Synthesis and sequence optimization of GFP mutants containing aromatic non-natural amino acids at the Tyr66 position

In order to alter the fluorescence properties of green fluorescent protein (GFP), aromatic non-natural amino acids were introduced into the Tyr66 position of GFP in a cell-free translation system using a four-base codon method. Two non-natural mutants (O-methyltyrosine and p-aminophenylalanine mutants) out of 18 mutants showed blue-shifted but weak fluorescence compared with wild-type GFP. Then the aminophenylalanine mutant was sequence optimized by introducing random mutations around the Tyr66 site. For this purpose, a method for random mutation of non-natural proteins in a cell-free system was developed. Three aminophenylalanine mutants with Y145F, Y145L and Y145 M mutations were obtained, which exhibited increased fluorescence by 1.5-, 3- and 4-fold, respectively. These results indicate that random mutation around non-natural amino acids is useful strategy in order to improve protein functions that are reduced by non-natural amino acid incorporation. The method described here will be applicable to other non-natural mutant proteins in a high-throughput manner.     

A new strategy for the synthesis of dinucleotides loaded with glycosylated amino acids--investigations on in vitro non-natural amino acid mutagenesis for glycoprotein synthesis

The in vitro non-natural amino acid mutagenesis method provides the opportunity to introduce non-natural amino acids site-specifically into proteins. To this end, a chemically synthesised aminoacylated dinucleotide is enzymatically ligated to a truncated suppressor transfer RNA. The loaded suppressor tRNA is then used in translation reactions to read an internal stop codon. Here we report an advanced and general strategy for the synthesis of the aminoacyl dinucleotide. The protecting group pattern developed for the dinucleotide facilitates highly efficient aminoacylation, followed by one-step global deprotection. The strategy was applied to the synthesis of dinucleotides loaded with 2-acetamido-2-deoxy-glycosylated amino acids, including N- and O-beta-glycosides and O- and C-alpha-glycosides of amino acids, thus enabling the extension of in vitro non-natural amino acid mutagenesis towards the synthesis of natural glycoproteins of high biological interest. We demonstrate the incorporation of the glycosylamino acids--although with low suppression efficiency--into the human interleukin granulocyte-colony stimulating factor (hG-CSF), as verified by the ELISA technique.     

Red fluorescent protein variants with incorporated non-natural amino acid analogues

Fluorescent proteins are important tools in biotechnology applications and biosensing. DsRed, a red fluorescent protein, has expanded the colors of fluorescent proteins beyond the more commonly used green fluorescent protein. Many genetic modifications have been performed on DsRed to overcome some of its drawbacks. These primarily focused on overcoming the oligomerization detrimental to DsRed activity, and the parasitic green fluorescence caused by the immature chromophore. One such variant, DsRed-monomer, has minimal green fluorescence and no oligomerization. A few traditional mutagenesis studies have been done with DsRed and its mutants to shift the fluorescence wavelengths creating additions to the pallet of fluorescent protein colors. We have explored incorporation of non-natural amino acid analogues into DsRed-Monomer, obtaining variants with differing emission properties. In this work, two such analogues of tyrosine have been incorporated into DsRed-Monomer: 3-amino-l-tyrosine and 3-fluoro-l-tyrosine. Tyrosine analogues were chosen due to the role of tyrosine in the formation and structure of the protein's chromophore. The variants obtained in our study showed altered emission wavelengths and spectral characteristics. Our study demonstrates that incorporation of non-natural analogues into DsRed-Monomer is a viable approach to alter the spectral characteristics of the protein. We envision that this study will open up the door to non-natural mutagenesis studies with red fluorescent proteins and its mutants.     

