bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural bases of lectin‐carbohydrate affinities: Comparison with protein‐folding energetics
Authors: García‐Hernández Enrique , Hernández‐Arana Andrés
Published: February 2009

We have made a comparative structure based analysis of the thermodynamics of lectin‐carbohydrate (L‐C) binding and protein folding. Examination of the total change in accessible surface area in those processes revealed a much larger decrease in free energy per unit of area buried in the case of L‐C associations. According to our analysis, this larger stabilization of L‐C interactions arises from a more favorable enthalpy of burying a unit of polar surface area, and from higher proportions of polar areas. Hydrogen bonds present at 14 L‐C interfaces were identified, and their overall characteristics were compared to those reported before for hydrogen bonds in protein structures. Three major factors might explain why polar–polar interactions are stronger in L‐C binding than in protein folding: (1) higher surface density of hydrogen bonds; (2) better hydrogen‐bonding geometry; (3) larger proportion of hydrogen bonds involving charged groups. Theoretically, the binding entropy can be partitioned into three main contributions: entropy changes due to surface desolvation, entropy losses arising from freezing rotatable bonds, and entropic effects that result from restricting translation and overall rotation motions. These contributions were estimated from structural information and added up to give calculated binding entropies. Good correlation between experimental and calculated values was observed when solvation effects were treated according to a parametrization developed by other authors from protein folding studies. Finally, our structural parametrization gave calculated free energies that deviate from experimental values by 1.1 kcal/mol on the average; this amounts to an uncertainty of one order of magnitude in the binding constant.
Hydrophobic photolabeling as a new method for structural characterization of molten globule and related protein folding intermediates
Authors: D'Silva Patrick R. , Lala Anil K.
Published: February 2009

Recent advances in attempts to unravel the protein folding mechanism have indicated the need to identify the folding intermediates. Despite their transient nature, in a number of cases it has been possible to detect and characterize some of the equilibrium intermediates, for example, the molten globule (MG) state. The key features of the MG state are retention of substantial secondary structure of the native state, considerable loss of tertiary structure leading to increased hydrophobic exposure, and a compact structure. NMR, circular dichroism, and fluorescence spectroscopies have been most useful in characterizing such intermediates. We report here a new method for structural characterization of the MG state that involves probing the exposed hydrophobic sites with a hydrophobic photoactivable reagent—2[3H]diazofluorene. This carbene‐based reagent binds to hydrophobic sites, and on photolysis covalently attaches itself to the neighboring amino acid side chains. The reagent photolabels α‐lactalbumin as a function of pH (3–7.4), the labeling at neutral pH being negligible and maximal at pH 3. Chemical and proteolytic fragmentation of the photolabeled protein followed by peptide sequencing permitted identification of the labeled residues. The results obtained indicate that the sequence corresponding to B (23–34) and C (86–98) helix of the native structure are extensively labeled. The small β‐domain (40–50) is poorly labeled, Val42 being the only residue that is significantly labeled. Our data, like NMR data, indicate that in the MG state of α‐lactalbumin, the α‐domain has a greater degree of persistent structure than the β‐domain. However, unlike the NMR method, the photolabeling method is not limited by the size of the protein and can provide information on several new residues, for example, Leu115. The current method using DAF thus allows identification of stable and hydrophobic exposed regions in folding intermediates as the reagent binds and on photolysis covalently links to these regions.
From fold predictions to function predictions: Automation of functional site conservation analysis for functional genome predictions
Authors: Zhang Baohong , Rychlewski Leszek , Pawlowski Krzysztof , Fetrow Jacquelyn S. , Skolnick Jeffrey , Godzik Adam
Published: February 2009

A database of functional sites for proteins with known structures, SITE, is constructed and used in conjunction with a simple pattern matching program SiteMatch to evaluate possible function conservation in a recently constructed database of fold predictions for Escherichia coli proteins (Rychlewski L et al., 1999, Protein Sci 8:614–624). In this and other prediction databases, fold predictions are based on algorithms that can recognize weak sequence similarities and putatively assign new proteins into already characterized protein families. It is not clear whether such sequence similarities arise from distant homologies or general similarity of physicochemical features along the sequence. Leaving aside the important question of nature of relations within fold superfamilies, it is possible to assess possible function conservation by looking at the pattern of conservation of crucial functional residues. SITE consists of a multilevel function description based on structure annotations and structure analyses. In particular, active site residues, ligand binding residues, and patterns of hydrophobic residues on the protein surface are used to describe different functional features. SiteMatch, a simple pattern matching program, is designed to check the conservation of residues involved in protein activity in alignments generated by any alignment method. Here, this procedure is used to study conservation of functional features in alignments between protein sequences from the E. coli genome and their optimal structural templates. The optimal templates were identified and alignments taken from the database of genomic structural predictions was described in a previous publication (Rychlewski L et al., 1999, Protein Sci 8:614–624). An automated assessment of function conservation is used to analyze the relation between fold and function similarity for a large number of fold predictions. For instance, it is shown that identifying low significance predictions with a high level of functional residue conservations can be used to extend the prediction sensitivity for fold prediction methods. Over 100 new fold/function predictions in this class were obtained in the E. coli genome. At the same time, about 30% of our previous fold predictions are not confirmed as function predictions, further highlighting the problem of function divergence in fold superfamilies.
The effect of multiple binding modes on empirical modeling of ligand docking to proteins
Authors: Brem Rachel , Dill Ken A.
Published: February 2009

