bluuebunny/crossref_metadata_2025 huggingface dataset:
Mapping fatty acid binding to β‐lactoglobulin: Ligand binding is restricted by modification of Cys 121
Authors: Narayan Mahesh , Berliner Lawrence J.
Published: July 2010

Native β‐lactoglobulin (Blg) binds 1 mole of palmitic acid per mole of protein with a dissociation constant of 0.6 μM for the primary fatty acid binding site. Chemical modification of Cys 121, which lies at the external putative hydrophobic binding site of Blg, does not affect retinol or 4,4′‐bis 1‐(phenylamino)‐8‐naphthalenesulfonate (bis‐ANS) binding to the protein, indicating that the incorporated appendages do not perturb the internal hydrophobic site within the β‐barrel of Blg (i.e., the retinoid site is unaffected). On the other hand, methylation of Cys 121, reduces the affinity of Blg for palmitic acid by 10‐fold as monitored by intrinsic fluorescence. Modification of the Cys 121 with methyl‐methanethiosulfonate or a thiol‐specific spin label appears to either further weaken or totally eliminate fatty acid binding, respectively, due to steric hindrance. Furthermore, this binding pattern has been independently verified using a spin labeled fatty acid analog and monitoring ESR as well as by bis‐ANS fluorescence when bound to the protein. These results suggest that fatty acids bind at the “external site” of β‐lactoglobulin, between the sole a‐helix and the β‐barrel. In addition, structural stability studies of native and chemically modified Blg appear to confirm this observation as well.
Purification and characterization of the heteromeric transcriptional activator MvaT of the Pseudomonas mevalonii mvaAB operon
Authors: Rosenthal R. Scott , Rodwell Victor W.
Published: July 2010

The mvaAB operon of Pseudomonas mevalonii encodes HMG‐CoA reductase (EC 1.1.1.88) and HMG‐CoA lyase (EC 4.1.3.4), enzymes that catalyze the initial reactions of mevalonate catabolism in this organism. Expression of this operon is regulated by the constitutively expressed transcriptional activator protein MvaT that binds in vitro to an upstream regulatory element. Mevalonate is essential for activation of transcription in vivo, and in vitro data demonstrated that MvaT binds to the mvaAB cis‐regulatory element in the absence of mevalonate with a Kd,app of 2 nM. Purification of MvaT enriched for two polypeptides of approximate molecular mass 15 kDa and 16 kDa, designated P15 and P16. MvaT, assayed by its DNA‐binding activity, comigrated with P15 and P16 during DNA‐affinity chromatography, size‐exclusion chromatography, and sucrose density gradient centrifugation. P15 and P16 also comigrated during denaturing isoelectric focusing of purified MvaT. Treatment of MvaT with dimethylsuberimidate formed a 31‐kDa polypeptide complex that contained N‐terminal sequences from P15 and P16. The apparent association of P15 and P16 in solution and their copurification with MvaT activity strongly suggests that MvaT is comprised of these two subunits. Size‐exclusion chromatography gave an estimated molecular mass for MvaT of 33 kDa. A partial DNA sequence of the P16 gene was obtained using PCR employing degenerate primers directed against the N‐termini of P15 and P16. P16 appears to be comprised of at least 128 aminoacyl residues having a predicted molecular mass of 14.3 kDa.
Conformation, stability, and active‐site cysteine titrations of Escherichia coli D26A thioredoxin probed by Raman spectroscopy
Authors: Vohník Stanislav , Tuma Roman , Thomas George J. , Hanson Chad , Fuchs James A. , Woodward Clare
Published: July 2010

The active‐site cysteines (Cys 32 and Cys 35) of Escherichia coli thioredoxin are oxidized to a disulfide bridge when the protein mediates substrate reduction. In reduced thioredoxin, Cys 32 and Cys 35 are characterized by abnormally low pKa values. A conserved side chain, Asp 26, which is sterically accessible to the active site, is also essential to oxidoreductase activity. pKa values governing cysteine thiol‐thiolate equilibria in the mutant thioredoxin, D26A, have been determined by direct Raman spectrophotometric measurement of sulfhydryl ionizations. The results indicate that, in D26A thioredoxin, both sulfhydryls titrate with apparent pKa values of 7.5 ± 0.2, close to values measured previously for wild‐type thioredoxin. Sulfhydryl Raman markers of D26A and wild‐type thioredoxin also exhibit similar band shapes, consistent with minimal differences in respective cysteine side‐chain conformations and sulfhydryl interactions. The results imply that neither the Cys 32 nor Cys 35 SH donor is hydrogen bonded directly to Asp 26 in the wild‐type protein. Additionally, the thioredoxin main‐chain conformation is largely conserved with D26A mutation. Conversely, the mutation perturbs Raman bands diagnostic of tryptophan (Trp 28 and Trp 31) orientations and leads to differences in their pH dependencies, implying local conformational differences near the active site. We conclude that, although the carboxyl side chain of Asp 26 neither interacts directly with active‐site cysteines nor is responsible for their abnormally low pKa values, the aspartate side chain may play a role in determining the conformation of the enzyme active site.
How many membrane proteins are there?
Authors: Boyd Dana , Schierle Clark , Beckwith Jon
Published: July 2010

