Intrahelical side chain-side chain contacts: the consequences of restricted rotameric states and implications for helix engineering and design

Intrahelical side chain–side chain (sc–sc) interactionsare assumed to play a crucial role in the formation and stabilityof -helices, yet it was found that only 37.2% of all helicalresidues are involved in such close contacts, assuming a specificminimum contact distance. The majority (58.0%) of these weredetected between residues with amino acid sequence spacing i,i + 4. The low frequency of intrahelical sc–sc contactswith sequence separations i, i + 1 and ii, i + 3, each observedwith only about one-third of the i, i + 4 counts, can be directlyand generally attributed to the absence of the g^- conformationin helices for the dihedral angle X₁- However, if it was assumedthat each side chain may maximally make only one sc–sccontact, as most commonly observed, the percentage of contactingpairs increased relative to the maximum possible pairs for agiven sequence spacing by a factor of {small tilde}4, e.g. from20.9 to 81.7% for i, i + 4 contacts. Stereochemical reasonsare also given for the observation that i, i + 3 contacts arecomposed largely of ion or polar pairs, while hydrophobic residuesdominate the i, i + 4 contacts. No significantly increased densityof intrahelical sc–sc contacts with increasing helix lengthwas found. Although there were generally fewer intrahelicalcontacts between buried helical residues when more contactswere made to the tertiary protein environment, the number ofintrahelical contacts did not increase with increasing solventexposure of the helices. Implications for helix design and thepacking of helices are discussed.     

A pore-forming protein with a metal-actuated switch

Staphylococcal -hemolysin, a pore-forming exotoxin, is a polypeptideof 293 amino acids that is secreted by Staphylococcus aureusas a water-soluble monomer. It assembles to form hexameric poresin lipid bilayers. Previous studies of pore formation have establishedthe involvement of a central glycine-rich loop. Here, we showthat when five consecutive histidine residues replace aminoacids 130–134 at the midpoint of the loop, they providea switch with which pore activity can be (i) turned off by micromolarconcentrations of divalent zinc ions and (ii) turned back onwith the chelating agent EDTA. Planar bilayer recordings showthat Zn²⁺ and EDTA can act on open channels from either sideof the bilayer and thus demonstrate that the central loop linespart of the conductive pathway. Our results suggest that genetically-engineeredpore-forming proteins might make useful components of metalion sensors     

Geometry of interaction of metal ions with histidine residues in protein structures

An analysis of the geometry and the orientation of metal ionsbound to histidine residues in proteins is presented. Cationsare found to lie in the imidazole plane along the lone pairon the nitrogen atom. Out of the two tautomeric forms of theimidazole ring, the NE2-protonated form is normally preferred.However, when bound to a metal ion the ND1-protonated form ispredominant and NE2 is the ligand atom. When the metal coordinationis through ND1, steric interactions shift the side chain torsionalangle, X₂ from its preferred value of 90 or 270. The orientationof histidine residues is usually stabilized through hydrogenbonding; ND1-protonated form of a helical residue can form ahydrogen bond with the carbonyl oxygen atom in the precedingturn of the helix. A considerable number of ligands are foundin helices and ß-sheets. A helical residue hound toa heme group is usually found near the C-terminus of the helix.Two ligand groups four residues apart in a helix, or two residuesapart in a ß-strand are used in many proteins to bindmetal ions.     

Replacing the ({beta}{alpha})-unit 8 of E.coli TIM with its chicken homologue leads to a stable and active hybrid enzyme

In order to investigate how structural modifications interferewith protein stability, we modified a (ß)-unit inE.coli triosephosphate isomerase (TIM), a typical (ß)-barrelprotein, assuming that the pseudosymmetrical ß-barrelcan be divided into eight successive loop/ß-strand/loop/-helixmotifs. We replaced the eighth (ß)-unit of E.coliTIM with the corresponding chicken (ß)-unit. The substitution,involving the replacement of 10 of the 23 residues of this (ß)-unit, was evaluated first by modelling, then experimentally.Modelling by bomology suggests how the amino add replacementsmight be accommodated in the hybrid E.coli/chicken TIM (ETCM8CHI).Both natural and hybrid recombinant TIMs, overproduced in E.coli,were purified to homogeneity and characterized as to their stabilityand kinetics. Our kinetic studies show that the modificationperformed here leads to an active enzyme. The stability studiesindicate that the stability of ETIM8CHI is comparable to thatof the wild type TIM.     