A popular approach to the computational modeling of ligand/receptor interactions is to use an empirical free energy like model with adjustable parameters. Parameters are learned from one set of complexes, then used to predict another set. To improve these empirical methods requires an independent way to study their inherent errors. We introduce a toy model of ligand/receptor binding as a workbench for testing such errors. We study the errors incurred from the two state binding assumption—the assumption that a ligand is either bound in one orientation, or unbound. We find that the two state assumption can cause large errors in free energy predictions, but it does not affect rank order predictions significantly. We show that fitting parameters using data from high affinity ligands can reduce two state errors; so can using more physical models that do not use the two state assumption. We also find that when using two state models to predict free energies, errors are more severe on high affinity ligands than low affinity ligands. And we show that two state errors can be diagnosed by systematically adding new binding modes when predicting free energies: if predictions worsen as the modes are added, then the two state assumption in the fitting step may be at fault.
Amino acid substitutions at the subunit interface of dimeric escherichia coli alkaline phosphatase cause reduced structural stability
Authors: Martin Donna Carolan , Pastra‐Landis S. C. , Kantrowitz Evan R.
Published: February 2009

The consequences of amino acid substitutions at the dimer interface for the strength of the interactions between the monomers and for the catalytic function of the dimeric enzyme alkaline phosphatase from Escherichia coli have been investigated. The altered enzymes R10A, R10K, R24A, R24K, T59A, and R10A/R24A, which have amino acid substitutions at the dimer interface, were characterized using kinetic assays, ultracentrifugation, and transverse urea gradient gel electrophoresis. The kinetic data for the wild‐type and altered alkaline phosphatases show comparable catalytic behavior with kcat values between 51.3 and 69.5 s–1 and Km values between 14.8 and 26.3 μM. The ultracentrifugation profiles indicate that the wild‐type enzyme is more stable than all the interface‐modified enzymes. The wild‐type enzyme is dimeric in the pH range of pH 4.0 and above, and disassembled at pH 3.5 and below. All the interface‐modified enzymes, however, are apparently monomeric at pH 4.0, begin assembly at pH 5.0, and are not fully assembled into the dimeric form until pH 6.0. The results from transverse urea gradient gel electrophoresis show clear and reproducible differences both in the position and the shape of the unfolding patterns; all these modified enzymes are more sensitive to the denaturant and begin to unfold at urea concentrations between 1.0 and 1.5 M; the wild‐type enzyme remains in the folded high mobility form beyond 2.5 M urea. Alkaline phosphatase H370A, modified at the active site and not at the dimer interface, resembles the wild‐type enzyme both in ultracentrifugation and electrophoresis studies. The results obtained suggest that substitution of a single amino acid at the interface sacrifices not only the integrity of the assembled dimer, but also the stability of the monomer fold, even though the activity of the enzyme at optimal pH remains unaffected and does not appear to depend on interface stability.
Diversity of functions of proteins with internal symmetry in spatial arrangement of secondary structural elements
Authors: Kinoshita Kengo , Kidera Akinori , Go Nobuhiro
Published: February 2009

We carry out a systematic analysis of the correlation between similarity of protein three‐dimensional structures and their evolutionary relationships. The structural similarity is quantitatively identified by an all‐against‐all comparison of the spatial arrangement of secondary structural elements in nonredundant 967 representative proteins, and the evolutionary relationship is judged according to the definition of superfamily in the SCOP database. We find the following symmetry rule: a protein pair that has similar folds but belong to different superfamilies has (with a very rare exception) certain internal symmetry in its common similar folds. Possible reasons behind the symmetry rule are discussed.
Oligomeric integrity—the structural key to thermal stability in bacterial alcohol dehydrogenases
Authors: Korkhin Yakov , Joseph KALB(GILBOA) A. , Peretz Moshe , Bogin Oren , Burstein Yigal , Frolow Felix
Published: February 2009

Principles of protein thermostability have been studied by comparing structures of thermostable proteins with mesophilic counterparts that have a high degree of sequence identity. Two tetrameric NADP(H)‐dependent alcohol dehydrogenases, one from Clostridium beijerinckii (CBADH) and the other from Thermoanaerobacter brockii (TBADH), having exceptionally high (75%) sequence identity, differ by 30° in their melting temperatures. The crystal structures of CBADH and TBADH in their holo‐enzyme form have been determined at a resolution of 2.05 and 2.5 Å, respectively. Comparison of these two very similar structures (RMS difference in Ca = 0.8 Å) revealed several features that can account for the higher thermal stability of TBADH. These include additional ion pairs, “charged‐neutral” hydrogen bonds, and prolines as well as improved stability of α‐helices and tighter molecular packing. However, a deeper structural insight, based on the location of stabilizing elements, suggests that enhanced thermal stability of TBADH is due mainly to the strategic placement of structural determinants at positions that strengthen the interface between its subunits. This is also supported by mutational analysis of structural elements at critical locations. Thus, it is the reinforcement of the quaternary structure that is most likely to be a primary factor in preserving enzymatic activity of this oligomeric bacterial ADH at elevated temperatures.
The schiff base complex of yeast 5‐aminolaevulinic acid dehydratase with laevulinic acid
Authors: Erskine Peter T. , Newbold Richard , Roper Jenny , Coker Alun , Warren Martin J. , Shoolingin‐Jordan Peter M. , Wood Steve P. , Cooper Jon B.
Published: February 2009

