Knowledge-based design of a soluble bacteriorhodopsin

Much knowledge has been accrued from high resolution protein structures. This knowledge provides rules and guidelines for the rational design of soluble proteins. We have extracted these rules and applied them to redesigning the structure of bacteriorhodopsin and to creating blueprints for a monomeric, soluble seven-helix bundle protein. Such a protein is likely to have desirable properties, such as ready crystallization, which membrane proteins lack and an internal structure similar to that of the native protein. While preserving residues shown to be necessary for protein function, we made modifications to the rest of the sequence, distributing polar and charged residues over the surface of the protein to achieve an amino acid composition as akin to that of soluble helical proteins as possible. A secondary goal was to increase apolar contacts in the helix intercalation regions of the protein. The scheme used to design the model sequences requires knowledge of the number and orientation of helices and some information about interior contacts, but detailed structural knowledge is not required to use a scheme of this type.      

Minimal substrate features for Erm methyltransferases defined by using a combinatorial oligonucleotide library

Erm methyltransferases are prevalent in pathogenic bacteria and confer resistance to macrolide, lincosamide, and streptogramin B antibiotics by specifically methylating the 23S ribosomal RNA at nucleotide A2058. We have identified motifs within the rRNA substrate that are required for methylation by Erm. Substrate molecules were constructed in a combinatorial manner from two separate sets (top and bottom strands) of short RNA sequences. Modifications, including LNA monomers with locked sugar residues, were incorporated into the substrates to stabilize their structures. In functional substrates, the A2058 methylation target (on the 13- to 19-nucleotide top strand) was displayed in an unpaired sequence immediately following a conserved irregular helix, and these are the specific structural features recognized by Erm. Erm methylation was enhanced by stabilizing the top-strand conformation with an LNA residue at G2056. The bottom strand (nine to 19 nucleotides in length) was required for methylation and was still functional after extensive modification, including substitution with a DNA sequence. Although it remains possible that Erm makes some unspecific contact with the bottom strand, the main role played by the bottom strand appears to be in maintaining the conformation of the top strand. The addition of multiple LNA residues to the top strand impeded methylation; this indicates that the RNA substrate requires a certain amount of flexibility for accommodation into the active site of Erm. The combinatorial approach for identifying small but functional RNA substrates is a step towards making RNA-Erm complexes suitable for cocrystal determination, and for designing molecules that might block the substrate-recognition site of the enzyme.     

An intein-based genetic selection allows the construction of a high-quality library of binary patterned de novo protein sequences

Combinatorial libraries of synthetic DNA are increasingly being used to identify and evolve proteins with novel folds and functions. An effective strategy for maximizing the diversity of these libraries relies on the assembly of large genes from smaller fragments of synthetic DNA. To optimize library assembly and screening, it is desirable to remove from the synthetic libraries any sequences that contain unintended frameshifts or stop codons. Although genetic selection systems can be used to accomplish this task, the tendency of individual segments to yield misfolded or aggregated products can decrease the effectiveness of these selections. Furthermore, individual protein domains may misfold when removed from their native context. We report the development and characterization of an in vivo system to preselect sequences that encode uninterrupted gene segments regardless of the foldedness of the encoded polypeptide. In this system, the inserted synthetic gene segment is separated from an intein/thymidylate synthase (TS) reporter domain by a polyasparagine linker, thereby permitting the TS reporter to fold and function independently of the folding and function of the segment-encoded polypeptide. TS-deficient Escherichia coli host cells survive on selective medium only if the insert is uninterrupted and in-frame, thereby allowing selection and amplification of desired sequences. We demonstrate that this system can be used as a highly effective preselection tool for the production of large, diverse and high-quality libraries of de novo protein sequences.     