Context dependence of phenotype prediction and diversity in combinatorial mutagenesis

Two different combinatorial mutagenesis experiments on the light-harvestingII (LH2) protein of Rhodobacter capsulatus indicate that heuristicrules relating sequence directly to phenotype are dependenton which sets or groups of residues are mutated simultaneously.Previously reported combinatorial mutagenesis of this chromogenicprotein (based on both phylogenetic and structural models) showedthat substituting amino acids with large molar volumes at Gly^ß31caused the mutated protein to have a spectrum characteristicof light-harvesting I (LH1). The six residues that underwentcombinatorial mutagenesis were modeled to lie on one side ofa transmembrane -helix that binds bacteriochlorophyll. In asecond experiment described here, we have not used structuralmodels or phylogeny in choosing mutagenesis sites. Instead,a set of six contiguous residues was selected for combinatorialmutagenesis. In this latter experiment, the residue substitutedat Gly^ß31 was not a determining factor in whetherLH2 or LH1 spectra were obtained; therefore, we conclude thatthe heuristic rules for phenotype prediction are context dependent.While phenotype prediction is context dependent, the abilityto identify elements of primary structure causing phenotypediversity appears not to be. This strengthens the argument forperforming combinatorial mutagenesis with an arbitrary groupingof residues if structural models are unavailable.     

The 3.0 A crystal structure of xylose isomerase from Streptomyces olivochromogenes

The crystal structure of xylose isomerase [E.C. 5.3.1.5 [EC] ] fromStreptomyces olivochromogenes has been determined to 3.0 Åresolution. The crystals belong to space group P22₁2₁ with unitcell parameters a = 98.7, b = 93.9, c = 87.7. The asymmetricunit contains half of a tetrameric molecule of 222 symmetry.The two-fold axis relating the two molecules in the asymmetricunit is close to where a crystallographic two-fold would beif the space group were 1222. This causes the diffraction patternto have strong 1222 pseudo-symmetry, so all data were collectedin this pseudo-space group. Since the sequence of this enzymehas not been reported, a polyalanine backbone has been fittedto the electron density. Xylose isomerase has two domains: theN-terminal domain is an eight-stranded /ß barrel of299 residues. The C-terminal domain is a large loop of 50 residueswhich is involved in inter-molecular contacts. Comparison ofxylose isomerase with the archetypical /ß barrel protein,triose phosphate isomerase, reveals that the proteins overlapbest when the third (ß) strand of xylose isomeraseis superimposed on the first (ß) strand of triosephosphate isomerase. This same overlap has also been found betweenthe muconate lactonising enzyme and triose phosphate isomerase[Goldman et al. (1987) J. Mol. Biol., in press].     

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

A series of truncated proteins from a thermostable Bacillusstearothermophilus -amylase was prepared to study the importanceof the extension in the C-terminus compared with other liquefyingBacillus -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.     

Effects of amino acid substitutions in the hydrophobic core of {alpha}-lactalbumin on the stability of the molten globule state

Five mutant –lactalbumins, with one or two amino acidsubstitution(s) in the B helix, were engineered to examine therelation between the stability of the molten globule state andthe hydrophobicity of these amino acids. The mutation sites(Thr29, Ala30 and Thr33) have been chosen on the basis of comparisonof the amino acid sequences of goat, bovine and gunea pig –lactalbumin,in which the guinea pig protein shows a remarkably more stablemolten globule than the other proteins. The recombinant proteinswere expressed Escherichia coli and then purified and refoldedefficiently to produce the active proteins. The stability ofthe molten globule state of these engineered proteins has beeninvestigated by urea–induced unfolding transition underan acidic condition (pH 2.0), where the molten globule stateis stable in the absence of urea. The results show that themolten globule state is stabilized by the amino acid substitutionswhich raise the hydrophobicity of the residues, suggesting thatthe hydrophobic core in a globular protein plays an importantrole in the stability of the molten globule state. The changein stabilization free energy of the molten globule state causedby each amino acid substitution has been evaluated, and molecularmechanisms of stabilization of the molten globule state arediscussed.     