The X‐ray structure of the complex formed between yeast 5‐aminolaevulinic acid dehydratase (ALAD) and the inhibitor laevulinic acid has been determined at 2.15 Å resolution. The inhibitor binds by forming a Schiff base link with one of the two invariant lysines at the catalytic center: Lys263. It is known that this lysine forms a Schiff base link with substrate bound at the enzyme' so‐called P‐site. The carboxyl group of laevulinic acid makes hydrogen bonds with the side‐chain‐OH groups of Tyr329 and Ser290, as well as with the main‐chain >NH group of Ser290. The aliphatic moiety of the inhibitor makes hydrophobic interactions with surrounding aromatic residues in the protein including Phe219, which resides in the flap covering the active site. Our analysis strongly suggests that the same interactions will be made by P‐side substrate and also indicates that the substrate that binds at the enzyme' A‐site will interact with the enzyme' zinc ion bound by three cysteines (133, 135, and 143). Inhibitor binding caused a substantial ordering of the active site flap (residues 217–235), which was largely invisible in the native electron density map and indicates that this highly conserved yet flexible region has a specific role in substrate binding during catalysis.
The Ubp6 family of deubiquitinating enzymes contains a ubiquitin‐like domain: SUb
Authors: Wyndham Allison M. , Baker Rohan T. , Chelvanayagam Gareth
Published: February 2009

A sequence motif that is Similar to Ubiquitin (SUb) has been identified in the Saccharomyces cerevisiae ubiquitin‐specific protease Ubp6. SUb is conserved in all known Ubp6 homologues from a spectrum of eukaryotic species and is also present in a group of hypothetical proteins of unknown function (Unk1–3) present in sequence databases. An N‐terminal deletion mutant of Ubp6 that lacks SUb is still capable of cleaving α‐linked ubiquitin fusions, suggesting that SUb forms a separate domain to the catalytic core of Ubp6 and demonstrating that it is not required for in vitro cleavage activity. A homology model of the 78 N‐terminal amino acids of human Ubp6, based on the known fold of ubiquitin, is presented. In human Ubp6, SUb shares only 20% sequence identity with ubiquitin. Even weaker similarity occurs between S. cerevisiae SUb and ubiquitin. The homology model supports a ubiquitin‐like fold for SUb and suggests that two conserved Lys residues, corresponding to Lys48 and Lys63 of ubiquitin, are functionally important.
Probing the role of water in the tryptophan repressor‐operator complex
Authors: Brown Martha P. , Grillo Adeola O. , Boyer Mireille , Royer Catherine A.
Published: February 2009

The Escherichia coli tryptophan repressor protein (TR) represses the transcription of several genes in response to the concentration of tryptophan in the environment. In the co‐crystal structure of TR bound to a DNA fragment containing its target very few direct contacts between TR and the DNA were observed. In contrast, a number of solvent mediated contacts were apparent. NMR solution structures, however, did not resolve any solvent mediated bonds at the complex interface. To probe for the role of water in TR operator recognition, the effect of osmolytes on the interactions between TR and a target oligonucleotide bearing the operator site was examined. In the absence of specific solvent mediated hydrogen bonding interactions between the protein and the DNA, increasing osmolyte concentration is expected to strongly stabilize the TR operator interaction due to the large amount of macromolecular surface area buried upon complexation. The results of our studies indicate that xylose did not alter the binding affinity significantly, while glycerol and PEG had a small stabilizing effect. A study of binding as a function of betaine concentration revealed that this osmolyte at low concentration results in a stabilization of the 1:1 TR/operator complex, but at higher concentrations leads to a switching between binding modes to favor tandem binding. Analysis of the effects of betaine on the 1:1 complex suggest that this osmolyte has about 78% of the expected effect. If one accepts the analysis in terms of the number of water molecules excluded upon complexation, these results suggest that about 75 water molecules remain at the interface of the 1:1 dimer/DNA complex. This value is consistent with the number of water molecules found at the interface in the crystallographically determined structure and supports the notion that interfacial waters play an important thermodynamic role in the specific complexation of one TR dimer with its target DNA. However, the complexity of the effects of betaine and the small or negligible effects of the other osmolytes could also arise from osmolyte induced competition between antagonistic coupled reactions.
The 80s loop of the catalytic chain of Escherichia coli aspartate transcarbamoylase is critical for catalysis and homotropic cooperativity
Authors: Macol Christine , Dutta Malini , Stec Boguslaw , Tsuruta Hiro , Kantrowitz Evan R.
Published: February 2009