One of the basic issues that arises in functional genomics is the ability to predict the subcellular location of proteins that are deduced from gene and genome sequencing. In particular, one would like to be able to readily specify those proteins that are soluble and those that are inserted in a membrane. Traditional methods of distinguishing between these two locations have relied on extensive, time‐consuming biochemical studies. The alternative approach has been to make inferences based on a visual search of the amino acid sequences of presumed gene products for stretches of hydrophobic amino acids. This numerical, sequence‐based approach is usually seen as a first approximation pending more reliable biochemical data. The recent availability of large and complete sequence data sets for several organisms allows us to determine just how accurate such a numerical approach could be, and to attempt to minimize and quantify the error involved. We have optimized a statistical approach to protein location determination. Using our approach, we have determined that surprisingly few proteins are misallocated using the numerical method. We also examine the biological implications of the success of this technique.
Computation of electrostatic complements to proteins: A case of charge stabilized binding
Authors: Chong Lillian T. , Dempster Sara E. , Hendsch Zachary S. , Lee Lee‐Peng , Tidor Bruce
Published: July 2010

Recent evidence suggests that the net effect of electrostatics is generally to destabilize protein binding due to large desolvation penalties. A novel method for computing ligand‐charge distributions that optimize the tradeoff between ligand desolvation penalty and favorable interactions with a binding site has been applied to a model for barnase. The result is a ligand‐charge distribution with a favorable electrostatic contribution to binding due, in part, to ligand point charges whose direct interaction with the binding site is unfavorable, but which make strong intra‐molecular interactions that are uncloaked on binding and thus act to lessen the ligand desolvation penalty.
Alignment algorithm for homology modeling and threading
Authors: Alexandrov Nickolai N. , Luethy Roland
Published: July 2010

A DNA/protein sequence comparison is a popular computational tool for molecular biologists. Finding a good alignment implies an evolutionary and/or functional relationship between proteins or genomic loci. Sequential similarity between two proteins indicates their structural resemblance, providing a practical approach for structural modeling, when structure of one of these proteins is known. The first step in the homology modeling is a construction of an accurate sequence alignment. The commonly used alignment algorithms do not provide an adequate treatment of the structurally mismatched residues in locally dissimilar regions. We propose a simple modification of the existing alignment algorithm which treats these regions properly and demonstrate how this modification improves sequence alignments in real proteins.
Determination and analysis of antigenic epitopes of prostate specific antigen (PSA) and human glandular kallikrein 2 (hK2) using synthetic peptides and computer modeling
Authors: Piironen Timo , Villoutreix Bruno O. , Becker Charlotte , Lilja Hans , Hollingsworth Kelly , Vihinen Mauno , Bridon Dominique , Qiu Xiaoxing , Rapp John , Dowell Barry , Lövgren Timo , Pettersson Kim , Lilja Hans
Published: July 2010

Prostate specific antigen (PSA) and human glandular kallikrein 2 (hK2), produced essentially by the prostate gland, are 237‐amino acid monomeric proteins, with 79% identity in primary structure. Twenty‐five anti‐PSA monoclonal antibodies (Mabs) were studied for binding to a large array of synthetic linear peptides selected from computer models of PSA and hK2, as well as to biotinylated peptides covering the entire PSA sequence.

Sixteen of the Mabs were bound to linear peptides forming four independent binding regions (I‐IV). Binding region I was localized to amino acid residues 1‐13 (identical sequence for PSA and hK2), II (a and b) was localized to residues 53‐64, III (a and b) was localized to residues 80‐91 (= kallikrein loop), and IV was localized to residues 151‐164. Mabs binding to regions I and IIa were reactive with free PSA, PSA‐ACT complex, and with hK2; Mabs binding to regions IIb, IIIa, and IV were reactive with free PSA and PSA‐ACT complex, but unreactive with hK2; Mabs binding to region IIIb detected free PSA only. All Mabs tested (n = 7) specific for free PSA reacted with kallikrein loop (binding region IIIb). The presence of Mabs interacting with binding region I did not inhibit the catalytic activity of PSA, whereas Mabs interacting with other binding regions inhibited the catalysis. Theoretical model structures of PSA, hK2, and the PSA‐ACT complex were combined with the presented data to suggest an overall orientation of PSA with regard to ACT.
Substrate binding and conformational changes of Clostridium glutamicum diaminopimelate dehydrogenase revealed by hydrogen/deuterium exchange and electrospray mass spectrometry
Authors: Wang Fang , Scapin Giovanna , Blanchard John S. , Angeletti Ruth Hogue
Published: July 2010

C. glutamicum meso‐diaminopimelate dehydrogenase is an enzyme of the L‐lysine biosynthetic pathway in bacteria. The binding of NADPH and diaminopimelate to the recombinant, overexpressed enzyme has been analyzed using hydrogen/deuterium exchange and electrospray ionization/mass spectrometry. NADPH binding reduces the extent of deuterium exchange, as does the binding of diaminopimelate. Pepsin digestion of the deuterated enzyme and enzyme‐substrate complexes coupled with liquid chromatography/mass spectrometry have allowed the identification of eight peptides whose deuterium exchange slows considerably upon the binding of the substrates. These peptides represent regions known or thought to bind NADPH and diaminopimelate. One of these peptides is located at the interdomain hinge region and is proposed to be exchangeable in the “open,” catalytically inactive, conformation but nonexchangeable in the “closed,” catalytically active conformation formed after NADPH and diaminopimelate binding and domain osure. Furthermore, the dimerization region has been localized by this method, and this study provides an example of detecting protein‐protein interface regions using hydrogen/deuterium exchange and electrospray ionization.
Identification of a specific tyrosine residue in Bryodin 1 distinct from the active site but required for full catalytic and cytotoxic activity
Authors: Fryxell Daniel K. , Gawlak Susan L. , Dodge Robert W. , Siegall Clay B.
Published: July 2010