Deterministic features of side-chain main-chain hydrogen bonds in globular protein structures

A total of 19 835 polar residues from a data set of 250 non-homologousand highly resolved protein crystal structures were used toidentify side-chain main-chain (SC-MC) hydrogen bonds. The ratioof the number of SC-MC hydrogen bonds to the total number ofpolar residues is close to 1:2, indicating the ubiquitous natureof such hydrogen bonds. Close to 56% of the SC-MC hydrogen bondsare local involving side-chain acceptor/donor (`i') and a main-chaindonor/acceptor within the window i–5 to i+5. These short-rangehydrogen bonds form well defined conformational motifs characterizedby specific combinations of backbone and side-chain torsionangles. (a) The Ser/Thr residues show the greatest preferencein forming intra-helical hydrogen bonds between the atoms O_iand O_i–4. More than half the examples of such hydrogenbonds are found at the middle of -helices rather than at theirends. The most favoured motif of these examples is _RRRR(g^–).(b) These residues also show great preference to form hydrogenbonds between O_i and O_i–3, which are closely related tothe previous type and though intra-helical, these hydrogen bondsare more often found at the C-termini of helices than at themiddle. The motif represented by _RRRR(g⁺) is most preferredin these cases. (c) The Ser, Thr and Glu are the most frequentlyfound residues participating in intra-residue hydrogen bonds(between the side-chain and main-chain of the same residue)which are characterized by specific motifs of the form ß(g⁺)for Ser/Thr residues and _R(g^–g⁺t) for Glu/Gln. (d) Theside-chain acceptor atoms of Asn/Asp and Ser/Thr residues showhigh preference to form hydrogen bonds with acceptors two residuesahead in the chain, which are characterized by the motifs ß (tt')Rand ß(t)_R, respectively. These hydrogen bonded segments,referred to as Asx turns, are known to provide stability totype I and type I' ß-turns. (e) Ser/Thr residues oftenform a combination of SC-MC hydrogen bonds, with the side-chaindonor hydrogen bonded to the carbonyl oxygen of its own peptidebackbone and the side-chain acceptor hydrogen bonded to an amidehydrogen three residues ahead in the sequence. Such motifs arequite often seen at the beginning of -helices, which are characterizedby the ß(g⁺)_RR motif. A remarkable majority of all thesehydrogen bonds are buried from the protein surface, away fromthe surrounding solvent. This strongly indicates the possibilityof side-chains playing the role of the backbone, in the proteininteriors, to satisfy the potential hydrogen bonding sites andmaintaining the network of hydrogen bonds which is crucial tothe structure of the protein.     

The structure of E.coli soluble inorganic pyrophosphatase at 2.7 A resolution

Hie structure of E.coli soluble inorganic pyrophosphatase hasbeen refined at 2.7 resolution to an R-factor of 20.9. Theoverall fold of the molecule is essentially the same as yeastpyrophosphatase, except that yeast pyrophosphatase is longerat both the N- and C-termini. Escherichia coli pyrophosphataseis a mixed +ß protein with a complicated topology.The active site cavity, which is also very similar to the yeastenzyme, is formed by seven ß-strands and an -helixand has a rather asymmetric distribution of charged residues.Our structure-based alignment extends and improves upon earliersequence alignment studies; it shows that probably no more than14, not 15–17 charged and polar residues are part of theconserved enzyme mechanism of pyrophosphatases. Six of theseconserved residues, at the bottom of the active site cavity,form a tight group centred on Asp70 and probably bind the twoessential Mg⁺ ions. The others, more spreadout and more positivelycharged, presumably bind substrate. Escherichia coli pyrophosphatasehas an extra aspartate residue in the active site cavity, whichmay explain why the two enzymes bind divalent cation differently.Based on the structure, we have identified a sequence motifthat seems to occur only in soluble inorganic pyrophosphatases.     

Protein secretion controlled by a synthetic gene in Escherichia coli

The inability of Escherichia coli to secrete proteins in growthmedium is one of the major drawbacks in its use in genetic engineering.A synthetic gene, homologous to the one coding for the kil peptideof pColE1, was made and cloned under the control of the lacpromoter, in order to obtain the inducible secretion of homologousor heterologous proteins by E.coli. The efficiency of this syntheticgene to promote secretion was assayed by analysing the productionand secretion of two proteins, the R-TEM1 ß-lactamase,and the -amylase from Bacillus licheniformis. This latter proteinwas expressed in E.coli from its gene either on the same plasmidas the kil gene or on a different plasmid. The primary effectof the induction of the kil gene is the overproduction of thesecreted proteins. When expressed at a high level, the kil genepromotes the overproduction of all periplasmic proteins andthe total secretion in the culture medium of both the ß-lactamaseor the -amylase. This secretion is semi-selective for most periplasmkproteins are not secreted. The kil peptide induces the secretionof homologous or heterologous proteins in two steps, first actingon the cytoplasmic membrane, then permeabilizing the outer membrane.This system, which is now being assayed at the fermentor scale,is the first example of using a synthetic gene to engineer anew property into a bacterial strain.     