The X‐ray structure of the Escherichia coli aspartate transcarbamoylase with the bisubstrate analog phosphonacetyl‐L‐aspartate (PALA) bound shows that PALA interacts with Lys84 from an adjacent catalytic chain. To probe the function of Lys84, site‐specific mutagenesis was used to convert Lys84 to alanine, threonine, and asparagine. The K84N and K84T enzymes exhibited 0.08 and 0.29% of the activity of the wild‐type enzyme, respectively. However, the K84A enzyme retained 12% of the activity of the wild‐type enzyme. For each of these enzymes, the affinity for aspartate was reduced 5‐ to 10‐fold, and the affinity for carbamoyl phosphate was reduced 10‐ to 30‐fold. The enzymes K84N and K84T exhibited no appreciable cooperativity, whereas the K84A enzyme exhibited a Hill coefficient of 1.8. The residual cooperativity and enhanced activity of the K84A enzyme suggest that in this enzyme another mechanism functions to restore catalytic activity. Modeling studies as well as molecular dynamics simulations suggest that in the case of only the K84A enzyme, the lysine residue at position 83 can reorient into the active site and complement for the loss of Lys84. This hypothesis was tested by the creation and analysis of the K83A enzyme and a double mutant enzyme (DM) that has both Lys83 and Lys84 replaced by alanine. The DM enzyme has no cooperativity and exhibited 0.18% of wild‐type activity, while the K83A enzyme exhibited 61% of wild‐type activity. These data suggest that Lys84 is not only catalytically important, but is also essential for binding both substrates and creation of the high‐activity, high‐affinity active site. Since low‐angle X‐ray scattering demonstrated that the mutant enzymes can be converted to the R‐structural state, the loss of cooperativity must be related to the inability of these mutant enzymes to form the high‐activity, high‐affinity active site characteristic of the R‐functional state of the enzyme.
New insight into the pH‐dependent conformational changes in bovine β‐lactoglobulin from Raman optical activity
Authors: Blanch Ewan W. , Hecht Lutz , Barron Laurence D.
Published: February 2009

We have studied the conformation of β‐lactoglobulin in aqueous solution at room temperature over the pH range ∼2.0–9.0 using vibrational Raman optical activity (ROA). The ROA spectra clearly show that the basic up and down β‐barrel core is preserved over the entire pH range, in agreement with other studies. However, from the shift of a sharp positive ROA band at ∼1268 to ∼1294 cm−1 on going from pH values below that of the Tanford transition, which is centered at pH ∼7.5, to values above, the Tanford transition appears to be associated with changes in the local conformations of residues in loop sequences possibly corresponding to a migration into the α‐helical region of the Ramachandran surface from a nearby region. These changes may be related to those detected in X‐ray crystal structures which revealed that the Tanford transition is associated with conformational changes in loops which form a doorway to the interior of the protein. The results illustrate how the ability of ROA to detect loop and turn structure separately from secondary structure is useful for studying conformational plasticity in proteins.
Selective association of protein molecules followed by mass spectrometry
Authors: Vis Hans , Dobson Christopher M. , Robinson Carol V.
Published: February 2009

Nanoflow electrospray mass spectrometry was used to monitor the formation of protein heterodimers of HU proteins from Bacillus stearothermophilus and Bacillus subtilis. This has enabled us to analyze both thermodynamic and kinetic features associated with the dissociation of homodimeric HU proteins. The results obtained correlate well with the kinetics of the protein dissociation process and the free energy difference between homo‐ and heterodimeric species anticipated from other studies. We suggest that this approach will have general applicability in studying protein association and dissociation under near‐equilibrium conditions and will be relevant to a wide range of biological systems.
Electrostatic interactions in the GCN4 leucine zipper: Substantial contributions arise from intramolecular interactions enhanced on binding
Authors: Hendsch Zachary S. , Tidor Bruce
Published: February 2009

The GCN4 leucine zipper is a peptide homodimer that has been the subject of a number of experimental and theoretical investigations into the determinants of affinity and specificity. Here, we utilize this model system to investigate electrostatic effects in protein binding using continuum calculations. A particularly novel feature of the computations made here is that they provide an interaction‐by‐interaction breakdown of the electrostatic contributions to the free energy of docking that includes changes in the interaction of each functional group with solvent and changes in interactions between all pairs of functional groups on binding. The results show that (1) electrostatic effects disfavor binding by roughly 15 kcal/mol due to desolvation effects that are incompletely compensated in the bound state, (2) while no groups strongly stabilize binding, the groups that are most destabilizing are charged and polar side chains at the interface that have been implicated in determining binding specificity, and (3) attractive intramolecular interactions (e.g., backbone hydrogen bonds) that are enhanced on binding due to reduced solvent screening in the bound state contribute significantly to affinity and are likely to be a general effect in other complexes. A comparison is made between the results obtained in an electrostatic analysis carried out calculationally and simulated results corresponding to idealized data from a scanning mutagenesis experiment. It is shown that scanning experiments provide incomplete information on interactions and, if overinterpreted, tend to overestimate the energetic effect of individual side chains that make attractive interactions. Finally, a comparison is made between the results available from a continuum electrostatic model and from a simpler surface‐area dependent solvation model. In this case, although the simpler model neglects certain interactions, on average it performs rather well.
Long‐range effects in protein–ligand interactions mediate peptide specificity inl the human major histocompatibility antigen HLA‐B27 (B*2701)
Authors: Krebs Stefan , Rognan Didier , Castro JOSÉ A.LÓPez De
Published: February 2009