Fidelity of seryl-tRNA synthetase to binding of natural amino acids from HierDock first principles computations

Seryl-tRNA synthetase (SerRS) charges serine to tRNA(Ser) following the formation of a seryl adenylate intermediate, but the extent to which other non-cognate amino acids compete with serine to bind to SerRS or for the formation of the activated seryl adenylate intermediate is not known. To examine the mechanism of discrimination against non-cognate amino acids, we calculated the relative binding energies of the 20 natural amino acids to SerRS. Starting with the crystal structure of SerRS from Thermus thermophilus with seryl adenylate bound, we used the HierDock and SCREAM (Side-Chain Rotamer Energy Analysis Method) computational methods to predict the binding conformation and binding energy of each of the 20 natural amino acids in the binding site in the best-binding mode and the activating mode. The ordering of the calculated binding energies in the activated mode agrees with kinetic measurements in yeast SerRS that threonine will compete with serine for formation of the activated intermediate while alanine and glycine will not compete significantly. In addition, we predict that asparagine will compete with serine for formation of the activated intermediate. Experiments to check the accuracy of this prediction would be useful in further validating the use of HierDock and SCREAM for designing novel amino acids to incorporate into proteins and for determining mutations in aminoacyl-tRNA synthetase design to facilitate the incorporation of amino acid analogs into proteins.     

Lipid modification of proteins and their membrane transport

An effective method for artificial attachment of lipid anchorsto water-soluble proteins has been developed. To this end, aprotein molecule is modified in a system of reversed micellesby a water-insoluble reagent, e.g. fatty acid chloride. Fattyacylated proteins acquire an ability to translocate across lipidmembranes and penetrate intact cells. This principle of impartingtransmembrane properties to water-soluble proteins makes itpossible to realize in vivo a direct transport of antibodiesacross the hemato-encephalic barrier into the brain and to developa method for virus suppression by fatty acylated anti-viralantibodies capable of penetrating infected cells. The effectof a drastic increase in the biological activity of exogenousprotein factors, e.g. Staphylococcus aureus enterotoxin A, asa result of their artificial fatty acylation has been discovered.The above-mentioned phenomena are discussed in relation to thein vivo data, indicating that post-translational modificationof proteins by fatty acids and phospholipids is very widespreadin nature and evidently plays an important role in protein transportand sorting. In this connection, lipid modification of proteinsis regarded as a possible general step of protein transportin vivo.     

Independence divergence-generated binary trees of amino acids

The discovery of the relationship between amino acids is importantin terms of the replacement ability, as used in protein engineeringhomology studies, and gaining a better understanding of theroles which various properties of the residues play in the creationof a unique, stable, 3-D protein structure. Amino acid sequencesof proteins edited by evolution are anything but random. Themeasure of nonrandomness, i.e. the level of editing, can becharacterized by an independence divergence value. This parameteris used to generate binary tree relationships between aminoacids. The relationships of residues presented in this paperare based on protein building features and not on the physico-chemicalcharacteristics of amino acids. This approach is not biasedby the tautology present in all sequence similarity-based relationshipstudies. The roles which various physico-chemical characteristicsplay in the determination of the relationships between aminoacids are also discussed.     

Searching for common sequence patterns among distantly related proteins

We have developed a program Gap Allowing Pattern Explorer (GAPE)to extract amino acid sequence motifs conserved among distantlyrelated proteins. The GAPE program is designed to allow gapsin the sequences. First, this program generates all possibleamino acid patterns comprising up to five amino acids. Sequencescontaining the amino acid residues in the same order as a generatedpattern are selected as subsequences, where the differencesin the distances between two consecutive amino acids are ignored.Next, the motifs are extracted from the subsequences under conditionsin which all four distances between the five amino acids arefixed. At this stage, motifs with gaps in their subsequenceare also found by relaxing one of the four fixed distances.The statistical significance for a motif obtained is calculatedbased on the amino acid composition of the sequences under consideration.When the GAPE program was applied to 59 pyridoxal-phosphaterelatedsequences and 64 ATP (AMP-forming)-related sequences, motifsextracted with a low expectation of occurrence contained someof the amino acid residues chemically proved to be involvedin the ligand recognition.     

Two-dimensional surface display of functional groups on a beta-helical antifreeze protein scaffold

We tested a disulfide-rich antifreeze protein as a potential scaffold for design or selection of proteins with the capability of binding periodically organized surfaces. The natural antifreeze protein is a beta-helix with a strikingly regular two-dimensional grid of threonine side chains on its ice-binding face. Amino acid substitutions were made on this face to replace blocks of native threonines with other amino acids spanning the range of beta-sheet propensities. The variants, displaying arrays of distinct functional groups, were studied by mass spectrometry, reversed-phase high performance liquid chromatography, thiol reactivity and circular dichroism and NMR spectroscopies to assess their structures and stabilities relative to wild type. The mutants are well expressed in bacteria, despite the potential for mis-folding inherent in these 84-residue proteins with 16 cysteines. We demonstrate that most of the mutants essentially retain the native fold. This disulfide bonded beta-helical scaffold, thermally stable and remarkably tolerant of amino acid substitutions, is therefore useful for design and engineering of macromolecules with the potential to bind various targeted ordered material surfaces.     