An 8-fold {beta}{alpha} barrel protein with redundant folding possibilities

Protein sequences containing redundant segments of secondarystructure at both termini have the choice a priori of foldinginto several possible circularly permuted variants of the wild-typetertiary structure. To test this hypothesis the gene of phosphoribosylanthranilate isomerase from yeast, which is a single-domain8-fold ß barrel protein, was modified to produce a10-fold ß homologue in Escherichia coli. It containeda duplicate of the two C-terminal ß units of supersecondarystructure fused to its N-terminus. Most of the protein was recoveredfrom the insoluble fraction of disrupted cells by dissolutionin guanidinium chloride solutions and refolding. Pristine proteinwas purified from the soluble fraction. The purified (ß)₁₀proteins were enzymically almost fully active. Absorbance, fluorescenceand circular dichroism spectra as well as the reversible unfoldingbehaviour of both proteins were also very similar to the propertiesof the original (ß)₈ protein. Digestion with endopeptidasesconverted both the pristine and the refolded (ß)₁₀variant to the same large fragment that had the N-terminal sequenceand mol. wt of the wild-type ß)₈ protein. The datasuggest that the folding of the (ß)₁₀ variant is controlledthermodynamically both in vivo and in vitro.     

Construction of multiple copy of {alpha}-domain gene fragment of human liver metallothionein IA in tandem arrays and its expression in transgenic tobacco plants

Metallothioneins (MT) are low molecular weight, cysteinerich,metal-binding proteins. An MT molecule contains two domainswhich appear to act independently—an -domain, which ischaracterized by cadmium-binding, and a ß-domain,which binds preferentially to copper. Based on this conception,DNA duplex encoding the -domain (106 bp) of human MT-I_A wasconstructed from a chemically-synthesized oligomer by repairsynthesis and enzymatic ligation and cloned into pUC19. Thegenes cloned were sequenced and found to be in the correct orderas designed. Synthetic directional adapters were attached tothe terminals of the -domain gene fragment of human MT-I_A toestablish complete control over fragment orientation duringligation. The use of these directional adapters thereby ensuredthe production of multiple copies of the -domain in tandem arrays.The successive -domains were linked by a peptide linker consistingof 10 residues. A chimeric gene containing 12 cloned tandemlyrepeated copies of the 106 bp -domain DNA was introduced intotobacco cells on a disarmed Ti-plasmid of Agrobacterium tumefaciens.A total of 10 different transgenic tobacco plants were generated,of which two showed root and shoot growth unaffected by up to200 mg/l kanamycin and 100 µM cadmium, whereas root growthof control plants was severely inhibited and leaf chlorosisdeveloped on media containing only 10 µM cadmium     

Interactions of (Ala*Ala*Lys*Pro)n and (Lys*Lys*Ser*Pro)n with DNA. Proposed coiled-coil structure of AlgR3 and AlgP from Pseudomonas aeruginosa

The proteins, AlgR3 and AlgP, are involved in the regulationof alginate synthesis in Pseudomonas. They contain multiplerepeats of Ala^*Ala^*Lys^*Pro as do several other proteins thatresemble histones. The interactions of synthesis oligopeptidescomposed of repeated Ala^*Ala^*Lys^*Pro or Lys^*Lys^*Ser^*Pro unitswith DNA were studied by fluorescence of the Fmoc (9-fluorenylmethyloxycarbonyl)group attached to the N-termini of the peptides. DNA quenchingof the Fmoc fluorescence of the peptides was used to estimatethe apparent association constants for the interaction of Fmoc(AAKP)_nOH(n = 2, 4, 8, 18, 32) and of Fmoc(KKSP)_nOH (n = 2, 4, 8, 16,20, 32) with DNA. The Fmoc(AAKP)_nOH peptides bind to DNA onlyat low ionic strength; the Fmoc(KKSP)_n OH peptides interactwith DNA at both low (0.05 M KCl) and high (0.2 M KCl) saltAt low ionic strength an increase in the number of the repeatunits causes an increase in the apparent association constantup to {small tilde}2 x 10⁶ M^–1 for both types of peptidesat N 24. The insertion of an AAKTA unit into the middle ofthe Fmoc(AAKP)₈OH peptide increases its affinity to DNA. Wepropose a model of (AAKP)_n and of its interaction with DNA.The repeat unit consists of a single turn of -helix followedby a bend necessitated by Pro. The resultant coiled-coil formsa right-handed superhelix with 10 AAKPs per repeat distanceof {small tilde}33 Å. With only slight modification ofthe canonical parameters of this model the AAKP super helixfits into the major groove of B-form DNA with one AAKP tetramerper base pair repeat of 3.4 Å. The -amine nitrogen ofLys can form a polar hydrogen bond with a phosphate oxygen atomof the DNA backbone. A better fit is obtained when the modelis modified to accommodate [(AAKP)₅AAKTA]_n as actually observedin AlgR3. We suggest that this coiled-coil represents a generalmotif for other protein–DNA interactions.     