B*2701 differs from all other HLA‐B27 subtypes of known peptide specificity in that, among its natural peptide ligands, arginine is not the only allowed residue at peptide position 2. Indeed, B*2701 is unique in binding many peptides with Gln2 in vivo. However, the mutation (Asp74Tyr) responsible for altered selectivity is far away from the B pocket of the peptide binding site to which Gln/Arg2 binds. Here, we present a model that explains this effect. It is proposed that a new rotameric state of the conserved Lys70 is responsible for the unique B*2701 binding motif. This side chain should be either kept away from pocket B through its interaction with Asp74 in most HLA‐B27 subtypes, or switched to this pocket if residue 74 is Tyr as in B*2701. Involvement of Lys70 in pocket B would thus allow binding of peptides with Gln2. Binding of Arg2‐containing peptides to B*2701 is also possible because Lys70 could adopt another conformation, H‐bonded to Asn97, which preserves the same binding mode of Arg2 as in B*2705. This model was experimentally validated by mutating Lys70 into Ala in B*2701. Edman sequencing of the B*2701(K70A) peptide pool showed only Arg2, characteristic of HLA‐B27‐bound peptides, and no evidence for Gln2. This supports the computational model and demonstrates that allowance of B*2701 for peptides with Gln2 is due to the long‐range effect of the polymorphic residue 74 of HLA‐B27, by inducing a conformational switch of the conserved Lys70.
Antibody variable region binding by Staphylococcal protein A: Thermodynamic analysis and location of the Fv binding site on E‐domain
Authors: Starovasnik Melissa A. , O'Connell Mark P. , Fairbrother Wayne J. , Kelley Robert F.
Published: February 2009

Immunoglobulins of human heavy chain subgroup III have a binding site for Staphylococcal protein A on the heavy chain variable domain (VH), in addition to the well‐known binding site on the Fc portion of the antibody. Thermodynamic characterization of this binding event and localization of the Fv‐binding site on a domain of protein A is described. Isothermal titration calorimetry (ITC) was used to characterize the interaction between protein A or fragments of protein A and variants of the hu4D5 antibody Fab fragment. Analysis of binding isotherms obtained for titration of hu4D5 Fab with intact protein A suggests that 3–4 of the five immunoglobulin binding domains of full length protein A can bind simultaneously to Fab with a Ka of 5.5 ± 0.5 × 105 M–1. A synthetic single immunoglobulin binding domain, Z‐domain, does not bind appreciably to hu4D5 Fab, but both the E and D domains are functional for hu4D5 Fab binding. Thermodynamic parameters for titration of the E‐domain with hu4D5 Fab are n = 1.0 ± 0.1, Ka = 2.0 ± 0.3 × 105 M–1, and ΔH = –7.1 ± 0.4 kcal mol–1. Similar binding thermodynamics are obtained for titration of the isolated VH domain with E‐domain indicating that the E‐domain binding site on Fab resides within VH. E‐domain binding to an IgG1 Fc yields a higher affinity interaction with thermodynamic parameters n = 2.2 ± 0.1, Ka > 1.0 × 107 M–1, and ΔH = –24.6 ± 0.6 kcal mol–1. Fc does not compete with Fab for binding to E‐domain indicating that the two antibody fragments bind to different sites. Amide 1H and 15N resonances that undergo large changes in NMR chemical shift upon Fv binding map to a surface defined by helix‐2 and helix‐3 of E‐domain, distinct from the Fc‐binding site observed in the crystal structure of the B‐domain/Fc complex. The Fv‐binding region contains negatively charged residues and a small hydrophobic patch which complements the basic surface of the region of the VH domain implicated previously in protein A binding.
Relating structure to thermodynamics: The crystal structures and binding affinity of eight OppA‐peptide complexes
Authors: Davies Thomas G. , Hubbard Roderick E. , Tame JEREMY R.H.
Published: February 2009

The oligopeptide‐binding protein OppA provides a useful model system for studying the physical chemistry underlying noncovalent interactions since it binds a variety of readily synthesized ligands. We have studied the binding of eight closely related tripeptides of the type Lysine‐X‐Lysine, where X is an abnormal amino acid, by isothermal titration calorimetry (ITC) and X‐ray crystallography. The tripeptides fall into three series of ligands, which have been designed to examine the effects of small changes to the central side chain. Three ligands have a primary amine as the second side chain, two have a straight alkane chain, and three have ring systems. The results have revealed a definite preference for the binding of hydrophobic residues over the positively charged side chains, the latter binding only weakly due to unfavorable enthalpic effects. Within the series of positively charged groups, a point of lowest affinity has been identified and this is proposed to arise from unfavorable electrostatic interactions in the pocket, including the disruption of a key salt bridge. Marked entropy‐enthalpy compensation is found across the series, and some of the difficulties in designing tightly binding ligands have been highlighted.
Conformational changes in the NS3 protease from hepatitis C virus strain Bk monitored by limited proteolysis and mass spectrometry
Authors: Orrù Stefania , Piaz Fabrizio Dal , Casbarra Annarita , Biasiol Gabriella , Francesco Raffaele De , Steinkühler Christian , Pucci Piero
Published: February 2009

