Differential effects of zinc on functionally distinct human growth hormone mutations

Human growth hormone (hGH) binds and activates lactogenic receptorsby a sequential receptor dimerization mechanism. The affinityfor the first lactogenic receptor is increased due to one zincmolecule linking hGH residues H18 and E174, located in helices1 and 4, respectively, with two adjacent residues in the lactogenicreceptor (D187 and H188). Two functionally unique groups ofmutant hGHs have been identified. Addition of 25 µM zincto lactogenic bioassays differentially affects mutant activitiesbased on which group they belong to. One mutation (G120R) islocated within the binding surface of hGH that interacts withthe second lactogenic receptor. In the presence of endogenouszinc, G120R reduces the maximal activity of hGH without alteringeither the agonist or antagonist phases of the bell-shaped dose–responsecurve. Addition of zinc to this assay further reduces the activityof this protein. In contrast, mutations within a hydrophobicmotif in hGH that functionally couples the two lactogenic receptorbinding sites decrease the sensitivity (right-shift) of theagonist phase of the dose–response curve without similarlyaffecting the antagonist phase. The addition of zinc to theselactogenic assays increases the sensitivity (left-shifts) ofthe dose–response curves, largely negating the effectof these mutations. The effects of zinc differentiate betweenmutations within these two distinct functional motifs by limitingthe pool of potential conformations that are available for bindingwithin either of the receptor binding sites of this ligand. Received April 10, 2003; revised June 7, 2003; accepted June 8, 2003.     

Vicinal disulfide turns

The formation of a disulfide bond between adjacent cysteineresidues is accompanied by the formation of a tight turn ofthe protein backbone. In nearly 90% of the structures analyzeda type VIII turn was found. The peptide bond between the twocysteines is in a distorted trans conformation, the omega torsionangle ranges from 159 to –133°, with an average valueof 171°. The constrained nature of the vicinal disulfideturn and the pronounced difference observed between the oxidizedand reduced states, suggests that vicinal disulfides may beemployed as a ‘redox-activated’ conformational switch. Received December 16, 2002; revised June 30, 2003; accepted July 30, 2003.     

A grafting approach to obtain site-specific metal-binding properties of EF-hand proteins

The EF-hand calcium-binding loop III from calmodulin was insertedwith glycine linkers into the scaffold protein CD2.D1 at threelocations to study site-specific calcium binding propertiesof EF-hand motifs. After insertion, the host protein retainsits native structure and forms a 1:1 metal–protein complexfor calcium and its analog, lanthanum. Tyrosine-sensitized Tb³⁺energy transfer exhibits metal binding and La³⁺ and Ca²⁺ competefor the metal binding site. The grafted EF-loop III in differentenvironments has similar La³⁺ binding affinities, suggestingthat it is largely solvated and functions independently fromthe host protein. Received May 25, 2002; revised January 23, 2003; accepted April 25, 2003.     

Fine mapping and structural analysis of immunodominant IgE allergenic epitopes in chicken egg ovalbumin

Ovalbumin is a major allergen in hen egg white that causes IgE-mediatedfood allergic reactions in children. In this study, the immunodominantIgE-binding epitopes of ovalbumin were mapped using arrays ofoverlapping peptides synthesized on activated cellulose membranes.Pooled human sera from 18 patients with egg allergy were usedto probe the membrane. Five distinct regions were found to containdominant allergic IgE epitopes, these being L38T49, D95A102,E191V200, V243E248 and G251N260. The critical amino acids involvedin IgE antibody binding were also determined. These epitopeswere composed primarily of hydrophobic amino acids, followedby polar and charged residues and being comprised of ß-sheetand ß-turn structures. One epitope, D95A102, consistedof a single -helix. These results provide useful informationon the functional role of amino acid residues to evaluate thestructure–function relationships and structural propertiesof allergic epitopes in ovalbumin. They also provide a strategicapproach for engineering ovalbumin to reduce its allergenicity. Received January 9, 2003; revised April 23, 2003; accepted August 28, 2003.     

