bluuebunny/crossref_metadata_2025 huggingface dataset:
Conformational analysis and clustering of short and medium size loops connecting regular secondary structures: A database for modeling and prediction
Authors: Donate Luis E. , Rufino Stephen D. , Canard Luc H.J. , Blundell Tom L.
Published: February 2009

Loops are regions of nonrepetitive conformation connecting regular secondary structures. We identified 2,024 loops of one to eight residues in length, with acceptable main‐chain bond lengths and peptide bond angles, from a database of 223 protein and protein‐domain structures. Each loop is characterized by its sequence, main‐chain conformation, and relative disposition of its bounding secondary structures as described by the separation between the tips of their axes and the angle between them. Loops, grouped according to their length and type of their bounding secondary structures, were superposed and clustered into 161 conformational classes, corresponding to 63% of all loops. Of these, 109 (51% of the loops) were populated by at least four nonhomologous loops or four loops sharing a low sequence identity. Another 52 classes, including 12% of the loops, were populated by at least three loops of low sequence similarity from three or fewer nonhomologous groups. Loop class suprafamilies resulting from variations in the termini of secondary structures are discussed in this article. Most previously described loop conformations were found among the classes. New classes included a 2:4 type IV hairpin, a helix‐capping loop, and a loop that mediates dinucleotide‐binding. The relative disposition of bounding secondary structures varies among loop classes, with some classes such as β‐hairpins being very restrictive. For each class, sequence preferences as key residues were identified; those most frequently at these conserved positions than in proteins were GIy, Asp, Pro, Phe, and Cys. Most of these residues are involved in stabilizing loop conformation, often through a positive ø conformation or secondary structure capping. Identification of helix‐capping residues and β‐breakers among the highly conserved positions supported our decision to group loops according to their bounding secondary structures. Several of the identified loop classes were associated with specific functions, and all of the member loops had the same function; key residues were conserved for this purpose, as is the case for the parvalbumin‐like calcium‐binding loops. A significant number, but not all, of the member loops of other loop classes had the same function, as is the case for the helix‐turn‐helix DNA‐binding loops. This article provides a systematic and coherent conformational classification of loops, covering a broad range of lengths and all four combinations of bounding secondary structure types, and supplies a useful basis for modelling of loop conformations where the bounding secondary structures are known or reliably predicted.
Deglycosylation of proteins for crystallization using recombinant fusion protein glycosidases
Authors: Grueninger‐Leitch Fiona , D'Arcy Allan , D'Arcy Brigitte , Chène Christiane
Published: February 2009

Obtaining high quality protein crystals remains a rate‐limiting step in the determination of three‐dimensional X‐ray structures. A frequently encountered problem in this respect is the high or heterogeneous carbohydrate content of many eukaryotic proteins. A number of reports have demonstrated the use of enzymatic deglycosylation in the crystallization of certain glycoproteins. Although this is an attractive tool, there are some problems that hinder the more widespread use of glycosidases in crystallization. First, commercially available glycosidases are relatively expensive, which virtually prohibits their use on a large scale. Second, the glycosidase must be removed from the glycoprotein of interest following deglycosylation, which is not always straightforward. To circumvent these problems we have cloned the two most generally useful glycosidases, peptide‐N‐glycosidase F and endoglycosidase F1 from Flavobacterium meningosepticum, as fusion proteins with glutathione S‐transferase. The fusion not only allows rapid purification of these enzymes from Escherichia coli cell extracts, but also permits rapid removal from target proteins following deglycosylation. We have used these enzymes to obtain crystals of phytase from Aspergillus ficuum and acid phosphatase from Aspergillus niger and to obtain a new crystal form of recombinant human renin.
The N‐terminal domains of tensin and auxilin are phosphatase homologues
Authors: Haynie Donald T. , Ponting Christopher P.
Published: February 2009

Tensin, an actin filament capping protein, and auxilin, a component of receptor‐mediated endocytosis, are known to have 350 residue regions of significant sequence similarity near their N‐termini (Schröder et al., 1995, Eur J Biochem 228:297–304). Here we demonstrate that these regions are homologous, not only to each other, but also to the catalytic domain of a putative protein tyrosine phosphatase (PTP) from Saccharomyces cerevisiae and to other PTPs. We propose that the PTP‐like portion of the homology region of tensin and auxilin represents a distinct domain. A detailed sequence comparison indicates that the PTP‐like domain in tensin is unlikely to exhibit phosphatase activity, whereas in auxilin it may possess a different phosphatase specificity from tyrosine phosphatases. It is probable that the PTP‐like domains in tensin and auxilin mediate binding interactions with phosphorylated polypeptides; they may therefore represent members of a distinct class of phosphopeptide recognition domain.
Insights into the local residual entropy of proteins provided by NMR relaxation
Authors: Li Zhigang , Raychaudhuri Soumya , Wand A. Joshua
Published: February 2009