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 -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 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 (/ß)₈ barrel domain of-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.     

Increasing the thermostability of a neutral protease by replacing positively charged amino acids in the N-terminal turn of {alpha}-helices

The 247–260 and 289–299 -helices of Bacillus subtilisneutral protease have a lysine in their N-terminal turn. Theselysines were replaced by Ser or Asp in order to improve electrostaticinteractions with the -helix dipole. After replacing Lys bySer at positions 249 or 290, the thermostability of the enzymewas increased by 0.3 and 1.0°C, respectively. The Asp249and Asp290 mutants exhibited a stabilization of 0.6 and 1.2°C,respectively. The results show the feasibility of stabilizingenzymes by introducing favourable residues at the end of -helices.     

Second-generation octarellins: two new de novo ({beta}/{alpha})8 polypeptides designed for investigating the influence of {beta}-residue packing on the {alpha}/{beta}-barrel structure stability

The sequence of octarellin I, the first de novo (ß/)₈polypeptide, was revised according to several criteria, amongothers the symmetry of the sequence, ß-residue volumeand hydrophobicity, and charge distribution. These considerationsand the overall conclusions drawn from the first design ledto two new sequences, corresponding to octarellins II and III.Octarellin II retains perfect 8-fold symmetry. Octarellin IIIhas the same sequence as octarellin II, except for the ß-strandswhich exhibit a 4-fold symmetry. The two proteins were producedin Escherichia coli. Infrared and CD spectral analyses of octarellinsII and III reveal a high secondary structure content. Non-denaturinggel electrophoresis, molecular sieve chromatography and analyticalultracentrifugation suggest that both of these second-generationartificial polypeptides exist as a mixture of a monomer anda dimer form. Octarellins II and III are at least 10 times moresoluble than octarellin I. Ureainduced unfolding followed byfluorescence emission suggests that the tryptophan residues,designed to be buried in the (ß/)₈, are indeed packedin the hydrophobic core of both proteins. However, octarellinIII displays a higher stability towards urea denaturation, indicatingthat introducing 4-fold symmetry into the ß-barrelmight be important for stability of the overall folding.     

Molecular model-building of amylin and {alpha}-calcitonin gene-related polypeptide hormones using a combination of knowledge sources

Amylin is the major component of the amyloid found in the pancreasesof noninsulin-dependent diabetics (type 2 diabetes). It is a37 amino acid polypeptide and has been shown to have 46% sequenceidentity with the neuropeptide -calcitonin gene-related peptide(-CGRP). Both amylin and -CGRP are known to be potent inhibitorsof glycogen synthesis in stripped rat soleus muscle. Secondarystructure prediction and tertiary structure model-building showthe two polypeptides to have an -helix/ß-strand motifsimilar to that observed in the insulin B-chain. The resultshave been supported by CD spectroscopy, although there is nosequence similarity between insulin and amylin/-CGRP. Aggregationstates have been predicted based on the dimeric and hexamericarrangements seen in porcine insulin. Rat and hamster amylinhave a changed sequence motif in the ß-strand whichresults in lack of amyloid formation and type 2 diabetes. This,we propose, is caused by disruption of hydrogen bonding whichprevents the formation of the dimer.     

Expression of goat {alpha}-lactalbumin in Escherichia coli and its refolding to biologically active protein

A cDNA encoding the mature region of goat -lactalbumin and the3'-non-coding region was fused to cDNA of the N-terminal halfof porcine adenylate kinase which had been placed under thecontrol of the tac promoter in an expression vector in Escherichiacoli. In addition, a methionine codon was inserted between thetwo cDNAs. When the plasmid carried the full-length 3'-non-codingregion, little accumulation of the fused protein was observed.However, the deletion of two-thirds of the 3'-non-coding regionproduced significant expression of the fused protein in E.colistrain JM105. Since goat -lactalbumin contains no methionineresidue, the mature goat -lactalbumin was isolated by CNBr digestionof the fused insoluble protein and refolded using thioredoxin.The homogeneous and biologically active goat -lactalbumin waspurified by Ca²⁺ ion-dependent hydrophobic chromatography.