Optimal protein library design using recombination or point mutations based on sequence-based scoring functions

In this paper, we introduce and test two new sequence-based protein scoring systems (i.e. S1, S2) for assessing the likelihood that a given protein hybrid will be functional. By binning together amino acids with similar properties (i.e. volume, hydrophobicity and charge) the scoring systems S1 and S2 allow for the quantification of the severity of mismatched interactions in the hybrids. The S2 scoring system is found to be able to significantly functionally enrich a cytochrome P450 library over other scoring methods. Given this scoring base, we subsequently constructed two separate optimization formulations (i.e. OPTCOMB and OPTOLIGO) for optimally designing protein combinatorial libraries involving recombination or mutations, respectively. Notably, two separate versions of OPTCOMB are generated (i.e. model M1, M2) with the latter allowing for position-dependent parental fragment skipping. Computational benchmarking results demonstrate the efficacy of models OPTCOMB and OPTOLIGO to generate high scoring libraries of a prespecified size.     

Novel dominant-negative prion protein mutants identified from a randomized library

Prion diseases are untreatable neurodegenerative disorders characterized by accumulation of PrP(Sc), an aggregated isoform of the cellular prion protein (PrP(C)). We generated a library of PrP variants with random mutations in the helix-3 domain and screened for dominant-negative mutants (DNMs) that would inhibit replication of prions (the Rocky Mountain Laboratory strain) in infected N2a cells. Two of the identified PrP mutants, Q167R and Q218K, were already known to counteract prion replication, thereby validating the effectiveness of this approach. In addition, novel DNMs were found efficiently to antagonize PrP(Sc) propagation in cells. In contrast to Q167R and Q218K, the newly identified DNMs S221P and Y217C resided on the cell surface at a substantially lower level, suggesting that robust cell surface display of DNM might not be a general prerequisite for efficient prion antagonism. The newly identified DNMs point to useful target-selective therapeutic tools for the treatment of prion diseases.     

Cholesterol oxidases: a study of nature's approach to protein design

Cholesterol oxidases are important as clinical reagents, potential larvicides, and tools in cell biology, and they are implicated in bacterial pathogenesis. Here we review chemical aspects of their function. We describe our current structural and mechanistic understanding of the type I and II cholesterol oxidases, our identification of an NH-pi hydrogen bond motif for stabilization of reduced flavins, our structural hypothesis of how O(2) gains access to the flavin, and our present understanding of type I cholesterol oxidase-lipid bilayer interactions.     

The SBASE domain library: a collection of annotated protein segments

SBASE is a database of annotated protein domain sequences representingvarious structural, functional, ligand binding and topogenicsegments of proteins. The current release of SBASE contains27 211 entries which are provided with standardized names inorder to facilitate retrieval. SBASE is cross-referenced tothe major protein and nucleic acid databanks as well as to thePROSITE catalog of protein sequence patterns [Bairoch, A. (1992)Nucleic Acids Res., 20, Suppl., 2013–2118]. SBASE canbe used to establish domain homologies through database searchusing programs such as FASTA [Lipman and Pearson (1985) Science,227, 1436–1441], FASTDB [Brutlag et al. (1990) Comp. Appl.Biosci., 6, 237–245] or BLAST3 [Altschul and Lipman (1990)Proc. Natl. Acad. Sci. USA, 87, 5509–5513], which is especiallyuseful in the case of loosely defined domain types for whichefficient consensus patterns cannot be established. The useof SBASE is illustrated on the DNA binding protein Brain-4.The database and a set of search and retrieval tools are freelyavailable on request to the authors or by anonymous ‘ftp’file transfer from <ftp.icgeb.trieste.it>.     

Mushroom tyrosinase oxidizes tyrosine-rich sequences to allow selective protein functionalization

We show that mushroom tyrosinase catalyzes the formation of reactive o‐quinones on unstructured, tyrosine‐rich sequences such as hemagglutinin (HA) tags (YPYDVPDYA). In the absence of exogenous nucleophiles and at low protein concentrations, the o‐quinone decomposes with fragmentation of the HA tag. At higher protein concentrations (>5 mg mL^?1), crosslinking is observed. Besthorn's reagent intercepts the o‐quinone to give a characteristic pink complex that can be observed directly on a denaturing SDS‐PAGE gel. Similar labeled species can be formed by using other nucleophiles such as Cy5‐hydrazide. These reactions are selective for proteins bearing HA and other unstructured poly‐tyrosine‐containing tags and can be performed in lysates to create specifically tagged proteins.     