A simple model is used to illustrate the relationship between the dynamics measured by NMR relaxation methods and the local residual entropy of proteins. The expected local dynamic behavior of well‐packed extended amino acid side chains are described by employing a one‐dimensional vibrator that encapsulates both the spatial and temporal character of the motion. This model is then related to entropy and to the generalized order parameter of the popular “model‐free” treatment often used in the analysis of NMR relaxation data. Simulations indicate that order parameters observed for the methyl symmetry axes in, for example, human ubiquitin correspond to significant local entropies. These observations have obvious significance for the issue of the physical basis of protein structure, dynamics, and stability.
Interleukin‐6: Structure‐function relationships
Authors: Simpson Richard J. , Hammacher Annet , Smith David K. , Matthews Jacqueline M. , Ward Larry D.
Published: February 2009

Interleukin‐6 (IL‐6) is a multifunctional cytokine that plays a central role in host defense due to its wide range of immune and hematopoietic activities and its potent ability to induce the acute phase response. Overexpression of IL‐6 has been implicated in the pathology of a number of diseases including multiple myeloma, rheumatoid arthritis, Castleman's disease, psoriasis, and post‐menopausal osteoporosis. Hence, selective antagonists of IL‐6 action may offer therapeutic benefits. IL‐6 is a member of the family of cytokines that includes interleukin‐11, leukemia inhibitory factor, oncostatin M, cardiotrophin‐1, and ciliary neurotrophic factor. Like the other members of this family, IL‐6 induces growth or differentiation via a receptor‐system that involves a specific receptor and the use of a shared signaling subunit, gp130. Identification of the regions of IL‐6 that are involved in the interactions with the IL‐6 receptor and gp130 is an important first step in the rational manipulation of the effects of this cytokine for therapeutic benefit. In this review, we focus on the sites on IL‐6 which interact with its low‐affinity specific receptor, the IL‐6 receptor, and the high‐affinity converter gp130. A tentative model for the IL‐6 hexameric receptor ligand complex is presented and discussed with respect to the mechanism of action of the other members of the IL‐6 family of cytokines.
Homology modeling using simulated annealing of restrained molecular dynamics and conformational search calculations with CONGEN: Application in predicting the three‐dimensional structure of murine homeodomain Msx‐1
Authors: Li Haicheng , Tejero Roberto , Monleon Daniel , Bassolino‐Klimas Donna , Abate‐Shen Cory , Bruccoleri Robert E. , Montelione Gaetano T.
Published: February 2009

We have developed an automatic approach for homology modeling using restrained molecular dynamics and simulated annealing procedures, together with conformational search algorithms available in the molecular mechanics program CONGEN (Bruccoleri RE, Karplus M, 1987, Biopolymers 26:137–168). The accuracy of the method is validated by “predicting” structures of two homeodomain proteins with known three‐dimensional structures, and then applied to predict the three‐dimensional structure of the homeodomain of the murine Msx‐1 transcription factor. Regions of the unknown protein structure that are highly homologous to the known template structure are constrained by “homology distance constraints,” whereas the conformations of nonhomologous regions of the unknown protein are defined only by the potential energy function. A full energy function (excluding explicit solvent) is employed to ensure that the calculated structures have good conformational energies and are physically reasonable. As in NMR structure determinations, information on the consistency of the structure prediction is obtained by superposition of the resulting family of protein structures. In this paper, our homology modeling algorithm is described and compared with related homology modeling methods using spatial constraints derived from the structures of homologous proteins. The software is then used to predict the DNA‐bound structures of three homeodomain proteins from the X‐ray crystal structure of the engrailed homeodomain protein (Kissinger CR et al., 1990, Cell 63:579–590). The resulting backbone and side‐chain conformations of the modeled yeast Matα2 and D. melanogaster Antennapedia homeodomains are excellent matches to the corresponding published X‐ray crystal (Wolberger C et al., 1991, Cell 67:517–528) and NMR (Billeter M et al., 1993, J Mol Biol 234:1084–1097) structures, respectively. Examination of these structures of Msx‐1 reveals a network of highly conserved surface salt bridges that are proposed to play a role in regulating protein–protein interactions of homeodomains in transcription complexes.
Three‐dimensional structures of glycolate oxidase with bound active‐site inhibitors
Authors: Stenberg Kaj , Lindqvist Ylva
Published: February 2009