Bryodin 1 (BD1) is a type I ribosome‐inactivating protein (RIP) with low inherent animal toxicity. It has been cloned recently and the recombinant protein (rBDl) has been produced and crystallized. To gain insight into the relationship of rBDl structure and function, we investigated the role of sequences in a region (residues 128‐156) that exhibits homology with membrane interactive sequences and is not part of the enzymatically defined active site. Progressive deletions representing α‐helical turns within these residues were generated; mutant rBDl proteins were expressed in Escherichia coli and demonstrated increasing losses of enzymatic activity. Point mutations were also generated within this region to replace Y140, Y141, and Y142 with either alanine or lysine. Mutants at position 140 or 142 retained full enzymatic activity, whereas A141 and K141 mutants were > 19‐fold less potent. In cytotoxicity assays, the rBDl point mutants at Y141 were > 80‐fold less potent than either rBDl or mutants at residues 140 or 142. However, when introduced into the anti‐CD40 single‐chain immunotoxin rBDl‐G28‐5 sFv, the A140 and A141 point mutations led to decreased cytotoxicity toward CD40 positive cell lines. These data indicate that Y141 plays an important role in the enzymatic activity of BD1 and that Y140, although not essential for catalytic activity, is required for full BD1 function. Because residues 140 and 141 are distinct from residues implicit in the active site, they may be involved in ribosomal and/or membrane interactions or in intracellular trafficking of the toxin and immunotoxin.
Interhelical contacts are required for the helix bundle fold of apolipophorin III and its ability to interact with lipoproteins
Authors: Wang Jianjun , Narayanaswami Vasanthy , Ryan Robert O. , Sykes Brian D.
Published: July 2010

Apolipophorin‐III (apoLp‐III) from the insect, Manduca sexta, is a 166‐residue exchangeable apolipoprotein that plays a critical role in the dynamics of plasma lipoprotein interconversions. Our previous work indicated that a 36‐residue C‐terminal peptide fragment, generated by cyanogen bromide digestion of apoLp‐III, was unable to bind to lipid surfaces (Narayanaswami V, Kay CM, Oikawa K, Ryan RO, 1994, Biochemistry 33:13312‐13320), and showed no secondary structure in aqueous solution. In this paper, we have performed structural studies of this peptide (E131‐Q166) complexed with SDS detergent micelles, or in the presence of the helix‐inducing solvent trifluoroethanol (TFE), by two‐dimensional 1H NMR spectroscopy. The peptide adopts an α‐helical structure in the presence of both SDS and 50% TFE. The lipid‐bound structure of the peptide, generated from the NMR NOE data, showed an elongated, slightly curved α‐helix. Despite its high α‐helix forming propensity, the peptide requires a helix‐promoting environment to adopt an α‐helical structure. This indicates the importance of the surrounding chemical environment and implies that, in the absence of lipid, tertiary contacts in the folded protein play a role in maintaining its structural integrity. Furthermore, the data suggest that the amphipathic helix bundle organization serves as a prerequisite structural motif for the reversible lipoprotein‐binding activity of M. sexta apoLp‐III.
Solid‐state NMR studies of the membrane‐bound closed state of the colicin E1 channel domain in lipid bilayers
Authors: Kim Yongae , Valentine Kathleen , Opella Stanley J. , Schendel Sharon L. , Cramer William A.
Published: July 2010

The colicin El channel polypeptide was shown to be organized anisotropically in membranes by solid‐state NMR analysis of samples of uniformly 15N‐labeled protein in oriented planar phospholipid bilayers. The 190 residue C‐terminal colicin E1 channel domain is the largest polypeptide to have been characterized by 15N solid‐state NMR spectroscopy in oriented membrane bilayers. The 15N‐NMR spectra of the colicin E1 show that: (1) the structure and dynamics are independent of anionic lipid content in both oriented and unoriented samples; (2) assuming the secondary structure of the polypeptide is helical, there are both trans‐membrane and in‐plane helical segments; (3) trans‐membrane helices account for approximately 20‐25% of the channel polypeptide, which is equivalent to 38‐48 residues of the 190‐residue polypeptide. The results of the two‐dimensional PISEMA spectrum are interpreted in terms of a single trans‐membrane helical hairpin inserted into the bilayer from each channel molecule. These data are also consistent with this helical hairpin being derived from the 38‐residue hydrophobic segment near the C‐terminus of the colicin E1 channel polypeptide.
Three solutions of the protein solubility problem
Authors: Terry Jenkins W.
Published: July 2010

Three simple equations are presented, which describe the variation of protein solubility (S) with changes in salt concentration, in terms of either the salt molality (M), the salt activity (ax), or the water activity (aw). Each equation yields, essentially independent, estimates of the numbers of salt ions (Δvx) and water molecules (Δvw) involved in the dissolution of a mol of the protein. The equations can be used to elucidate the physical significance of the parameters in other empirical equations for protein solubility.
15N backbone dynamics of the S‐peptide from ribonuclease A in its free and S‐protein bound forms: Toward a site‐specific analysis of entropy changes upon folding
Authors: Alexandrescu Andrei T. , Rathgeb‐Szabo Klara , Jahnke Wolfgang , Schulthess Therese , Kammerer Richard A. , Rumpel Klaus
Published: July 2010

