Solution conformation of endothelin determined by means of 1H-NMR spectroscopy and distance geometry calculations

The structure of endothelin-1 (ET-1), an endothelial cell-derivedpeptide with vasoconstricting activity, was determined in anaqueous solution by means of a combination of NMR and distancegeometry calculations. The resulting structure is characterizedby an <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-helical conformation in the sequence region, Lys9-Cys15.Furthermore, an extended structure and a turn structure existin the Cys1-Ser4 and Ser5-Asp8 regions respectively, and nopreferred conformation was found for the C-terminal part ofthe peptide which was not uniquely constrained by the NMR data.These structural elements, the <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-helical structure in the sequenceportion, Cys-X-X-X-Cys, and the extended structure in Cys-X-Cys,are homologous to those found commonly in several neurotoxicpeptides.     

The solution structure and conformational dynamics of tumor necrosis factor-{alpha} and a (Cys69 - Asp; Cys101 - Arg) analog as examined by IR spectroscopy and hydrogen exchange

An analog of human tumor necrosis factor-<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0"> (TNF-<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">) was createdwith Cys69 and CyslOl replaced with Asp and Arg respectively.We have undertaken a comparative study of the solution conformationand dynamics of the native and analog molecules using a combinationof Fourier transform IR spectroscopy and hydrogen-deuterium(H-D) exchange kinetics. IR spectroscopic results indicate thatthe analog molecule adopts a gross structure similar to thatof the native molecule but significant differences in the conformationof the ß-sheets are observed. Increased bandwidthsobserved for several of the amide I components also suggesta less rigid structure for the analog molecule. Further, bymonitoring the frequency shifts of the individual amide I componentbands as a function of hydrogen exchange, we have enhanced ourability to assign these components to individual protein secondarystructures, particularly the high frequency ß-strandmode. Hydrogen exchange kinetic studies indicate that the Asp–Arganalog adopts a looser, more flexible solution structure relativeto the natural sequence molecule.     

Chemical synthesis, expression and product assessment of a gene coding for biologically active human tumour necrosis factor {alpha}

A gene encoding human tumour necrosis factor <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0"> (TNF-<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">) has beenchemically synthesized, cloned and expressed to yield a biologicallyactive protein in Escherichia coli. The 480-bp gene was assembledby enzymic ligation of 32 oligonucleotides, cloned directlyinto M13mp18 for sequence verification and expressed in thebroad host range high-level expression vector pMMB66EHST. Expressedrecombinant TNF-<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0"> was shown to have the correct molecular weight,processed N-terminal sequence, antibody cross-reactivity andtumour cell killing activity. The expression product of thesynthetic gene has been purified to homogeneity by a two-stepion-exchange procedure and the purified material shown to beactive.     

Construction, expression, purification and functional analysis of recombinant NF{kappa}B p50/p65 heterodimer

NF<IMG SRC="http://peds.oxfordjournals.org/math/kappa.gif" ALT="{kappa}" BORDER="0">B plays an important role in mediating the gene expressionof numerous cellular processes such as growth, development,the inflammatory response and virus proliferation. The p50/p65heterodimer is the most abundant form of the NF<IMG SRC="http://peds.oxfordjournals.org/math/kappa.gif" ALT="{kappa}" BORDER="0">B dimers andplays a more elaborate role in gene regulation. Biochemicalresearch on p50/p65 NF<IMG SRC="http://peds.oxfordjournals.org/math/kappa.gif" ALT="{kappa}" BORDER="0">B has not benefited however from the availabilityof easily purified recombinant protein. We report two methodsfor the large scale expression and purification of recombinantNF<IMG SRC="http://peds.oxfordjournals.org/math/kappa.gif" ALT="{kappa}" BORDER="0">B p50/p65 heterodimer. The first utilizes a bacterial doubleexpression vector which contains two ribosomal binding sitesto facilitate the coexpression of the polypeptides in the p50/p65NF<IMG SRC="http://peds.oxfordjournals.org/math/kappa.gif" ALT="{kappa}" BORDER="0">B heterodimer. The second method uses a mixed protein refoldingstrategy. Both methods yield crystallizable protein. Electrophoreticmobility shift assays confirm that the DNA binding affinityis independent of the method used to purify the protein. Thesemethods will facilitate the numerous studies on various NF<IMG SRC="http://peds.oxfordjournals.org/math/kappa.gif" ALT="{kappa}" BORDER="0">B/Relfamily members.     