Evidence that elongation of the catalytic loop of the Azotobacter vinelandii rhodanese changed selectivity from sulfur- to phosphate-containing substrates

Recent investigations have shown that the rhodanese domains,ubiquitous structural modules which might represent an exampleof conserved structures with possible functional diversity,are structurally related to the catalytic subunit of Cdc25 phosphataseenzymes. The major difference characterizing the active-siteof the Azotobacter vinelandii rhodanese RhdA, with respect tothe closely related Cdc25s (A, B, C), is that in Cdc25 phosphatasesthe active site loop [His–Cys–(X)₅–Arg] isone residue longer than in RhdA [His–Cys–(X)₄–Arg].According to the hypothesis that the length of the RhdA active-siteloop should play a key role in substrate recognition and catalyticactivity, RhdA scaffold was the starting point for producingmutants with single-residue insertion to generate the catalyticloop HCQTHAHR (in RhdA-Ala) and HCQTHSHR (in RhdA-Ser). Analysesof the catalytic performances of the engineered RhdAs revealedthat elongation of the catalytic loop definitely compromisedthe ability to catalyze sulfur transfer reactions, while itgenerated ‘phosphatase’ enzymes able to interactproductively with the artificial substrate 3-O-methylfluoresceinphosphate. Although this study is restricted to an example ofrhodanese modules (RhdA), it provided experimental evidenceof the hypothesis that a specific mutational event (a single-residueinsertion or deletion in the active-site loop) could changethe selectivity from sulfur- to phosphate-containing substrates(or vice versa). Received February 17, 2003; revised May 30, 2003; accepted June 6, 2003.     

Specificity grafting of human antibody frameworks selected from a phage display library: generation of a highly stable humanized anti-CD22 single-chain Fv fragment

A prerequisite for the enrichment of antibodies screened fromphage display libraries is their stable expression on a phageduring multiple selection rounds. Thus, if stringent panningprocedures are employed, selection is simultaneously drivenby antigen affinity, stability and solubility. To take advantageof robust pre-selected scaffolds of such molecules, we graftedsingle-chain Fv (scFv) antibodies, previously isolated froma human phage display library after multiple rounds of in vitropanning on tumor cells, with the specificity of the clinicallyestablished murine monoclonal anti-CD22 antibody RFB4. We showthat a panel of grafted scFvs retained the specificity of themurine monoclonal antibody, bound to the target antigen withhigh affinity (6.4–9.6 nM), and exhibited exceptionalbiophysical stability with retention of 89–93% of theinitial binding activity after 6 days of incubation in humanserum at 37°C. Selection of stable human scaffolds withhigh sequence identity to both the human germline and the rodentframeworks required only a small number of murine residues tobe retained within the human frameworks in order to maintainthe structural integrity of the antigen binding site. We expectthis approach may be applicable for the rapid generation ofhighly stable humanized antibodies with low immunogenic potential. Received June 10, 2003; accepted August 27, 2003.     

On the rotational operators in protein structure simulations

The reduction of the computational complexity of the algorithmsdealing with protein structure analysis and conformation predictionsis of prime importance. One common element in most of thesealgorithms is the process of transforming geometrical informationbetween dihedral angles and Cartesian coordinates of the atomsin the protein using rotational operators. In the literature,the operators used in protein structures are rotation matrices,quaternions in vector and matrix forms and the Rodrigues–Gibbsformula. In the protein structure-related literature, the mostwidely promoted rotational operator is the quaternions operator.In this work, we studied the computational efficiency of themathematical operations of the above rotational operators appliedto protein structures. A similar study applied to protein structureshas not been reported previously. We concluded that the computationalefficiency of these rotational operators applied to proteinchains is different from those reported for other applications(such as mechanical machinery) and the conclusions are not analogous.Rotation matrices are the most efficient mathematical operatorsin the protein chains. We examined our findings in two proteinmolecules: Ab1 tyrosine kinase and heparin-binding growth factor2. We found that the rotation matrix operator has between 2and 187% fewer mathematical operations than the other rotationaloperators. Received June 6, 2003; revised July 17, 2003; accepted August 20, 2003.     