Backbone 15N relaxation parameters (Rl, R2, 1H‐15N NOE) have been measured for a 22‐residue recombinant variant of the S‐peptide in its free and S‐protein bound forms. NMR relaxation data were analyzed using the “model‐free” approach (Lipari & Szabo, 1982). Order parameters obtained from “model‐free” simulations were used to calculate 1H‐15N bond vector entropies using a recently described method (Yang & Kay, 1996), in which the form of the probability density function for bond vector fluctuations is derived from a diffusion‐in‐a‐cone motional model. The average change in 1H‐15N bond vector entropies for residues T3‐S15, which become ordered upon binding of the S‐peptide to the S‐protein, is ‐12.6 ± 1.4 J/mol.residue.K. 15N relaxation data suggest a gradient of decreasing entropy values moving from the termini toward the center of the free peptide. The difference between the entropies of the terminal and central residues is about ‐ 12 J/mol.residue.K, a value comparable to that of the average entropy change per residue upon complex formation. Similar entropy gradients are evident in NMR relaxation studies of other denatured proteins. Taken together, these observations suggest denatured proteins may contain entropic contributions from non‐local interactions. Consequently, calculations that model the entropy of a residue in a denatured protein as that of a residue in a di ‐ or tri‐peptide, might over‐estimate the magnitude of entropy changes upon folding.
Chemical synthesis and structural characterization of the RGD‐protein decorsin: A potent inhibitor of platelet aggregation
Authors: Laureto Patrizia Polverino De , Scaramella Elena , Filippis Vincezo De , Marin Oriano , Doni Maria Gabriella , Fontana Angelo
Published: July 2010

Decorsin is a 39‐residue RGD‐protein crosslinked by three disulfide bridges isolated from the leech Macrobdella decora belonging to the family of GPIIb‐IIIa antagonists and acting as a potent inhibitor of platelet aggregation. Here we report the solid‐phase synthesis of decorsin using the Fmoc strategy. The crude polypeptide was purified by reverse‐phase HPLC in its reduced form and allowed to refold in the presence of glutathione. The homogeneity of the synthetic oxidized decorsin was established by reverse‐phase HPLC and capillary zone electrophoresis. The results of amino acid analysis after acid hydrolysis of the synthetic protein, NH2‐terminal sequencing and mass determination (4,377 Da) by electrospray mass spectrometry were in full agreement with this theory. The correct pairing of the three disulfide bridges in synthetic decorsin was determined by a combined approach of both peptide mapping using proteolytic enzymes and analysis of the disulfide chirality by CD spectroscopy in the near‐UV region. Synthetic decorsin inhibited human platelet aggregation with an IC50 of 0.1μM, a figure quite similar to that determined utilizing decorsin from natural source. In particular, the synthetic protein was 2,000‐fold more potent than a model RGD‐peptide (e.g., Arg‐Gly‐Asp‐Ser) in inhibiting platelet aggregation. Thermal denaturation experiments of synthetic decorsin, monitored by CD spectroscopy, revealed its high thermal stability (Tm 74 °C). The features of the oxidative refolding process of reduced decorsin, as well as the thermal stability of the oxidized species, were compared with those previously determined for the NH2‐terminal core domain fragment 1‐41 or 1‐43 from hirudin. This fragment shows similarity in size, pairing of the three be disulfides and three‐dimensional structure with those of decorsin, even if very low sequence similarity. It is suggested that the less efficient oxidative folding and the enhanced thermal stability of decorsin in respect to those of hirudin core domain likely can be ascribed to the presence of the six Pro residues in the decorsin chain, whereas none is present in the hirudin domain. The results of this study indicate that decorsin can be obtained by solid‐phase methodology in purity and quantities suitable for structural and functional studies and thus open the way to prepare by chemical methods novel decorsin derivatives containing unusual amino acids or even non‐peptidic moieties.
Stability and folding properties of a model β‐sheet protein, Escherichia coli CspA
Authors: Reid Katherine L. , Rodriguez Hector M. , Hillier Brian J. , Gregoret Lydia M.
Published: July 2010

Although β‐sheets represent a sizable fraction of the secondary structure found in proteins, the forces guiding the formation of β‐sheets are still not well understood. Here we examine the folding of a small, all β‐sheet protein, the E. coli major cold shock protein CspA, using both equilibrium and kinetic methods. The equilibrium denaturation of CspA is reversible and displays a single transition between folded and unfolded states. The kinetic traces of the unfolding and refolding of CspA studied by stopped‐flow fluorescence spectroscopy are monoexponential and thus also consistent with a two‐state model. In the absence of denaturant, CspA refolds very fast with a time constant of 5 ms. The unfolding of CspA is also rapid, and at urea concentrations above the denaturation midpoint, the rate of unfolding is largely independent of urea concentration. This suggests that the transition state ensemble more closely resembles the native state in terms of solvent accessibility than the denatured state. Based on the model of a compact transition state and on an unusual structural feature of CspA, a solvent‐exposed cluster of aromatic side chains, we propose a novel folding mechanism for CspA. We have also investigated the possible complications that may arise from attaching polyhistidine affinity tags to the carboxy and amino termini of CspA.
The importance of dynamic light scattering in obtaining multiple crystal forms of trypanosoma brucei PGK
Authors: Bernstein Bradley E. , Michels Paul A. M. , Kim Hidong , Petra Philip H. , Hol Wim G. J.
Published: July 2010