Selenocysteine's mechanism of incorporation and evolution revealed in cDNAs of three glutathione peroxidases

The nonsense codon, UGA, has for the first time recently beenshown to encode selenocysteine in two proteins, mouse glutathioneperoxidase (GSH-Px) (EC 1.11.1.9 [EC] ) and bacterial formate dehydrogenase.A co-translational rather than post-translational selenium-incorporationmechanism has been implicated. Furthermore, high expressionlevels of GSH-Px have suggested that suppression of terminationis efficient and specific. We have isolated and characterizedpituitary, kidney and placenta cDNAs for bovine, human and mouseGSH-Px respectively. It is demonstrated that this novel suppressionevent occurs in diverse tissues, in at least three mammalianspecies and at the translational step. Surprisingly, GSH-Pxis shown to be extramitochondrially encoded, indicating a cytosolicsuppression event rather than one utilizing the mitochondria'swell-documented extended codon-reading ability. Sequence analysisreveals that a simple proximal contextual pattern responsiblefor readthrough does not exist. Analysis of predicted secondarystrucutres of mRNAs, however, has revealed a conformation whichmay be unique to selenocysteine proteins and may prove usefulas a tool for artificial incorporation of selenocysteines. Ahuman intron for GSH-Px from an unspliced mRNA has been isolatedwhose position indicates an ancient, divergent evolutionaryrelationship with thioredoxin-S₂, rather than an independentconvergent one.     

Amino acid composition of protein termini are biased in different manners

An exhaustive statistical analysis of the amino acid sequencesat the carboxyl (C) and amino (N) termini of proteins and ofcoding nucleic acid sequences at the 5' side of the stop codonswas undertaken. At the N ends, Met and Ala residues are over-representedat the first (+1) position whereas at positions 2 and 5 Thris preferred. These peculiarities at N-termini are most probablyrelated to the mechanism of initiation of translation (for Met)and to the mechanisms governing the life-span of proteins viaregulation of their degradation (for Ala and Thr). We assumethat the C-terminal bias facilitates fixation of the C endson the protein globule by a preference for charged and Cys residues.The terminal biases, a novel feature of protein structure, haveto be taken into account when molecular evolution, three-dimensionalstructure, initiation and termination of translation, proteinfolding and life-span are concerned. In addition, the bias ofprotein termini composition is an important feature which shouldbe considered in protein engineering experiments.     

Local polarity analysis: a sensitive method that discriminates between native proteins and incorrectly folded models

The evaluation of calculated protein structures is an importantstep in the protein design cycle. Known criteria for this assessmentof proteins are the polar and apolar, accessible and buriedsurface area, electrostatic interactions and other interactionsbetween the protein atoms (e.g. HO, S-S),atomic packing, analysisof amino acid environment and surface charge distribution. Weshow that a powerful test of accuracy of protein structure canbe derived by analysing the water contact of atoms and additionallytaking into account their polarity. On the basis of estimatedreference values of the polar fraction of typical globular proteinswith known structure (mean, SD and distribution), the evaluationof misfolded structures can be improved significantly. The referencevalues are derived by moving windows of different length (3–99amino acid residues) over the amino acid sequence. Model proteins,which are included in the Brookhaven protein structure databank,deliberately misfolded proteins, hypothetical proteins and predictedprotein structures are diagnosed as at least partially incorrectlyfolded. The local fault, mostly observed, is that polar groupsare buried too frequently in the interior of the protein. Thedatabase-derived quantities are useful in screening the designedproteins prior to experimentation and may also be useful inthe assessment of errors in the experimentally determined proteinstructures.     