Increased thermal stability against irreversible inactivation of 3-isopropylmalate dehydrogenase induced by decreased van der Waals volume at the subunit interface

We have investigated factors affecting stability at the subunit–subunitinterface of the dimeric enzyme 3-isopropylmalate dehydrogenase(IPMDH) from Bacillus subtilis. Site-directed mutagenesis wasused to replace methionine 256, a key residue in the subunitinteraction, with other amino acids. Thermal stability againstirreversible inactivation of the mutated enzymes was examinedby analyzing the residual activity after heat treatment. Themutations M256V and M256A increased thermostability by 2.0 and6.0°C, respectively, whereas the mutations M256L and M256Ihad no effect. Thermostability of the M256F mutated enzyme was4.0°C lower than that of the wild-type enzyme. To our surprise,increasing the hydrophobicity of residue 256 within the hydrophobiccore of the enzyme resulted in a lower thermal stability. Themutated enzymes showed an inverse correlation between thermostabilityand the volume of the side chain at position 256. Based on theX-ray crystallographic structure of Escherichia coli IPMDH,the environment around M256 in the B.subtilis homolog is predictedto be sterically crowded. These results suggest that Met256prevents favorable packing. Introduction of a smaller aminoacid at position 256 improves the packing and stabilizes thedimeric structure of IPMDH. The van der Waals volume of theamino acid residue at the hydrophobic subunit interface is animportant factor for maintaining the stability of the subunit–subunitinterface and is not always optimized in the mesophilic IPMDHenzyme. Received September 3, 2002; revised June 13, 2003; accepted June 20, 2003.     

Change in the crystal packing of soybean {beta}-amylase mutants substituted at a few surface amino acid residues

In spite of the high similarity of amino acid sequence and three-dimensionalstructure between soybean ß-amylase (SBA) and sweetpotato ß-amylase (SPB), their quaternary structureis quite different, being a monomer in SBA and a tetramer inSPB. Because most of the differences in amino acid sequencesare found in the surface region, we tested the tetramerizationof SBA by examining mutations of residues located at the surface.We designed the SBA tetramer using the SPB tetramer structureas a model and calculating the change of accessible surfacearea (ASA) for each residue in order to select sites for themutation. Two different mutant genes encoding SB3 (D374Y/L481R/P487D)and SB4 (K462S added to SB3), were constructed for expressionin Escherichia coli and the recombinant proteins were purified.They existed as a monomer in solution, but gave completely differentcrystals from the native SBA. The asymmetric unit of the mutantscontains four molecules, while that of native SBA contains one.The interactions of the created interfaces revealed that therewere more intermolecular interactions in the SB3 than in theSB4 tetramer. The substituted charged residues on the surfaceare involved in interactions with adjacent molecules in a differentway, forming a new crystal packing pattern. Received June 9, 2003; revised September 11, 2003; accepted September 16, 2003.     

Chimeric ribonuclease as a source of human adapter protein for targeted drug delivery

Assembled modular complexes for targeted drug delivery can bebased on strong non-covalent interactions between a cargo modulecontaining an adapter protein and a docking tag fused to a targetingprotein. We have recently constructed a completely humanizedadapter/docking tag system based on interactions between 15amino acid (Hu-tag) and 110 amino acid (HuS) fragments of humanribonuclease I (RNase I). Although recombinant HuS can be expressedand refolded into a functionally active form, the purificationprocedure is cumbersome and expensive, and more importantly,it yields a significant proportion of improperly folded proteins.Here we describe engineering, high-yield expression, and purificationof a chimeric bovine/human RNase (BH-RNase) comprising 1–29N-terminal amino acids of bovine ribonuclease A and 30–127amino acids of human RNase I. Unlike RNase I, the chimeric BH-RNasecan be cleaved by either subtilisin or proteinase K betweenA20 and S21, providing a functionally active HuS. The HuS obtainedfrom chimeric BH-RNase differs from wild-type HuS by an N24Tsubstitution; therefore, we have reverted this substitutionby mutating N24 to T24 in BH-RNase. This BH-RNase mutant canalso be cleaved by subtilisin or proteinase K yielding wild-typeHuS. The affinity of HuS obtained from BH-RNase to Hu-tag isapproximately five times higher than that for recombinant HuS,reflecting a higher percentage of properly folded proteins. Received June 9, 2003; revised August 4, 2003; accepted August 28, 2003.     