Conformational changes occurring within the NS3 protease domain from the hepatitis C virus Bk strain (NS31–180) under different physico‐chemical conditions either in the absence or in the presence of its cofactor Pep4A were investigated by limited proteolysis experiments. Because the surface accessibility of the protein is affected by conformational changes, when comparative experiments were carried out on NS31–180 either at different glycerol concentrations or in the presence of Pep4A, differential peptide maps were obtained from which protein regions involved in the structural changes could be inferred. The surface topology of isolated NS31–180 in solution was essentially consistent with the crystal structure of the protein with the N‐terminal segment showing a high conformational flexibility. At higher glycerol concentration, the protease assumed a more compact structure showing a decrease in the accessibility of the N‐terminal segment that either was forced to interact with the protein or originate intermolecular interactions with neighboring molecules. Binding of the cofactor Pep4A caused the displacement of the N‐terminal arm from the protein moiety, leading this segment to again adopt an open and flexible conformation, thus suggesting that the N‐terminus of the protease contributes only marginally to the stability of the complex. The observed conformational changes might be directly correlated with the activation mechanism of the protease by either the cosolvent or the cofactor peptide because they lead to tighter packing of the substrate binding site.
X‐ray structure of human stromelysin catalytic domain complexed with nonpeptide inhibitors: Implications for inhibitor selectivity
Authors: Pavlovsky Alexander G. , Williams Mark G. , Ye Qi‐Zhuang , Ortwine Daniel F. , Purchase Claude F. , White Andrew D. , Dhanaraj V. , Roth Bruce D. , Johnson Linda L. , Hupe Donald , Humblet Christine , Blundell Tom L.
Published: February 2009

Effective inhibitors of matrix metalloproteinases (MMPs), a family of connective tissue‐degrading enzymes, could be useful for the treatment of diseases such as cancer, multiple sclerosis, and arthritis. Many of the known MMP inhibitors are derived from peptide substrates, with high potency in vitro but little selectivity among MMPs and poor bioavail‐ability. We have discovered nonpeptidic MMP inhibitors with improved properties, and report here the crystal structures of human stromelysin‐1 catalytic domain (SCD) complexed with four of these inhibitors. The structures were determined and refined at resolutions ranging from 1.64 to 2.0 Å. Each inhibitor binds in the active site of SCD such that a bulky diphenyl piperidine moiety penetrates a deep, predominantly hydrophobic S1' pocket. The active site structure of the SCD is similar in all four inhibitor complexes, but differs substantially from the peptide hydroxamate complex, which has a smaller side chain bound in the S1' pocket. The largest differences occur in the loop forming the “top” of this pocket. The occupation of these nonpeptidic inhibitors in the S1' pocket provides a structural basis to explain their selectivity among MMPs. An analysis of the unique binding mode predicts structural modifications to design improved MMP inhibitors.
A new protein folding screen: Application to the ligand binding domains of a glutamate and kainate receptor and to lysozyme and carbonic anhydrase
Authors: Armstrong Neali , Lencastre Alexandre De , Gouaux Eric
Published: February 2009

Production of folded and biologically active protein from Escherichia coli derived inclusion bodies can only be accomplished if a scheme exists for in vitro naturation. Motivated by the need for a rapid and statistically meaningful method of determining and evaluating protein folding conditions, we have designed a new fractional factorial protein folding screen. The screen includes 12 factors shown by previous experiments to enhance protein folding and it incorporates the 12 factors into 16 different folding conditions. By examining a 10256th fraction of the full factorial, multiple folding conditions were determined for the ligand binding domains from glutamate and kainate receptors, and for lysozyme and carbonic anhydrase B. The impact of each factor on the formation of biologically active material was estimated by calculating factor main effects. Factors and corresponding levels such as pH (8.5) and L‐ginine (0.5 M) consistently had a positive effect on protein folding, whereas detergent (0.3 mM lauryl maltoside) and nonpolar additive (0.4 M sucrose) were detrimental to the folding of these four proteins. One of the 16 conditions yielded the most folded material for three out of the four proteins. Our results suggest that this protein folding screen will be generally useful in determining whether other proteins will fold in vitro and, if so, what factors are important. Furthermore, fractional factorial folding screens are well suited to the evaluation of previously untested factors on protein folding.
Conformational properties of native sperm whale apomyoglobin in solution
Authors: Lecomte JULIETTE T.J. , Sukits Steven F. , Bhattacharya Shibani , Falzone Christopher J.
Published: February 2009

Apomyoglobin from sperm whale is often used for studies of ligand binding, protein folding, and protein stability. In an effort to describe its conformational properties in solution, homonuclear and heteronuclear (13C and 15N) NMR methods were applied to the protein in its native state. Assignments were confirmed for nuclear Overhauser effects (NOEs) involving side chain and backbone protons in the folded regions of the structure. These NOEs were used to derive distance restraints. The shifts induced by the hydrophobic dye 8‐anilino‐1‐naphthalenesulfonic acid (ANS) were inspected in the regions remote from its binding site and served as an indicator of conformational flexibility. 3JαH‐NH values were obtained to assess dihedral angle averaging and to provide additional restraints. A family of structures was calculated with X‐PLOR and an ab initio simulated annealing protocol using holomyoglobin as a template. Where the structure appeared well defined by chemical shift, line width, ANS perturbation, and density of NOEs, the low resolution model of apomyoglobin provides a valid approximation for the structure. The new model offers an improved representation of the folded regions of the protein, which encompass the A, B, E, helices as well as parts of the G and H helices. Regions that are less well defined at this stage of calculations include the CD corner and the end of the H‐helix. The EF‐F‐FG segment remains uncharacterized.
Positional preference of proline in α‐helices
Authors: Kim Mee Kyoung , Kang Young Kee
Published: February 2009