Crystal structure of a synthetic cyclodecapeptide for template-assembled synthetic protein design

The structural prototype of a new generation of regioselectively addressable functionalized templates (RAFTs) for use in protein de novo design has been synthesized and crystallized. The structure of the aromatically substituted cyclodecapeptide was determined by X-ray diffraction; it consists of an antiparallel beta sheet spanned by heterochirally induced type IIprime prime or minute beta turns, similar to that observed in gramicidin S. The three-dimensional structure of the artificial template was also examined by an NMR spectroscopic analysis in solution and shown to be compatible with a beta-sheet plane suitable for accommodating secondary functional peptide fragments for the synthesis of template-assembled synthetic proteins (TASPs).     

Prediction of protein side chain conformations: a study on the influence of backbone accuracy on conformation stability in the rotamer space

We have studied the effect of backbone inaccuracy on the efficiency of protein side chain conformation prediction using rotamer libraries. The backbones were generated by randomly perturbing the crystallographic conformation of 12 proteins and exhibit C alpha r.m.s.d.s of up to 2 A. Our results show that, even for a perturbation of the backbone fully compatible with the temperature factors of the proteins, the predicted side chain conformations of approximately 10% of the buried side chains remain variable. This fraction increases further for larger backbone deviations. However, for backbone deviations of up to 2 A r.m.s.d., the predicted side chain r.m.s.d. varies only in a ratio of < 1.4. Moreover, a possible strategy for obtaining side chain conformations close to the experimental ones consists of extracting the consensus conformations of the side chains from a series of backbone conformations. Such a procedure allows the computation of the side chain conformations with no loss of accuracy for backbones exhibiting r.m.s.d.s of up to 1 A from the crystallographic coordinates. For larger backbone deviations (up to 2 A r.m.s.d.) the r.m.s.d. of the buried side chains increases from 1.33 up to 1.60 A. We also discuss the influence of the size of the rotamer library on the quality of the prediction.      

Polarity as a criterion in protein design

Hypothetical proteins can be tested computationally by determiningwhether or not the designed sequence-structure pair has thecharacteristics of a typical globular protein. We have developedsuch a test by deriving quantities with approximately constantvalue for all globular proteins, based on empirical analysisof the exposed and buried surfaces of 128 structurally knownproteins. The characteristic quantities that best appear tosegregate badly designed or deliberately misfolded proteinsfrom their properly folded natural relatives are the polar fractionof side chains on the protein surface and, independently, inthe protein interior. Three of the seven hypothetical structurestested here can be rejected as having too many polar side-chaingroups in the interior or too few on the protein surface. Inaddition, a recently designed nutritional protein is identifiedas being very much unlike globular proteins. These database-derivedcharacteristic quantities are useful in screening designed proteinsprior to experiment and may be useful in screening experimentallydetermined (X-ray, NMR) protein structures for possible errors.     

Synthetic potential of molluscan sulfatases for the library synthesis of regioselectively O-sulfonated D-galacto-sugars