Structure-based substitutions for increased solubility of a designed protein

Manipulation of protein solubility is important for many aspectsof protein design and engineering. Previously, we designed aseries of consensus ankyrin repeat proteins containing one,two, three and four identical repeats (1ANK, 2ANK, 3ANK and4ANK). These proteins, particularly 4ANK, are intended for useas a universal scaffold on which specific binding sites canbe constructed. Despite being well folded and extremely stable,4ANK is soluble only under acidic conditions. Designing interactionswith naturally occurring proteins requires the designed proteinto be soluble at physiological pH. Substitution of six leucineswith arginine on exposed hydrophobic patches on the surfaceof 4ANK resulted in increased solubility over a large pH range.Study of the pH dependence of stability demonstrated that 4ANKis one of the most stable ankyrin repeat proteins known. Inaddition, analogous leucine to arginine substitutions on thesurface of 2ANK allowed the partially folded protein to assumea fully folded conformation. Our studies indicate that replacementof surface-exposed hydrophobic residues with positively chargedresidues can significantly improve protein solubility at physiologicalpH. Received June 23, 2003; revised August 22, 2003; accepted August 28, 2003.     

Role of the tyrosine corner motif in the stability of neocarzinostatin

Although the immunoglobulin-like ß-sandwich fold hasno specifically conserved function, some common structural featureshave been observed, in particular a structural motif, the tyrosinecorner. Such a motif was described in neocarzinostatin (NCS),a bacterial protein the structure of which is very similar tothat of the immunoglobulin domain. Compared with the other ß-sheetproteins, the NCS ‘tyrosine corner’ presents non-standardstructural features. To investigate the role of this motif inthe NCS structure and stability, we studied the properties ofa mutant where the H bond interaction had been eliminated byreplacing the tyrosine with a phenylalanine. This mutation costs4.0 kcal/mol showing that the NCS ‘tyrosine corner’is involved in protein stability as in the other Greek key proteins.This destabilization is accompanied by remote structural effects,including modification of the binding properties, suggestingan increase in the internal flexibility of the protein. Witha view to using this protein for drug targeting, these resultsalong with those obtained previously allow us to define clearlythe limitations of the modifications that can be performed onthis scaffold. Received December 3, 2002; revised June 6, 2003; accepted September 3, 2003.     

Prediction of protein secondary structure with a reliability score estimated by local sequence clustering

Most algorithms for protein secondary structure prediction arebased on machine learning techniques, e.g. neural networks.Good architectures and learning methods have improved the performancecontinuously. The introduction of profile methods, e.g. PSI-BLAST,has been a major breakthrough in increasing the prediction accuracyto close to 80%. In this paper, a brute-force algorithm is proposedand the reliability of each prediction is estimated by a z-scorebased on local sequence clustering. This algorithm is intendedto perform well for those secondary structures in a proteinwhose formation is mainly dominated by the neighboring sequencesand short-range interactions. A reliability z-score has beendefined to estimate the goodness of a putative cluster foundfor a query sequence in a database. The database for predictionwas constructed by experimentally determined, non-redundantprotein structures with <25% sequence homology, a list maintainedby PDBSELECT. Our test results have shown that this new algorithm,belonging to what is known as nearest neighbor methods, performedvery well within the expectation of previous methods and thatthe reliability z-score as defined was correlated with the reliabilityof prediction. This led to the possibility of making very accuratepredictions for a few selected residues in a protein with anaccuracy measure of Q₃ > 80%. The further development ofthis algorithm, and a nucleation mechanism for protein foldingare suggested. Received March 27, 2003; revised June 30, 2003; accepted August 22, 2003.     

Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution

To expand the functionality of lipase B from Candida antarctica(CALB) we have used directed evolution to create CALB mutantswith improved resistance towards irreversible thermal inactivation.Two mutants, 23G5 and 195F1, were generated with over a 20-foldincrease in half-life at 70°C compared with the wild-typeCALB (WT-CALB). The increase in half-life was attributed toa lower propensity of the mutants to aggregate in the unfoldedstate and to an improved refolding. The first generation mutant,23G5, obtained by error-prone PCR, had two amino acid mutations,V210I and A281E. The second generation mutant, 195F1, derivedfrom 23G5 by error-prone PCR, had one additional mutation, V221D.Amino acid substitutions at positions 221 and 281 were determinedto be critical for lipase stability, while the residue at position210 had only a marginal effect. The catalytic efficiency ofthe mutants with p-nitrophenyl butyrate and 6,8-difluoro-4-methylumbelliferyloctanoate was also found to be superior to that of WT-CALB. Received May 8, 2003; revised June 9, 2003; accepted June 23, 2003.     

The structural roles of conserved Pro196, Pro197 and His199 in the mechanism of thymidylate synthase

We generated replacement sets for three highly conserved residues,Pro196, Pro197 and His199, that flank the catalytic nucleophile,Cys198. Pro196 and Pro197 have restricted mobility that couldbe important for the structural transitions known to be essentialfor activity. To test this hypothesis we obtained and characterized13 amino acid substitutions for Pro196, 14 for Pro197 and 14for His199. All of the Pro196 and Pro197 variants, except P197R,and four of the His199 variants complemented TS-deficient Escherichiacoli cells, indicating they had at least 1% of wild-type activity.For all His199 mutations, k_cat/K_m for substrate and cofactordecreased more than 40-fold, suggesting that the conserved hydrogenbond network co-ordinated by His199 is important for catalysis.Pro196 can be substituted with small hydrophilic residues withlittle loss in k_cat, but 15- to 23-fold increases in K_m^dUMP.Small hydrophobic substitutions for Pro197 were most active,and the most conservative mutant, P197A, had only a 5-fold lowerk_cat/K_m^dUMP than wild-type TS. Several Pro196 and Pro197 variantswere temperature sensitive. The small effects of Pro196 or Pro197mutations on enzyme kinetics suggest that the conformationalrestrictions encoded by the Pro–Pro sequence are largelymaintained when either member of the pair is mutated. Received February 24, 2003; revised June 19, 2003; accepted June 20, 2003.     

Improvement of binding of Puumala virus neutralization site resembling peptide with a second-generation phage library

We have previously selected a peptide insert FPCDRLSGYWERGIPSPCVRrecognizing the Puumala virus (PUUV) G2-glycoprotein-specificneutralizing monoclonal antibody (MAb) 1C9 with K_d of 2.85x10^–8from a random peptide library X₂CX₁₄CX₂ expressed on the pIIIprotein of the filamentous phage fd-tet. We have now createda second-generation phage-displayed peptide library in whicheach amino acid of the peptide was mutated randomly to anotherwith a certain probability. Peptides were selected for higheraffinity for MAb 1C9 and for a common binding motif for MAb4G2 having an overlapping epitope with MAb 1C9 in G2 glycoprotein.The resulting peptides were synthesized as spots on cellulosemembrane. Amino acid changes which improved the reactivity ofthe peptides to MAb 1C9 were combined in the peptide ATCDKLFGYYERGIPLPCALwith K_d of 1.49x10^–9 in biosensor measurements. Our resultsshow that the binding properties of peptides, the affinity andthe specificity can be improved and the binding specificitydetermining amino acids and structural factors can be analyzedby combining binding assays with synthetic peptides on membranewith the use of second-generation phage display libraries. Received October 18, 2002; revised April 16, 2003; accepted May 26, 2003.     

Complex chimeras to map ligand binding sites of GPCRs

Family 1a GPCRs are thought to bind small molecule ligands ina pocket comprising sequences from non-contiguous transmembranehelices. In this study, receptor–ligand binding determinantswere defined by building a series of complex chimeras wheremultiple sequences were exchanged between related G-proteincoupled receptors. Regions of P2Y₁, P2Y₂ and BLT₁ predictedto interact with nucleotide and leukotriene ligands were identifiedand receptors were engineered within their transmembrane helicesto transpose the ligand binding site of one receptor on to anotherreceptor. Ligand-induced activation of chimeras was comparedwith wild-type receptor activation in a yeast reporter geneassay. Binding of ligand to a P2Y₂/BLT₁ chimera confirmed thatthe ligand binding determinants of BLT₁ are located in the upperregions of the helices and extracellular loops of this receptorand that they had been successfully transferred to a receptorthat normally binds unrelated ligands. Received April 20, 2002; revised March 31, 2003; accepted April 8, 2003.     