Structural identity between the iron- and manganese-containing superoxide dismutases

We have recently reported the first complete amino acid sequenceof an iron-containing superoxide dismutase. The iron enzymeis thought to be closely homologous to the manganese-containingsuperoxide dismutases. The availability of complete amino acidsequence information for four manganese superoxide dismutasesand the crystal structures for two iron and two manganese superoxidedismutases prompted us to investigate the degree of homologybetween the two proteins at various levels. We report that itis not possible to clearly distinguish the two proteins on thebasis of their secondary or tertiary structures. It would appearthat a small number of single site substitutions are responsiblefor conferring distinguishing properties between the two proteins.Substitution of glyclne 77 and glutamine 154 by a glutamineand an alanine respectively in Photobacterium leiognathi ironsuperoxide dismutase may distinguish the kinetic and other particularproperties of this protein from the manganese protein (and otheriron superoxide dismutases). Furthermore the primary structureof both the iron and manganese proteins does not appear to haveany homology with any other known amino acid sequence.     

Protein sequence entropy is closely related to packing density and hydrophobicity

We investigated the correlation between the Shannon information entropy, 'sequence entropy', with respect to the local flexibility of native globular proteins as described by inverse packing density. These are determined at each residue position for a total set of 130 query proteins, where sequence entropies are calculated from each set of aligned residues. For the accompanying aggregate set of 130 alignments, a strong linear correlation is observed between the calculated sequence entropy and the corresponding inverse packing density determined at an associated residue position. This region of linearity spans the range of C(alpha) packing densities from 12 to 25 amino acids within a sphere of 9 angstrom radius. Three different hydrophobicity scales all mimic the behavior of the sequence entropies. This confirms the idea that the ability to accommodate mutations is strongly dependent on the available space and on the propensity for each amino acid type to be buried. Future applications of these types of methods may prove useful in identifying both core and flexible residues within a protein.     

A mini-protein designed by removing a module from barnase: molecular modeling and NMR measurements of the conformation

A globular domain can be decomposed into compact modules consistingof contiguous 10–30 amino acid residues. The correlationbetween modules and exons observed in different proteins suggeststhat each module was encoded by an ancestral exon and that moduleswere combined into globular domains by exon fusion. Barnaseis a single domain RNase consisting of 110 amino acid residuesand was decomposed into six modules. We designed a mini-proteinby removing the second module, M2, from barnase in order togain an insight into the structural and functional roles ofthe module. In the molecular modeling of the mini-protein, weevaluated thermodynamic stability and aqueous solubility togetherwith mechanical stability of the model. We chemically synthesizeda mini-barnase with ¹⁵N-labeling at 10 residues, whose correspondingresidues in barnase are all found in the region around the hydrophobiccore. Circular dichroism and NMR measurements revealed thatmini-barnase takes a non-random specific conformation that hasa similar hydrophobic core structure to that of barnase. Thisresult, that a module could be deleted without altering thestructure of core region of barnase, supports the view thatmodules act as the building blocks of protein design.     

High solubility of random-sequence proteins consisting of five kinds of primitive amino acids

Searching for functional proteins among random-sequence librariesis a major challenge of protein engineering; the difficultiesinclude the poor solubility of many random-sequence proteins.A library in which most of the polypeptides are soluble andstable would therefore be of great benefit. Although modernproteins consist of 20 amino acids, it has been suggested thatearly proteins evolved from a reduced alphabet. Here, we haveconstructed a library of random-sequence proteins consistingof only five amino acids, Ala, Gly, Val, Asp and Glu, whichare believed to have been the most abundant in the prebioticenvironment. Expression and characterization of arbitrarilychosen proteins in the library indicated that five-alphabetrandom-sequence proteins have higher solubility than do 20-alphabetrandom-sequence proteins with a similar level of hydrophobicity.The results support the reduced-alphabet hypothesis of the primordialgenetic code and should also be helpful in constructing optimizedprotein libraries for evolutionary protein engineering.     