Phosphoglycerate kinase (PGK) catalyzes the phosphoryl transfer between 1,3 bis‐phosphoglycerate and ADP to form 3‐phosphoglycerate and ATP, undergoing significant conformational changes during catalysis. To more precisely document this reaction and the corresponding conformational changes, we have crystallized Trypanosoma brucei PGK in several crystal forms: (1) in the presence of 3‐phosphoglycerate and MgADP, PGK crystallizes with four molecules in the asymmetric unit; (2) in the presence of the ATP analog, AMP‐PNP, PGK crystallizes in a similar form; (3) in the presence of the bisubstrate analog, adenylyl 1,1,5,5‐tetrafluoropentane‐l,5‐bisphosphonate, PGK crystals grow with one molecule in the asymmetric unit. Large scale expression and purification of T. brucei PGK from an E. coli overexpression system was required to obtain sufficient enzyme yields. Results from dynamic light scattering experiments allowed us to identify substrates and analogs which were amenable for crystallization. Ease of crystal growth and diffraction quality for a particular PGK‐ligand complex is highly consistent with the apparent monodispersity of the complex in solution as judged by dynamic light scattering. The three‐dimensional structures of the various enzyme‐ligand complexes are currently being exploited to obtain a better understanding of PGK catalysis, as well as for structure based design of enzyme inhibitors to be used in the development of anti‐trypanosomal agents.
Mechanism and evolution of protein dimerization
Authors: Nussinov Ruth , Xu Dong , Tsai Chung‐Jung
Published: July 2010

We have investigated the mechanism and the evolutionary pathway of protein dimerization through analysis of experimental structures of dimers. We propose that the evolution of dimers may have multiple pathways, including (1) formation of a functional dimer directly without going through an ancestor monomer, (2) formation of a stable monomer as an intermediate followed by mutations of its surface residues, and (3), a domain swapping mechanism, replacing one segment in a monomer by an equivalent segment from an identical chain in the dimer. Some of the dimers which are governed by a domain swapping mechanism may have evolved at an earlier stage of evolution via the second mechanism. Here, we follow the theory that the kinetic pathway reflects the evolutionary pathway. We analyze the structure‐kinetics‐evolution relationship for a collection of symmetric homodimers classified into three groups: (1)14 dimers, which were referred to as domain swapping dimers in the literature; (2) nine 2‐state dimers, which have no measurable intermediates in equilibrium denaturation; and (3), eight 3‐state dimers, which have stable intermediates in equilibrium denaturation. The analysis consists of the following stages: (i) The dimer is divided into two structural units, which have twofold symmetry. Each unit contains a contiguous segment from one polypeptide chain of the dimer, and its complementary contiguous segment from the other chain, (ii) The division is repeated progressively, with different combinations of the two segments in each unit. (iii) The coefficient of compactness is calculated for the units in all divisions. The coefficients obtained for different cuttings of a dimer form a compactness profile. The profile probes the structural organization of the two chains in a dimer and the stability of the monomelic state. We describe the features of the compactness profiles in each of the three dimer groups. The profiles identify the swapping segments in domain swapping dimers, and can usually predict whether a dimer has domain swapping. The kinetics of dimerization indicates that some dimers which have been assigned in the literature as domain swapping cases, dimerize through the 2‐state kinetics, rather than through swapping segments of performed monomers. The compactness profiles indicate a wide spectrum in the kinetics of dimerization: dimers having no intermediate stable monomers; dimers having an intermediate with a stable monomer structure; and dimers having an intermediate with a stable structure in part of the monomer. These correspond to the multiple evolutionary pathways for dimer formation. The evolutionary mechanisms proposed here for dimers are applicable to other oligomers as well.
Crystallization of phycoerythrin 545 of Rhodomonas lens using detergents and unusual additives
Authors: Becker Michael , Stubbs Milton T. , Huber Robert
Published: July 2010

Phycoerythrin 545 from the cryptomonad alga, Rhodomonas lens, has been crystallized under a wide variety of conditions. Although this type of photosynthetic light‐harvesting protein is water soluble, detergents were always required for crystallization. The crystals were typically poorly ordered, or ordered in only two dimensions. However, crystals that were well‐ordered in three dimensions could be obtained under two different conditions. Both used polyethylene glycol as precipitant and the detergent lauryldimethylaminoxide, but the additives that were critical for obtaining well‐ordered crystals were propionamide in one case and Cs+/Br− in the other. Crystals obtained in the presence of propionamide have the space group P212121, with cell constants of a = 85.6 Å, b = 108.2 Å, and c = 131.0 Å, and contain two dimers [i.e., 2 × (α2β2)] in the asymmetric unit. They show diffraction to at least 3.0 Å resolution. The crystals grown with Cs+/Br− are nearly isomorphous. Both types of crystals show intense, strongly polarized fluorescence, suggesting that energy transfer in the crystals is highly efficient. This should provide a basis for quantitative investigation of the role of exciton interactions in energy transfer in cryptomonad phycobiliproteins.
A proposed architecture for lecithin cholesterol acyl transferase (LCAT): Identification of the catalytic triad and molecular modeling
Authors: Peelman F. , Vinaimont N. , Vanloo B. , Labeur C. , Rosseneu M. , Verhee A. , Verschelde J‐L. , Vanderckove J. , Tavernier J. , Seguret‐Mace S. , Duverger N. , Hutchinson G.
Published: July 2010