Immobilized oxidoreductase as an additive for refolding inclusion bodies: application to antibody fragments

Three foldases—the apical domain of GroEL (mini-chaperone)and two oxidoreductases (DsbA and DsbC) from Escherichia coli—werestudied in refolding a protein with immunoglobulin fold (immunoglobulin-foldedprotein) that had been produced as inclusion bodies in E.coli.The foldases promoted the refolding of single-chain antibodyfragments from denaturant-solubilized and reduced inclusionbodies in vitro, and also effectively functioned as alternativesfor labilizing agent and oxidizing reagent in the stepwise dialysissystem. Immobilization of the oxidoreductases enhanced refoldingand recovery of functional single-chain antibody in the dialysissystem, suggesting that immobilized oxidoreductases can be usedas an effective additive for refolding immunoglobulin-foldedproteins in vitro. Received April 7, 2003; revised June 5, 2003; accepted June 6, 2003.     

Structure-oriented rational design of chymotrypsin inhibitor models

Three peptides modelling a highly potent, 35-residue chymotrypsininhibitor (Schistocerca gregaria chymotrypsin inhibitor) weredesigned and synthesized by convergent peptide synthesis. Foreach model peptide, the inhibitory constant (K_i) on chymotrypsinand the solution structure were determined. In addition, moleculardynamics calculations were performed for all of them. Two modelscontaining approximately half of the parent inhibitor (17 of35 residues) were designed and subsequently found to have nosubstantial inhibitory activity (K_i values in the mM range).The third model composed of 24 amino acid residues proved tobe an effective (K_i 10^–7) inhibitor of bovine chymotrypsin.Both the solution structure properties determined by NMR spectroscopyand the dynamic behaviour of the latter model system are comparableto the native inhibitor. In contrast, the structure and dynamicsof the first two related model peptides show characteristicdifferences. We suggest that the conformation and flexibilityof the modelled protease inhibitor are crucial for its biologicalefficiency. Moreover, the structural and dynamic features ofthe binding loop (28–33) and those of the rest of themolecule appear to be interdependent. Most importantly, thesestructural characteristics can be rationally modified, at leastpartially, by peptide design. Received March 7, 2003; revised August 25, 2003; accepted August 26, 2003.     

PROSPECT II: protein structure prediction program for genome-scale applications

A new method for fold recognition is developed and added tothe general protein structure prediction package PROSPECT (http://compbio.ornl.gov/PROSPECT/).The new method (PROSPECT II) has four key features. (i) We havedeveloped an efficient way to utilize the evolutionary informationfor evaluating the threading potentials including singletonand pairwise energies. (ii) We have developed a two-stage threadingstrategy: (a) threading using dynamic programming without consideringthe pairwise energy and (b) fold recognition considering allthe energy terms, including the pairwise energy calculated fromthe dynamic programming threading alignments. (iii) We havedeveloped a combined z-score scheme for fold recognition, whichtakes into consideration the z-scores of each energy term. (iv)Based on the z-scores, we have developed a confidence index,which measures the reliability of a prediction and a possiblestructure–function relationship based on a statisticalanalysis of a large data set consisting of threadings of 600query proteins against the entire FSSP templates. Tests on severalbenchmark sets indicate that the evolutionary information andother new features of PROSPECT II greatly improve the alignmentaccuracy. We also demonstrate that the performance of PROSPECTII on fold recognition is significantly better than any othermethod available at all levels of similarity. Improvement inthe sensitivity of the fold recognition, especially at the superfamilyand fold levels, makes PROSPECT II a reliable and fully automatedprotein structure and function prediction program for genome-scaleapplications. Received March 20, 2003; revised June 28, 2003; accepted July 8, 2003.