Altered specificities of genetically engineered {alpha}1 antitrypsin variants

Seven active site variants of human <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">₁-antitrypsin (<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">₁AT) wereproduced in Escherichia coli following site-specific mutagenesisof the <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">₁AT complementary DNA. <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">₁AT (Ala ³⁵⁸), <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">₁AT (Ile³⁵⁸ and<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">₁AT (Val³⁵⁸), were efficient inhibitors of both neutrophil andpancreatic elastases, but not of cathepsin G. <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">₁AT (Ala³⁵⁸, Val³⁵⁸)and <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">₁AT (Phe³⁵⁸ specifically inhibited pancreatic elastase andcathepsin G respectively. The most potent inhibitor of neutrophilelastase was <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">₁AT (Leu³⁵⁸), which also proved to be effectiveagainst cathepsin G. The <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">₁AT (Arg³⁵⁸) variant inactivated thrombinwith kinetics similar to antithrombin III in the presence ofheparin. Electrophoretic analysis showed that SDS-stable highmol. wt complexes were formed between the mutant inhibitorsand the cognate proteases in each case. These data indicatethat effective inhibition occurs when the <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">₁AT P1 residue (position358) corresponds to the primary specificity of the target protease.Moreover, alteration of the P3 residue (position 356) can furthermodify the reactivity of the inhibitor. Two of the variantshave therapeutic potential: <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">₁AT (Leu³⁵⁸ may be more useful thanplasma <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">₁AT in the treatment of destructive lung disorders and<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">₁ (Arg³⁵⁸ could be effective in the control of thrombosis.     

Hyperthermostable mutants of Bacillus licheniformis {alpha}-amylase: multiple amino acid replacements and molecular modelling

We have identified previously two critical positions for thethermostability of the highly thermostable <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-amylase from Bacilluslicheniformis. We have now introduced all 19 possible aminoacid residues to these two positions, His 133 and Ala209. Themost favourable substitutions were to Ile and Val, respectively,which both increased the half-life of the enzyme at 80°Cby a factor of <IMG SRC="http://peds.oxfordjournals.org/math/bsim.gif" ALT="{bsim}" BORDER="0">3. At both positions a stabilizing effect ofhydrophobic residues was observed, although only in the caseof position 133 could a clear correlation be drawn between thehydrophobicity of the inserted amino acid and the gain in proteinstability. The construction of double mutants showed a cumulativeeffect of the most favourable and/or deleterious substitutions.Computer modelling was used to generate a 3-D structure of thewild-type protein and to model substitutions at position 209,which lies in the conserved (<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">/ß)₈ barrel domain of<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-amylase; Ala209 would be located at the beginning of the thirdhelix of the barrel, in the bottom of a small cavity facingthe fourth helix. The model suggests that replacement by, forexample, a valine could fill this cavity and therefore increaseintra- and interhelical compactness and hydrophobic interactions.     

Model complexes of tumor necrosis factor-{alpha} with receptors Rl and R2

The biological activities of tumor necrosis factor-<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0"> (TNF-<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">) aremediated by two different receptors, TNFR1 and TNFR2. To analyzethe receptor binding site(s) of TNF-<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">, molecular models havebeen built of the complexes of TNF-<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0"> with the extracellular regionsof receptors Rl and R2, based on the known crystal structuresof TNF-<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0"> and lymphotoxin bound to Rl. The model structure ofR2 from residues 18-160 was built by analogy to the crystalstructure of Rl in complex with lymphotoxin. The amino acidsequences of Rl and R2 show 27.5% identity over this regionand were aligned with five insertions and three deletions. Thereare 18 conserved cysteines that form disulfides. R2 has lostone pair of cysteines compared with Rl, but two new cysteineswere modeled as forming a new disulfide bond. Both symmetricand asymmetric trimers of TNF-<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0"> were used to model the complexeswith TNFR1 and R2. An analysis of differences in the model complexesshowed good agreement with data on the differential bindingof TNF mutants to its two receptors.     