The enzyme cholesterol lecithin acyl transferase (LCAT) shares the Ser/Asp‐Glu/His triad with lipases, esterases and proteases, but the low level of sequence homology between LCAT and these enzymes did not allow for the LCAT fold to be identified yet. We, therefore, relied upon structural homology calculations using threading methods based on alignment of the sequence against a library of solved three‐dimensional protein structures, for prediction of the LCAT fold. We propose that LCAT, like lipases, belongs to the α/β hydrolase fold family, and that the central domain of LCAT consists of seven conserved parallel beta‐strands connected by four α‐helices and separated by loops. We used the conserved features of this protein fold for the prediction of functional domains in LCAT, and carried out site‐directed mutagenesis for the localization of the active site residues. The wild‐type enzyme and mutants were expressed in Cos‐1 cells. LCAT mass was measured by ELISA, and enzymatic activity was measured on recombinant HDL, on LDL and on a monomelic substrate. We identified D345 and H377 as the catalytic residues of LCAT, together with F103 and L182 as the oxyanion hole residues. In analogy with lipases, we further propose that a potential “lid” domain at residues 50‐74 of LCAT might be involved in the enzyme‐substrate interaction. Molecular modeling of human LCAT was carried out using human pancreatic and Candida antarctica lipases as templates. The three‐dimensional model proposed here is compatible with the position of natural mutants for either LCAT deficiency or Fish‐eye disease. It enables moreover prediction of the LCAT domains involved in the interaction with the phospholipid and cholesterol substrates.
Comparisons between the structures of HCV and Rep helicases reveal structural similarities between SF1 and SF2 super‐families of helicases
Authors: Korolev Sergey , Lohman Timothy M. , Waksman Gabriel , Yao Nanhua , Weber Patricia C.
Published: July 2010

Three helicase structures have been determined recently: that of the DNA helicase PcrA, that of the hepatitis C virus RNA helicase, and that of the Escherichia coli DNA helicase Rep. PcrA and Rep belong to the same super‐family of helicases (SF1) and are structurally very similar. In contrast, the HCV helicase belongs to a different super‐family of helicases, SF2, and shows little sequence homology with the PcrA/Rep helicases. Yet, the HCV helicase is structurally similar to Rep/PcrA, suggesting preservation of structural scaffolds and relationships between helicase motifs across these two super‐families. The comparison study presented here also reveals the existence of a new helicase motif in the SF1 family of helicases.
Spider minor ampullate silk proteins contain new repetitive sequences and highly conserved non‐silk‐like “spacer regions”
Authors: Colgin Mark A. , Lewis Randolph V.
Published: July 2010

Spider minor ampullate silk is a strong non‐elastic deformably stretchable silk used in web formation. This silk from Nephila clavipes is composed of two proteins, MiSp 1 and 2, whose transcripts are 9.5 and 7.5 kb, respectively, as determined by northern blots. Both MiSp proteins are organized into a predominantly repetitive region and a small nonrepetitive carboxy terminal region. These highly repetitive regions are composed mainly of glycine and alanine, but also contain tyrosine, glutamine, and arginine. The sequences are mainly GGX and GA repeats. The repetitive regions are interrupted by nonrepetitive serine‐rich spacer regions. Although the sequences of the spacer regions differ from the repetitive regions, sequences of the spacers from different regions of the proteins are nearly identical. The sequence differences between major and minor ampullate silks may explain the differing mechanical properties of the fibers.
Multiple catalytic roles of His 287 of Rhodospirillum rubrum ribulose 1,5‐bisphosphate carboxylase/oxygenase
Authors: Harpel Mark R. , Larimer Frank W. , Hartman Fred C.
Published: July 2010

Active‐site His 287 of Rhodospirillum rubrum ribulose 1,5‐bisphosphate (RuBP) carboxylase/oxygenase interacts with the C3‐hydroxyl of bound substrate or reaction‐intermediate analogue (CABP), water molecules, and ligands for the activator metal‐ion (Andersson I, 1996, J Mol Biol 259:160‐174; Taylor TC, Andersson I, 1997, J Mol Biol 265:432‐444). To test structure‐based postulates of catalytic functionality, His 287 was replaced with Asn or Gln. The mutants are not affected adversely in subunit assembly, activation (binding of Mg2+ and carbamylation of Lys 191), or recognition of phosphorylated ligands; they bind CABP with even greater tenacity than does wild‐type enzyme. H287N and H287Q are severely impaired in catalyzing overall carboxylation (∼103‐fold and >105‐fold, respectively) and enolization (each mutant below threshold for detection) of RuBP. H287N preferentially catalyzes decarboxylation of carboxylated reaction intermediate instead of forward processing to phosphoglycerate. Analysis of RuBP turnover that occurs at high concentrations of mutants over extended time periods reveal > 10‐fold reduced CO2/O2 specificities, elevated misprotonation of the enediol intermediate, and misprocessing of the oxygenated intermediate of the oxygenase pathway. These results are consistent with multifaceted roles for His 287 in promoting enediol formation, enediol tautomerization, and forward‐processing of carboxylated intermediate.
Determinants of protein hydrogen exchange studied in equine cytochrome c
Authors: Milne J.S. , Mayne L. , Roder H. , Wand A.J. , Englander S. W.
Published: July 2010

The exchange of a large number of amide hydrogens in oxidized equine cytochrome c was measured by NMR and compared with structural parameters. Hydrogens known to exchange through local structural fluctuations and through larger unfolding reactions were separately considered. All hydrogens protected from exchange by factors greater than 103 are in defined H‐bonds, and almost all H‐bonded hydrogens including those at the protein surface were measured to exchange slowly. H‐exchange rates do not correlate with H‐bond strength (length) or crystallographic B factors. It appears that the transient structural fluctuation necessary to bring an exchangeable hydrogen into H‐bonding contact with the H‐exchange catalyst (OH−‐ion) involves a fairly large separation of the H‐bond donor and acceptor, several angstroms at least, and therefore depends on the relative resistance to distortion of immediately neighboring structure. Accordingly, H‐exchange by way of local fluctuational pathways tends to be very slow for hydrogens that are neighbored by tightly anchored structure and for hydrogens that are well buried. The slowing of buried hydrogens may also reflect the need for additional motions that allow solvent access once the protecting H‐bond is separated, although it is noteworthy that burial in a protein like cytochrome c does not exceed 4 Å. When local fluctuational pathways are very slow, exchange can become dominated by a different category of larger, cooperative, segmental unfolding reactions reaching up to global unfolding.
Structural and functional characterization of recombinant human cellular retinaldehyde‐binding protein
Authors: Crabb John W. , Chen Yang , Goldflam Steve , Intres Richard , West Karen A. , Hulmes Jeffery D. , Kapron James T. , Luck Linda A. , Carlson Anne , Bok Dean , Horwitz Joseph
Published: July 2010