Maintenance of the hydrophobic face of the diphtheria toxin amphipathic transmembrane helix 1 is essential for the efficient delivery of the catalytic domain to the cytosol of target cells

Abstract The transmembrane (T) domain of diphtheria toxin (DT) comprisesnine -helices and has been shown to play an essential role inthe efficent delivery of the catalytic (C) domain ofDT acrossthe eukaryotic cell membrane and into the cytosol. We have demonstratedrecently thatthe first three amphipathic helixes of the T domain,although not necessary for either channel formation or receptorbinding, are required for the efficient transmembrane deliveryof the Cdomain.In the present study,we have performed a detailedstructure-function analysis of T domainhelix 1 (TH1) of theDT-related fusion protein DAB₃₈₉lL-2. We performed exchangeandsite-directed mutagenesis of TH1 and the resulting mutantfusion toxins were analyzed by gel electrophoresis and testedfor their efficiencies in the delivery of the C domain to thecell cytosol. We demonstrate that the overall charge distributionand hydrophobicity of amino acids in the amphipathic helix TH1,rather than a specific amino acid sequence, are critical forthe function of this helix. The insertion of a charged residuein the hydrophobic face of TH1 abolishes cytotoxic activity,whereas replacement of a hydrophobic residue by a charged aminoacid in the hydrophilic face of the helix has little, if any,effect on cytotoxic activity. In addition,we have identifiedSer220 by site-directed mutagenesis as a residue that appearsto be criticalfor correct folding of the fusion toxin. Mutationsin this position result in fusion proteins that are extremelysensitive to proteolytic attack.     

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

The sequences of four--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.     

Conservation of mechanism, variation of rate: folding kinetics of three homologous four-helix bundle proteins

The amino acid sequence of a protein determines both its final folded structure and the folding mechanism by which this structure is attained. The differences in folding behaviour between homologous proteins provide direct insights into the factors that influence both thermodynamic and kinetic properties. Here, we present a comprehensive thermodynamic and kinetic analysis of three homologous homodimeric four-helix bundle proteins. Previous studies with one member of this family, Rop, revealed that both its folding and unfolding behaviour were interesting and unusual: Rop folds (k(0)(f) = 29 s(-1)) and unfolds (k(0)(u) = 6 x 10(-7) s(-1)) extremely slowly for a protein of its size that contains neither prolines nor disulphides in its folded structure. The homologues we discuss have significantly different stabilities and rates of folding and unfolding. However, the rate of protein folding directly correlates with stability for these homologous proteins: proteins with higher stability fold faster. Moreover, in spite of possessing differing thermodynamic and kinetic properties, the proteins all share a similar folding and unfolding mechanism. We discuss the properties of these naturally occurring Rop homologues in relation to previously characterized designed variants of Rop.     

Alignment of protein sequences using secondary structure: a modified dynamic programming method

A method for comparison of protein sequences based on theirprimary and secondary structure is described. Protein sequencesare annotated with predicted secondary structures (using a modifiedChou and Fasman method). Two lettered code sequences are generated(Xx, where X is the amino acid and x is its annotated secondarystructure). Sequences are compared with a dynamic programmingmethod (STRALIGN) that includes a similarity matrix for boththe amino acids and secondary structures. The similarity valuefor each paired two-lettered code is a linear combination ofsimilarity values for the paired amino acids and their annotatedsecondary structures. The method has been applied to eight globinproteins (28 pairs) for which the X-ray structure is known.For protein pairs with high primary sequence similarity (>45%),STRALIGN alignment is identical to that obtained by a dynamicprogramming method using only primary sequence information.However, alignment of protein pairs with lower primary sequencesimilarity improves significantly with the addition of secondarystructure annotation. Alignment of the pair with the least primarysequence similarity of 16% was improved from 0 to 37% ‘correct’alignment using this method. In addition, STRALIGN was successfullyapplied to seven pairs of distantly related cytochrome c proteins,and three pairs of distantly related picornavirus proteins.     

Understanding the relationship between the primary structure of proteins and their amyloidogenic propensity: clues from inclusion body formation

Amyloid formation is dependent to a considerable extent on the amino acid sequence of the protein. The present study delineates certain sequence-dependent features that are correlated with amyloidogenic propensity. The analyses indicate that amyloid formation is favored by lower thermostability and increased half-life of the protein. There seems to be a certain degree of bias in the composition of order-promoting amino acids in the case of amyloidogenic proteins. Based on these parameters, a prediction function for the amyloidogenic propensity of proteins has been created. The prediction function has been found to rationalize the reported effect of certain mutations on amyloid formation. It seems that a higher sheet propensity of residues that constitute the first seven residues of a helical structure in a protein might increase the propensity for a helix to sheet transition in that region under denaturing conditions.