A key step in plant photorespiration, the oxidation of glycolate to glyoxylate, is carried out by the peroxisomal flavoprotein glycolate oxidase (EC 1.1.3.15). The three‐dimensional structure of this α/β barrel protein has been refined to 2 Å resolution (Lindqvist Y. 1989. J Mol Biol 209:151‐166). FMN dependent glycolate oxidase is a member of the family of α‐hydroxy acid oxidases. Here we describe the crystallization and structure determination of two inhibitor complexes of the enzyme, TKP (3‐Decyl‐2,5‐dioxo‐4‐hydroxy‐3‐pyrroline) and TACA (4‐Carboxy‐5‐(1‐pentyl)hexylsulfanyl‐1,2,3‐triazole). The structure of the TACA complex has been refined to 2.6 Å resolution and the TKP complex, solved with molecular replacement, to 2.2 Å resolution. The Rfree for the TACA and TKP complexes are 24.2 and 25.1%, respectively. The overall structures are very similar to the unliganded holoenzyme, but a closer examination of the active site reveals differences in the positioning of the flavin isoalloxazine ring and a displaced flexible loop in the TKP complex. The two inhibitors differ in binding mode and hydrophobic interactions, and these differences are reflected by the very different Ki values for the inhibitors, 16 nM for TACA and 4.8 μM for TKP. Implications of the structures of these enzyme‐inhibitor complexes for the model for substrate binding and catalysis proposed from the holo‐enzyme structure are discussed.
The role of divalent cations in structure and function of murine adenosine deaminase
Authors: Cooper Bruce F. , Sideraki Vera , Wilson David K. , Dominguez David Y. , Clark Sandra W. , Quiocho Florante A. , Rudolph Frederick B.
Published: February 2009

For murine adenosine deaminase, we have determined that a single zinc or cobalt cofactor bound in a high affinity site is required for catalytic function while metal ions bound at an additional site(s) inhibit the enzyme. A catalytically inactive apoenzyme of murine adenosine deaminase was produced by dialysis in the presence of specific zinc chelators in an acidic buffer. This represents the first production of the apoenzyme and demonstrates a rigorous method for removing the occult cofactor. Restoration to the holoenzyme is achieved with stoichiometric amounts of either Zn2+ or Co2+ yielding at least 95% of initial activity. Far UV CD and fluorescence spectra are the same for both the apo‐ and holoenzyme, providing evidence that removal of the cofactor does not alter secondary or tertiary structure. The substrate binding site remains functional as determined by similar quenching measured by tryptophan fluorescence of apo‐ or holoenzyme upon mixing with the transition state analog, deoxycoformycin. Excess levels of adenosine or N6‐methyladenosine incubated with the apoenzyme prior to the addition of metal prevent restoration, suggesting that the cofactor adds through the substrate binding cleft. The cations Ca2+, Cd2+ Cr2+, Cu+, Cu2+, Mn2+, Fe2+, Fe3+, Pb2+, or Mg2+ did not restore adenosine deaminase activity to the apoenzyme. Mn2+, Cu2+, and Zn2+ were found to be competitive inhibitors of the holoenzyme with respect to substrate and Cd2+ and Co2+ were noncompetitive inhibitors. Weak inhibition (Ki ≥ 1000 μM) was noted for Ca2+, Fe2+, and Fe3+.
Energy profile of maltooligosaccharide permeation through maltoporin as derived from the structure and from a statistical analysis of saccharide‐protein interactions
Authors: Meyer Joachim E. W. , Schulz Georg E.
Published: February 2009