Cellular retinaldehyde‐binding protein (CRALBP) is abundant in the retinal pigment epithelium (RPE) and Miiller cells of the retina where it is thought to function in retinoid metabolism and visual pigment regeneration. The protein carries 11‐cis‐retinal and/or 11‐cis‐retinol as endogenous ligands in the RPE and retina and mutations in human CRALBP that destroy retinoid binding functionality have been linked to autosomal recessive retinitis pigmentosa. CRALBP is also present in brain without endogenous retinoids, suggesting other ligands and physiological roles exist for the protein. Human recombinant cellular retinaldehyde‐binding protein (rCRALBP) has been over expressed as non‐fusion and fusion proteins in Escherichia coli from pET3a and pET19b vectors, respectively. The recombinant proteins typically constitute 15‐20% of the soluble bacterial lysate protein and after purification, yield about 3‐8 mg per liter of bacterial culture. Liquid chromatography electrospray mass spectrometry, amino acid analysis, and Edman degradation were used to demonstrate that rCRALBP exhibits the correct primary structure and mass. Circular dichroism, retinoid HPLC, UV‐visible absorption spectroscopy, and solution state 19F‐NMR were used to characterize the secondary structure and retinoid binding properties of rCRALBP. Human rCRALBP appears virtually identical to bovine retinal CRALBP in terms of secondary structure, thermal stability, and stereoselective retinoid‐binding properties. Ligand‐dependent conformational changes appear to influence a newly detected difference in the bathochromic shift exhibited by bovine and human CRALBP when complexed with 9‐cis‐retinal. These recombinant preparations provide valid models for human CRALBP structure‐function studies.
Mass spectrometric analysis of integral membrane proteins: Application to complete mapping of bacteriorhodopsins and rhodopsin
Authors: Wang Jianyao , Ball Lauren E. , Oatis John E. , Dharmasiri Kuruppu , Busman Mark , Cowden Lloraine B. , Galijatovic Alema , Knapp Daniel R. , Chen Ning , Crouch Rosalie K.
Published: July 2010

Integral membrane proteins have not been readily amenable to the general methods developed for mass spectrometric (or internal Edman degradation) analysis of soluble proteins. We present here a sample preparation method and high performance liquid chromatography (HPLC) separation system which permits online HPLC‐electrospray ionization mass spectrometry (ESI‐MS) and ‐tandem mass spectrometry (MS/MS) analysis of cyanogen bromide cleavage fragments of integral membrane proteins. This method has been applied to wild type (WT) bacteriorhodopsin (bR), cysteine containing mutants of bR, and the prototypical G‐protein coupled receptor, rhodopsin (Rh).

In the described method, the protein is reduced and the cysteine residues pyridylethylated prior to separating the protein from the membrane. Following delipidation, the pyridylethylated protein is cleaved with cyanogen bromide. The cleavage fragments are separated by reversed phase HPLC using an isopropanol/acetonitrile/aqueous TFA solvent system and the effluent peptides analyzed online with a Finnigan LCQ Ion Trap Mass Spectrometer. With the exception of single amino acid fragments and the glycosylated fragment of Rh, which is observable by matrix assisted laser desorption ionization (MALDI)‐MS, this system permits analysis of the entire protein in a single HPLC run. This methodology will enable pursuit of chemical modification and crosslinking studies designed to probe the three dimensional structures and functional conformational changes in these proteins. The approach should also be generally applicable to analysis of other integral membrane proteins.
The N‐terminal segment of antithrombin acts as a steric gate for the binding of heparin
Authors: Fitton Hazel L. , Skinner Richard , Dafforn Timothy R. , Jin Lei , Pike Robert N.
Published: July 2010

The binding of heparin causes a conformational change in antithrombin to give an increased heparin binding affinity and activate the inhibition of thrombin and factor Xa. The areas of antithrombin involved in binding heparin and stabilizing the interaction in the high‐affinity form have been partially resolved through the study of both recombinant and natural variants. The role of a section of the N‐terminal segment of antithrombin, residues 22‐46 (segment 22‐46), in heparin binding was investigated using rapid kinetic analysis of the protein cleaved at residues 29‐30 by limited proteolysis with thermolysin. The cleaved antithrombin had 5.5‐fold lowered affinity for heparin pentasaccharide and 1.8‐fold for full‐length, high‐affinity heparin. It was shown that, although the initial binding of heparin is slightly enhanced by the cleavage, it dissociates much faster from the cleaved form, giving rise to the overall decrease in heparin affinity. This implies that the segment constituting residues 22‐46 in the N terminus of antithrombin hinders access to the binding site for heparin, hence the increased initial binding for the cleaved form, whereas, when heparin is bound, segment 22‐46 is involved in the stabilization of the binding interaction, as indicated by the increased dissociation constant. When the heparin pentasaccharide is bound to antithrombin prior to incubation with thermolysin, it protects the N‐terminal cleavage site, implying that segment 22‐46 moves to interact with heparin in the conformational change and thus stabilizes the complex.
Crystal structure of recombinant human tissue kallikrein at 2.0 Å resolution
Authors: Katz Bradley A. , Liu Beishan , Barnes Micael , Springman Eric B.
Published: July 2010