Conformational free energy calculations have been carried out for proline‐containing alanine‐based pentadecapeptides with the sequence Ac‐(Ala)n‐Pro‐(Ala)m‐NHMe, where n + m = 14, to figure out the positional preference of proline in α‐helices. The relative free energy of each peptide was calculated by subtracting the free energy of the extended conformation from that of the α‐helical one, which is used here as a measure of preference. The highest propensity is found for the peptide with proline at the N‐terminus (i.e., Ncap + 1 position), and the next propensities are found at Ncap, N' (Ncap — 1), and C' (Ccap + 1) positions. These computed results are reasonably consistent with the positional propensities estimated from X‐ray structures of proteins. The breaking in hydrogen bonds around proline is found to play a role in destabilizing α‐helical conformations, which, however, provides the favored hydration of the corresponding N‐H and C=O groups. The highest preference of proline at the beginning of α‐helix appears to be due to the favored electrostatic and nonbonded energies between two residues preceding proline and the intrinsic stability of α‐helical conformation of the proline residue itself as well as no disturbance in hydrogen bonds of α‐helix by proline. The average free energy change for the substitution of Ala by Pro in a α‐helix is computed to be 4.6 kcal/mol, which is in good agreement with the experimental value of ∼4 kcal/mol estimated for an oligopeptide dimer and proteins of barnase and T4 lysozyme.
Heat capacity change for ribonuclease A folding
Authors: Pace C. Nick , Grimsley Gerald R. , Thomas Susan T. , Makhatadze George I.
Published: February 2009

The change in heat capacity ΔCp for the folding of ribonuclease A was determined using differential scanning calorimetry and thermal denaturation curves. The methods gave equivalent results, ΔCp = 1.15 ± 0.08 kcal mol–1 K–1. Estimates of the conformational stability of ribonuclease A based on these results from thermal unfolding are in good agreement with estimates from urea unfolding analyzed using the linear extrapolation method.
Structural characterization of the membrane‐associated regulatory subunit of type I cAMP‐dependent protein kinase by mass spectrometry: Identification of Ser81 as the in vivo phosphorylation site of RIα
Authors: Boeshans Karen M. , Resing Katheryn A. , Hunt John B. , Ahn Natalie G. , Shabb John B.
Published: February 2009

The mechanism by which the type Iα regulatory subunit (RIα) of cAMP‐dependent protein kinase is localized to cell membranes is unknown. To determine if structural modification of RIα is important for membrane association, both beef skeletal muscle cytosolic RI and beef heart membrane‐associated RI were characterized by electrospray ionization mass spectrometry. Total sequence coverage was 98% for both the membrane‐associated and cytosolic forms of RI after digestion with AspN protease or trypsin. Sequence data indicated that membrane‐associated and cytosolic forms of RI were the same RIα gene product. A single RIα phosphorylation site was identified at Ser81 located near the autoinhibitory domain of both membrane‐associated and cytosolic RIα. Because both R subunit preparations were 30–40% phosphorylated, this post‐translational modification could not be responsible for the membrane compartmentation of the majority of RIα. Mass spectrometry also indicated that membrane‐associated RIα had a higher extent of disulfide bond formation in the amino‐terminal dimerization domain. No other structural differences between cytosolic and membrane‐associated RIα were detected. Consistent with these data, masses of the intact proteins were identical by LCQ mass spectrometry. Lack of detectable structural differences between membrane‐associated and cytosolic RIα strongly suggests an interaction between RIα and anchoring proteins or membrane lipids as more likely mechanisms for explaining RIα membrane association in the heart.
TRIADs: A new class of proteins with a novel cysteine‐rich signature
Authors: Van Der Reijden Bert A. , Erpelinck‐Verschueren Claudia A.J. , BOB LÖWENBERG  , Jansen Joop H.
Published: February 2009

Triad1 was recently identified as a nuclear RING finger protein, which is up‐regulated during retinoic acid induced granulocytic differentiation of acute leukemia cells. Here we show that a cysteine‐rich domain (C6HC), present in Triad1, is conserved in at least 24 proteins encoded by various eukaryotes. The C6HC consensus pattern C‐x(4)‐C‐x(14–30)‐C‐x(1–4)‐C‐x(4)‐C‐x(2)‐C‐x(4)‐H‐x(4)‐C defines this structure as the fourth family member of the zinc‐binding RING, LIM, and LAP/PHD fingers. Strikingly, in 22 of 24 proteins the C6HC domain is flanked by two RING finger structures. We have termed the novel C6HC motif DRIL (double RING finger linked). The strong conservation of the larger tripartite TRIAD (two RING fingers and DRIL) structure indicates that the three subdomains are functionally linked and identifies a novel class of proteins.
Distinguishing between sequential and nonsequentially folded proteins: Implications for folding and misfolding
Authors: Tsai Chung‐Jung , Maizel Jacob V , Nussinov Ruth
Published: February 2009