The crystal structure of the maltodextrin‐specific porin from Salmonella typhimurium ligated with a maltotrioside at the pore eyelet is known at 2.4 Å resolution. The three glucose units assume a conformation close to the natural amylose helix. The pore eyelet fits exactly the cross‐section of a maltooligosaccharide chain and thus functions as a constraining orifice. The oligomer permeates the membrane by screwing along the amylose helix through this orifice. Because each glucose glides along the given helix, its interactions can be sampled at any point along the pathway. The interactions are mostly hydrogen bonds, but also contacts to aromatic rings at one side of the pore. We have derived the energy profile of a gliding maltooligosaccharide by following formation and breakage of hydrogen bonds and by assessing the saccharide‐aromatics interactions from a statistical analysis of saccharide binding sites in proteins. The resulting profile indicates smooth permeation despite extensive hydrogen bonding at the orifice.
Kaj Ulrik Linderstrøm‐Lang (1896–1959)
Authors: Schellman John A. , Schellman Charlotte G.
Published: February 2009

The Carlsberg Laboratory in Copenhagen has had a long tradition of outstanding science. At the time covered by this discussion, Kjeldahl, Sørensen, and Linderstrøm–Lang had been consecutive directors of the Chemical Laboratory for 83 years. Lang's inspired leadership began in the 1930s with a number of innovations (study of metabolism in single cells, titrations in non–aqueous solvents, relation of proteolysis to structure) but it was not until the early 1950s that Denmark had sufficiently recovered from the war for the laboratory to enter world science again. During World War II, Lang had been active in the Danish resistance movement.

After the war, a number of major advances were being made that would revolutionize the field of protein chemistry (Pauling and Corey's H–bonded structures, Sanger's sequencing techniques, chromatography, Watson and Crick structures, modern instrumentation). The time for the new field of the physical biochemistry of proteins had arrived. Lang, with his broad experience, adventurous spirit, and genius for innovation, created an environment that was ideal for the convergence of these disconnected advances into a uniform science. The emphasis was to be on quantitative measurements on proteins in solution with interpretations based on molecular structures.

During an all‐too–brief period of time, Lang's laboratory attracted a large fraction of those who were destined to be the leaders of the next generation of protein chemists. At this time, the Carlsberg Laboratory was probably the most scientifically exciting environment for a protein chemist. The methods developed at that time–hydrogen exchange, limited proteolysis, optical rotatory dispersion, volume changes accompanying protein reactions, automatic titrations– are still all in common use and many of the visitors to the laboratory in that period and their students are still playing major roles in protein research.

Lang's other qualities should not be ignored. He was not only a great scientist but also a musician, raconteur, artist, and an exceptionally warm and compassionate human being.
Comparison of high‐resolution structures of the diphtheria toxin repressor in complex with cobalt and zinc at the cation‐anion binding site
Authors: Pohl Ehmke , Qiu Xiayang , Must Lisa M. , Holmes Randall K. , Hol Wim G. J.
Published: February 2009

The diphtheria toxin repressor (DtxR) from Coryne‐bacterium diphtheriae is a divalent‐metal activated repressor of chromosomal genes responsible for siderophore‐mediated iron‐uptake and of a gene on several corynebacteriophages that encodes diphtheria toxin. Even though DtxR is the best characterized iron‐dependent repressor to date, numerous key properties of the protein still remain to be explained. One is the role of the cation‐anion pair discovered in its first metal‐binding site. A second is the reason why zinc exhibits its activating effect only at a concentration 100‐fold higher than other divalent cations.

In the presently reported 1.85 Å resolution Co‐DtxR structure at 100K, the sulfate anion in the cation‐anion‐binding site interacts with three side chains that are all conserved in the entire DtxR family, which points to a possible physiological role of the anion.

A comparison of the 1.85 Å Cobalt‐DtxR structure at 100K and the 2.4 Å Zinc‐DtxR structure at room temperature revealed no significant differences. Hence, the difference in efficiency of Co2+ and Zn2+ to activate DtxR remains a mystery and might be hidden in the properties of the intriguing second metal‐binding site. Our studies do, however, provide a high resolution view of the cation‐anion‐binding site that has most likely evolved to interact not only with a cation but also with the anion in a very precise manner.
The chemistry and enzymology of the type I signal peptidases
Authors: Dalbey Ross E. , Lively Mark O. , Bron Sierd , Dijl Jan Maarten Van
Published: February 2009