Human tissue kallikrein, a trypsin‐like serine protease involved in blood pressure regulation and inflammation processes, was expressed in a deglycosylated form at high levels inPichia pastoris, purified, and crystallized. The crystal structure at 2.0 Å resolution is described and compared with that of porcine kallikrein and of other trypsin‐like proteases. The active and S1 sites (nomenclature of Schechter I, Berger A, 1967,Biochem Biophys Res Commun 27:157‐162) are similar to those of porcine kallikrein. Compared to trypsin, the S1 site is enlarged owing to the insertion of an additional residue,cis‐Pro 219. The replacement Tyr 228 → Ala further enlarges the S1 pocket. However, the replacement of Gly 226 in trypsin with Ser in human tissue kallikrein restricts accessibility of substrates and inhibitors to Asp 189 at the base of the S1 pocket; there is a hydrogen bond between Oδ1Asp189and Oγser226. These changes in the architecture of the S1 site perturb the binding of inhibitors or substrates from the modes determined or inferred for trypsin. The crystal structure gives insight into the structural differences responsible for changes in specificity in human tissue kallikrein compared with other trypsin‐like proteases, and into the structural basis for the unusual specificity of human tissue kallikrein in cleaving both an Arg‐Ser and a Met‐Lys peptide bond in its natural protein substrate, kininogen. A Zn+2‐dependent, small‐molecule competitive inhibitor of kallikrein (Ki= 3.3 μM) has been identified and the bound structure modeled to guide drug design.
The BPI/LBP family of proteins: A structural analysis of conserved regions
Authors: Beamer Lesa J. , Carroll Stephen F. , Eisenberg David
Published: July 2010

Two related mammalian proteins, bactericidal/permeability‐increasing protein (BPI) and lipopolysaccharide‐binding protein (LBP), share high‐affinity binding to lipopolysaccharide (LPS), a glycolipid found in the outer membrane of Gram‐negative bacteria. The recently determined crystal structure of human BPI permits a structure/function analysis, presented here, of the conserved regions of these two proteins sequences. In the seven known sequences of BPI and LBP, 102 residues are completely conserved and may be classified in terms of location, side‐chain chemistry, and interactions with other residues. We find that the most highly conserved regions lie at the interfaces between the tertiary structural elements that help create two apolar lipid‐binding pockets. Most of the conserved polar and charged residues appear to be involved in inter‐residue interactions such as H‐bonding. However, in both BPI and LBP a subset of conserved residues with positive charge (lysines 42, 48, 92, 95, and 99 of BPI) have no apparent structural role. These residues cluster at the tip of the NH2‐terminal domain, and several coincide with residues known to affect LPS binding; thus, it seems likely that these residues make electrostatic interactions with negatively charged groups of LPS. Overall differences in charge and electrostatic potential between BPI and LBP suggest that BPI's bactericidal activity is related to the high positive charge of its NH2‐terminal domain. A model of human LBP derived from the BPI structure provides a rational basis for future experiments, such as site‐directed mutagenesis and inhibitor design.
NMR determination of the global structure of the 113Cd derivative of desulforedoxin: Investigation of the hydrogen bonding pattern at the metal center
Authors: Goodfellow B.J. , Moura I. , Moura J.J.G. , Rusnak F. , Domke T.
Published: July 2010

Desulforedoxin (Dx) is a simple homodimeric protein isolated from Desulfovibrio gigas (Dg) containing a distorted rubredoxin‐like center with one iron coordinated by four cysteinyl residues (7.9 kDa with 36 amino acids per monomer). In order to probe the geometry and the H‐bonding at the active site of Dx, the protein was reconstituted with 113Cd and the solution structure determined using 2D NMR methods. The structure of this derivative was initially compared with the NMR solution structure of the Zn form (Goodfellow BJ et al., 1996, J Biol Inorg Chem 1:341‐353). Backbone amide protons for G4, D5, G13, L11 NH, and the Q14 NH side‐chain protons, H‐bonded in the X‐ray structure, were readily exchanged with solvent. Chemical shift differences observed for amide protons near the metal center confirm the H‐bonding pattern seen in the X‐ray model (Archer M et al., 1995, J Mol Biol 251:690‐702) and also suggest that H‐bond lengths may vary between the Fe, Zn, and 113Cd forms. The H‐bonding pattern was further probed using a heteronuclear spin echo difference (HSED) experiment; the results confirm the presence of NH‐S H‐bonds inferred from D2O exchange data and observed in the NMR family of structures. The presence of “H‐bond mediated” coupling in Dx indicates that the NH‐S H‐bonds at the metal center have significant covalent character. The HSED experiment also identified an intermonomer “through space” coupling for one of the L26 methyl groups, indicating its proximity to the 113Cd center in the opposing monomer. This is the first example of an intermonomer “through space” coupling. Initial structure calculations produced subsets of NMR families with the S of C28 pointing away from or toward the L26 methyl: only the subset with the C28 sulfur pointing toward the L26 methyl could result in a “through space” coupling. The HSED result was therefore included in the structure calculations. Comparison of the Fe, Zn, and 113Cd forms of Dx suggests that the geometry of the metal center and the global fold of the protein does not vary to any great extent, although the H‐bond network varies slightly when Cd is introduced. The similarity between the H‐bonding pattern seen at the metal center in Dx, Rd (including H‐bonded and through space‐mediated coupling), and many zinc‐finger proteins suggests that these H‐bonds are structurally vital for stabilization of the metal centers in these proteins.