Relationships between sequence and structure for the four-{alpha}-helix bundle tertiary motif in proteins

The sequences of four-<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-helical bundle proteins are characterizedby a pattern of hydrophilic and hydrophobic amino acids whichis repeated every seven residues. At each position of the heptadrepeat there are specific constraints on the amino acid propertieswhich result from the topology of the tertiary motif. Theseconstraints give rise to patterns of amino acid distributionwhich are distinct from those of other proteins. The distributionsin each of the heptad positions have been determined by a statisticalanalysis of structural and sequence data derived from sevenfamines of aligned protein sequences. The constitution of eachposition is dominated by a very small number of different aminoacids, with the core positions consisting overwhelmingly ofLeu and Ala. The positional preferences of the individual aminoacids can be generally interpreted in terms of residue propertiesand topological constraints. The potential for four-a-helixbundle folding is reflected primarily in the pattern of residueoccurrence in the heptad and not in the overall amino acid compositionof the protein. Possible applications of this analysis in structurepredictions, sequence alignments and in the rational designand engineering of four-a-helkal bundle proteins are discussed.     

C-terminal truncations of a thermostable Bacillus stearothermophilus {alpha}-amylase

A series of truncated proteins from a thermostable Bacillusstearothermophilus <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-amylase was prepared to study the importanceof the extension in the C-terminus compared with other liquefyingBacillus <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-amylases. The mutations introducing new translationtermination sites shortened the 515 amino acid residue-longwild type enzyme by 17, 32, 47, 73 or 93 residues. The longerthe truncation, the lower the specific activity of the enzyme.Only the two longest mutant proteins were active: the specificactivity of the 498 residue variant was 97% and protein 483was 36% that of the parental enzyme. The K_m values of starchhydrolysis changed from 1.09 for wild type enzyme to 0.35 and0.21 for mutants 498 and 483, respectively, indicating alteredsubstrate binding. The mutant enzymes had almost identical pHand temperature optima with the wild type amylase, but enhancedthermal stability and altered end product profile. The consequencesof the truncation to the structure and function of the enzymeswere explored with molecular modeling. The liquefying amylasesseem to require {small tilde}480 residues to be active, whereasthe C-terminal end of B.stearothermophilus amylase is requiredfor increased activity.     

Structure-function validation of high lysine analogs of {alpha}-hordothionin designed by protein modeling

Cereal grains and legume seeds, which are key protien sourcesfor the vegetarian diet, are generally deficient in essentialamino acids. Maize, in particular, is deficient in lysine. Theinherent lack of lysine-rich protiens in maize has necessitatedthe search for heterologous protiens enriched in this aminoacid, the isolation of the corresponding gene and its ultimateintroduction into maize through plant transformation techniques.However, a rate-limiting step to this strategy has been theavailability of plant-derived lysine-rich protiens. An appealingsolution to the problem is to artificially increase the lysinecontent of a given protein by mutating appropriate residuesto lysine. Here, we expound this strategy, starting with theprotein <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0"> hordothionin that is derived from barley seeds andconsists of five lysine residues in a total of 45 amino acids(11 % lysine). To facilitate rational substitutions, the 3-Dstructure of the protein has been determined by homology modelingwith crambin. based on this model, we have identified surfaceresidues amenable to substitution with lysine. Furthermore,the acceptability of the mutations has been validated throughthe syntheses and characterization of the derivatives. To thisend, our approach has permitted the creation of a modified <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-hordothionin protein that has a lysine content of 27 % andretains the antifungal activity of the wild-type protein.