Yang Yanwu , Sambasiva Rao N. , Qin Jun , Walker Eldon , Sen Subha 
Published: February 2009

AbstractMultidimensional heteronuclear NMR has been applied to the structural analysis of myotrophin, a novel protein identified from spontaneously hypertensive rat hearts and hypertrophic human hearts. Myotrophin has been shown to stimulate protein synthesis in myocytes and likely plays an important role in the initiation of cardiac hypertrophy, a major cause of mortality in humans. Recent cDNA cloning revealed that myotrophin has 118 amino acids containing 2.5 contiguous ANK repeats, a motif known to be involved in a wide range of macromolecular recognition. A series of two‐ and three‐dimensional heteronuclear bond correlation NMR experiments have been performed on uniformly 15N‐labeled or uniformly 15N/13C‐labeled protein to obtain the 1H, 15N, and 13C chemical shift assignments. The secondary structure of myotrophin has been determined by a combination of NOEs, NH exchange data, 3JHNα coupling constants, and chemical shifts of 1Hα, 13Cα, and 13Cβ. The protein has been found to consist of seven helices, all connected by turns or loops. Six of the seven helices (all but the C‐terminal helix) form three separate helix‐turn‐helix motifs. The two full ANK repeats in myotrophin are characteristic of multiple turns followed by a helix‐turn‐helix motif. A hairpin‐like turn involving L32‐R36 in ANK repeat #1 exhibits slow conformational averaging on the NMR time scale and appears dynamically different from the corresponding region (D65‐I69) of ANK repeat #2.
Regulation and crystallization of phosphorylated and dephosphorylated forms of truncated dimeric phenylalanine hydroxylase
Authors: Kobe Bostjan , House Colin M. , Feil Susanne C. , Michell Belinda J. , Tiganis Tony , Parker Michael W. , Kemp Bruce E. , Cotton Richard G.H. , Jennings Ian G. 
Published: February 2009

AbstractPhenylalanine hydroxylase is regulated in a complex manner, including activation by phosphorylation. It is normally found as an equilibrium of dimeric and tetrameric species, with the tetramer thought to be the active form. We converted the protein to the dimeric form by deleting the C‐terminal 24 residues and show that the truncated protein remains active and regulated by phosphorylation. This indicates that changes in the tetrameric quaternary structure of phenylalanine hydroxylase are not required for enzyme activation. Truncation also facilitates crystallization of both phosphorylated and dephosphorylated forms of the enzyme.
Conformational substates in enzyme mechanism: The 120 K structure of α‐lytic protease at 1.5 Å resolution
Authors: Rader Stephen D. , Agard David A. 
Published: February 2009

AbstractInsight into the dynamic properties of α‐lytic protease (αP) has been obtained through the use of low‐temperature X‐ray crystallography and multiple‐conformation refinement. Previous studies of αLP have shown that the residues around the active site are able to move significantly to accommodate substrates of different sizes. Here we show a link between the ability to accommodate ligands and the dynamics of the binding pocket. Although the structure of αLP at 120 K has B‐factors with a uniformly low value of 4.8 Å2 for the main chain, four regions stand out as having significantly higher B‐factors. Because thermal motion should be suppressed at cryogenic temperatures, the high B‐factors are interpreted as the result of trapped conformational substates. The active site residues that are perturbed during accommodation of different substrates are precisely those showing conformational substates, implying that substrate binding selects a subset of conformations from the ensemble of accessible states. To better characterize the precise nature of these substates, a protein model consisting of 16 structures has been refined and evaluated. The model reveals a number of features that could not be well‐described by conventional B‐factors: for example, 40% of the main‐chain residue conformations are distributed asymmetrically or in discrete clusters. Furthermore, these data demonstrate an unexpected correlation between motions on either side of the binding pocket that we suggest is a consequence of “dynamic close packing.” These results provide strong evidence for the role of protein dynamics in substrate binding and are consistent with the results of dynamic studies of ligand binding in myoglobin and ribonuclease A.
Conformational stability of ribonuclease T1 determined by hydrogen‐deuterium exchange
Authors: Mullins Leisha S. , Raushel Frank M. , Pace C.N. 
Published: February 2009

AbstractThe hydrogen‐deuterium exchange kinetics of 37 backbone amide residues in RNase T1 have been monitored at 25, 40, 45, and 50°C at pD 5.6 and at 40 and 45 °C at pD 6.6. The hydrogen exchange rate constants of the hydrogen‐bonded residues varied over eight orders of magnitude at 25°C with 13 residues showing exchange rates consistent with exchange occurring as a result of global unfolding. These residues are located in strands 2‐4 of the central β‐pleated sheet. The residues located in the α‐helix and the remaining strands of the β‐sheet exhibited exchange behaviors consistent with exchange occurring due to local structural fluctuations. For several residues at 25°C, the global free energy change calculated from the hydrogen exchange data was over 2 kcal/mol greater than the free energy of unfolding determined from urea denaturation experiments. The number of residues showing this unexpected behavior was found to increase with temperature. This apparent inconsistency can be explained quantitatively if the cis‐trans isomerization of the two cis prolines, Pro‐39 and Pro‐55, is taken into account. The cis‐trans isomerization equilibrium calculated from kinetic data indicates the free energy of the unfolded state will be 2.6 kcal/mol higher at 25°C when the two prolines are cis rather than trans (Mayr LM, Odefey CO, Schutkowski M, Schmid FX. 1996. Kinetic analysis of the unfolding and refolding of ribonuclease T1 by a stopped‐flow double‐mixing technique. Biochemistry 35:5550‐5561). The hydrogen exchange results are consistent with the most slowly exchanging hydrogens exchanging from a globally higher free energy unfolded state in which Pro‐55 and Pro‐39 are still predominantly in the cis conformation. When the conformational stabilities determined by hydrogen exchange are corrected for the proline isomerization equilibrium, the results are in excellent agreement with those from an analysis of urea denaturation curves.
Design and characterization of the anion‐sensitive coiled‐coil peptide
Authors: Hoshino Masaru , Goto Yuji , Yumoto Noboru , Yoshikawa Susumu 
Published: February 2009

AbstractAs a model for analyzing the role of charge repulsion in proteins and its shielding by the solvent, we designed a peptide of 27 amino acid residues that formed a homodimeric coiled‐coil. The interface between the coils consisted of hydro‐phobic Leu and Val residues, and 10 Lys residues per monomer were incorporated into the positions exposed to solvent. During the preparation of a disulfide‐linked dimer in which the two peptides were linked in parallel by the two disulfide bonds located at the N and C terminals, a cyclic monomer with an intramolecular disulfide bond was also obtained. On the basis of CD and 1H‐NMR, the conformational stabilities of these isomers and several reference peptides were examined. Whereas all these peptides were unfolded in the absence of salt at pH 4.7 and 20°C, the addition of NaCIO4cooperatively stabilized the α‐helical conformation. The crosslinking of the peptides by disulfide bonds significantly decreased the midpoint salt concentration of the transition. The 1H‐NMR spectra in the presence of NaCIO4 suggested that, whereas the disulfide‐bonded dimer assumed a native‐like conformation, the cyclic monomer assumed a molten globule‐like conformation with disordered side chains. However, the cyclic monomer exhibited cooperative transitions against temperature and Gdn‐HCl that were only slightly less cooperative than those of the disulfide‐bonded parallel dimer. These results indicate that the charge repulsion critically destabilizes the native‐like state as well as the molten globule‐like state, and that the solvent‐dependent charge repulsion may be useful for controlling the conformation of designed peptides.