The discovery that proteins exported from the cytoplasm are typically synthesized as larger precursors with cleavable signal peptides has focused interest on the peptidases that remove the signal peptides. Here, we review the membrane‐bound peptidases dedicated to the processing of protein precursors that are found in the plasma membrane of prokaryotes and the endoplasmic reticulum, the mitochondrial inner membrane, and the chloroplast thylakoidal membrane of eukaryotes. These peptidases are termed type I signal (or leader) peptidases. They share the unusual feature of being resistant to the general inhibitors of the four well‐characterized peptidase classes. The eukaryotic and prokaryotic signal peptidases appear to belong to a single peptidase family. This review emphasizes the evolutionary concepts, current knowledge of the catalytic mechanism, and substrate specificity requirements of the signal peptidases.
Structural basis for the negative allostery between Ca2+‐ and Mg2+ ‐binding in the intracellular Ca2+ ‐receptor calbindin D9k
Authors: Andersson Maria , Ivarsson Ianrik , Anders Svensson L. , Malmendal Anders , Linse Sara , Forsén Sture
Published: February 2009

The three‐dimensional structures of the magnesium‐ and manganese‐bound forms of calbindin D9% were determined to 1.6 Å and 1.9 Å resolution, respectively, using X‐ray crystallography. These two structures are nearly identical but deviate significantly from both the calcium bound form and the metal ion‐free (apo) form. The largest structural differences are seen in the C‐terminal EF‐hand, and involve changes in both metal ion coordination and helix packing. The N‐terminal calcium binding site is not occupied by any metal ion in the magnesium and manganese structures, and shows little structural deviation from the apo and calcium bound forms. 1 H‐NMR and UV spectroscopic studies at physiological ion concentrations show that the C‐terminal site of the protein is significantly populated by magnesium at resting cell calcium levels, and that there is a negative allosteric interaction between magnesium and calcium binding. Calcium binding was found to occur with positive cooperativity at physiological magnesium concentration.
Structural studies of the streptavidin binding loop
Authors: Stenkamp Ronald E. , Trong Isolde Le , Klumb Lisa , Stayton Patrick S. , Freitag Stefanie
Published: February 2009

The 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

We 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

Compactness 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

The 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

The 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

A 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

The 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

Using 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

Streptavidin 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

The 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

Multidimensional 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

Phenylalanine 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

Insight 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

The 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

As 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.
Synthesis and characterization of histidine‐phosphorylated peptides
Authors: Medzihradszky Katalin F. , Phillipps Nancy J. , Senderowicz Lionel , Wang Ping , Turck Christoph W.
Published: February 2009

Posttranslational phosphorylation of proteins is an important event in many cellular processes. Whereas phosphoesters of serine, threonine, and tyrosine have been studied extensively, only limited information is available for other amino acids modified by a phosphate group. The formation of phosphohistidine residues in proteins was discovered originally in prokaryotic organisms, but also has been found recently in eukaryotic cells. We describe methods for the synthesis and analysis of phosphohistidine‐containing peptides, a prerequisite for the investigation of the role of this posttranslational modification in cellular processes.
The crystal structure of human α‐thrombin complexed with LY178550, a nonpeptidyl, active site‐directed inhibitor
Authors: Chirgadze Nickolay Y. , Sall Daniel J. , Klimkowski Valentine J. , Clawson David K. , Briggs Steve L. , Hermann Robert , Smith Gerald F. , Gifford‐Moore Donetta S. , Wery Jean‐Pierre
Published: February 2009

The crystal structure of human α‐thrombin in complex with LY178550, a nonpeptidyl, active site‐directed inhibitor, has been solved to 2.07 α resolution by the method of X‐ray crystallography. The final model of the complex has a crystallographic R‐value of 21.5% (Rfree = 23.1%) with 0.014 Å and 2.4° standard deviation from ideal bond lengths and angles, respectively. Well‐defined electron density was observed for the inhibitor in the active site. The inhibitor binds to the active site in an L‐shaped manner, mimicking the bound conformation of the tripeptide arginal series of thrombin inhibitors (Chirgadze NY et al., 1992, American Crystallographic Association Meeting 20:116 [Abstr. PB311]). The basic amidine of LY178550 forms a salt bridge with Asp 189 within the specificity pocket, while the 4‐benzylpiperidine side chain engages in a number of hydrophobic interactions at the S2 and S3 binding sites. The inhibitor does not interact in any fashion with the active site sequence Ser 214‐Gly 216, as occurs with many of the inhibitors studied previously. The indole N‐H of the inhibitor forms a hydrogen bond to the γ‐oxygen of the catalytic serine (Ser 195).