We describe here an algorithm for distinguishing sequential from nonsequentially folding proteins. Several experiments have recently suggested that most of the proteins that are synthesized in the eukaryotic cell may fold sequentially. This proposed folding mechanism in vivo is particularly advantageous to the organism. In the absence of chaperones, the probability that a sequentially folding protein will misfold is reduced significantly. The problem we address here is devising a procedure that would differentiate between the two types of folding patterns. Footprints of sequential folding may be found in structures where consecutive fragments of the chain interact with each other. In such cases, the folding complexity may be viewed as being lower. On the other hand, higher folding complexity suggests that at least a portion of the polypeptide backbone folds back upon itself to form three‐dimensional (3D) interactions with noncontiguous portion(s) of the chain. Hence, we look at the mechanism of folding of the molecule via analysis of its complexity, that is, through the 3D interactions formed by contiguous segments on the polypeptide chain. To computationally splice the structure into consecutively interacting fragments, we either cut it into compact hydrophobic folding units or into a set of hypothetical, transient, highly populated, contiguous fragments (“building blocks” of the structure). In sequential folding, successive building blocks interact with each other from the amino to the carboxy terminus of the polypeptide chain. Consequently, the results of the parsing differentiate between sequentially vs. nonsequentially folded chains. The automated assessment of the folding complexity provides insight into both the likelihood of misfolding and the kinetic folding rate of the given protein. In terms of the funnel free energy landscape theory, a protein that truly follows the mechanism of sequential folding, in principle, encounters smoother free energy barriers. A simple sequentially folded protein should, therefore, be less error prone and fold faster than a protein with a complex folding pattern.
The progressive development of structure and stability during the equilibrium folding of the α subunit of tryptophan synthase from Escherichia coli
Authors: Gualfetti Peter J. , Bilsel Osman , Matthews C. Robert
Published: February 2009

The urea‐induced equilibrium unfolding of the α subunit of tryptophan synthase (aTS), a single domain α/β barrel protein, displays a stable intermediate at ˜3.2 M urea when monitored by absorbance and circular dichroism (CD) spectroscopy (Matthews CR, Crisanti MM, 1981, Biochemistry 20:784–792). The same experiment, monitored by one‐dimensional proton NMR, shows another cooperative process between 5 and 9 M urea that involves His92 (Saab‐Rincón G et al., 1993, Biochemistry 52:13981–13990). To further test and quantify the implied four‐state model, N ⇄ I1 ⇄ I2 ⇄ U, the urea‐induced equilibrium unfolding process was followed by tyrosine fluorescence total intensity, tyrosine fluorescence anisotropy and far‐UV CD. All three techniques resolve the four stable states, and the transitions between them when the FL total intensity and CD spectroscopy data were analyzed by the singular value decomposition method. Relative to U, the stabilities of the N, I1, and I2 states are 15.4, 9.4, and 4.9 kcal mol−1, respectively. I2 partially buries one or more of the seven tyrosines with a noticeable restriction of their motion; it also recovers ∼6% of the native CD signal. This intermediate, which is known to be stabilized by the hydrophobic effect, appears to reflect the early coalescence of nonpolar side chains without significant organization of the backbone. I1 recovers an additional 43% of the CD signal, further sequesters tyrosine residues in nonpolar environments, and restricts their motion to an extent similar to N. The progressive development of a higher order structure as the denaturant concentration decreases implies a monotonic contraction in the ensemble of conformations that represent the U, I2, I1, and N states of αTS.
Structure and dynamics of the fatty acid binding cavity in apo rat intestinal fatty acid binding protein
Authors: Likić Vladimir A. , Prendergast Franklyn G.
Published: February 2009

The structure and dynamics of the fatty acid binding cavity in I‐FABP (rat intestinal fatty acid binding protein) were analyzed. In the crystal structure of apo I‐FABP, the probe occupied cavity volume and surface are 539 ± 8 Å3 and 428 Å2, respectively (1.4 Å probe). A total of 31 residues contact the cavity with their side chains. The side‐chain cavity surface is partitioned according to the residue type as follows: 36–39% hydrophobic, 21–25% hydrophilic, and 37–43% neutral or ambivalent. Thus, the cavity surface is neither like a typical protein interior core, nor is like a typical protein external surface. All hydrophilic residues that contact the cavity—with the exception of Asp74—are clustered on the one side of the cavity. The cavity appears to expand its hydrophobic surface upon fatty acid binding on the side opposite to this hydrophilic patch. In holo I‐FABP the fatty acid chain interactions with the hydrophilic side chains are mediated by water molecules. Molecular dynamics (MD) simulation of fully solvated apo I‐FABP showed global conformational changes of I‐FABP, which resulted in a large, but seemingly transient, exposure of the cavity to the external solvent. The packing density of the side chains lining the cavity, studied by Voronoi volumes, showed the presence of two distinctive small hydrophobic cores. The MD simulation predicts significant structural perturbations of the cavity on the subnano‐second time scale, which are capable of facilitating exchange of I‐FABP internal water.