The substrate specificities of three molluscan sulfatases (E.C. 3.1.6.1; snail, abalone, and limpet origins) were investigated with assorted p-nitrophenyl (pNP) di-O-sulfonated beta-D-galactopyranosides and beta-lactosides [3,6-SO(3) Gal (1), 3',6'-SO(3) Lac (2), 4, 6SO(3) Gal (3), 2,6-SO(3) Gal (4), 3,4-SO(3) Gal (5), and 3,6-SO(3) GalNAc (6); Ac, acetyl; Gal, galactose; Lac, lactose] together with mono-O-sulfonated beta-D-galactopyranoside [pNP 3SO(3)-Gal (7)] and tri-O-sulfonated alpha-D-galactopyranoside [2,3,6-SO(3)-alpha-Gal (11)]. Some notable differences between the substrate specificity of the three sulfatases were disclosed; snail sulfatase hydrolyzed the 3O- and 2O-sulfo groups of 1 and 4, respectively, to afford 6SO(3) Gal (9) in high yields, while the abalone enzyme did not act on 4. Only the limpet enzyme could cleave the 3O-sulfo groups of 7 to give pNP beta-galactoside. In contrast, every enzyme could utilize 11 as a good substrate to afford a mixture of 6SO(3)-alpha-Gal (13) and 2,6-SO(3) alpha-Gal (12). None of the enzymes could cleave the O-sulfo groups of 5 and 6, which indicates that a primary 6O-sulfo group tends to promote the enzymatic hydrolysis of O-sulfo groups at the secondary positions.     

Microbial reactor design for synthetic protein production

The physical factors that may affect the performance of a microbial reactor for producing syntlietic protein are reviewed in ternis of chemical engineering principles. In particular, it is shown that an appreciation of these principles can be used to maximize the productivity of synthetic protein from liquid-hydrocarbon and cellulose fermentations.     

LINKER: a program to generate linker sequences for fusion proteins

The construction of functional fusion proteins often requiresa linker sequence that adopts an extended conformation to allowfor maximal flexibility. Linker sequences are generally selectedbased on intuition. Without a reliable selection criterion,the design of such linkers is often difficult, particularlyin situations where longer linker sequences are required. Herewe describe a program called LINKER which can automaticallygenerate a set of linker sequences that are known to adopt extendedconformations as determined by X-ray crystallography and NMR.The only required input to the program is the desired linkersequence length. The program is specifically designed to assistin fusion protein construction. A number of optional input parametershave been incorporated so that users are able to enhance sequenceselection based on specific applications. The program outputsimply contains a set of sequences with a specified length.This program should be a useful tool in both the biotechnologyindustry and biomedical research. It can be accessed throughthe Web page http://www.fccc.edu/research/labs/feng/linker.html.     

优化整流设计方案挖掘装置节电潜力

阐述了目前整流装置设计中挖掘节电潜力的途径、方案及预期效果,指出了在方案选择中应注意的问题。     

Using support vector machine and evolutionary profiles to predict antifreeze protein sequences

Antifreeze proteins (AFPs) are ice-binding proteins. Accurate identification of new AFPs is important in understanding ice-protein interactions and creating novel ice-binding domains in other proteins. In this paper, an accurate method, called AFP_PSSM, has been developed for predicting antifreeze proteins using a support vector machine (SVM) and position specific scoring matrix (PSSM) profiles. This is the first study in which evolutionary information in the form of PSSM profiles has been successfully used for predicting antifreeze proteins. Tested by 10-fold cross validation and independent test, the accuracy of the proposed method reaches 82.67% for the training dataset and 93.01% for the testing dataset, respectively. These results indicate that our predictor is a useful tool for predicting antifreeze proteins. A web server (AFP_PSSM) that implements the proposed predictor is freely available.     

Probabilistic approach to the design of symmetric protein quaternary structures

Probabilistic methods have been developed that estimate thesite-specific probabilities of the amino acids in sequenceslikely to fold to a particular target structure, and such informationcan be used to guide the de novo design of proteins and to probesequence variability. An extension of these methods for thedesign of symmetric homo-oligomeric quaternary structures ispresented. The theory is in excellent agreement with the resultsof studies on exactly solvable lattice models. Application toan atomically detailed representation of proteins verifies theutility of a symmetry assumption, which greatly simplifies andaccelerates the calculations. The method may be applied to awide variety of symmetric and periodic protein structures. Received August 20, 2003; revised October 24, 2003; accepted October 28, 2003     

From DNA sequence to improved functionality: using protein sequence comparisons to rapidly design a thermostable consensus phytase

Naturally-occurring phytases having the required level of thermostabilityfor application in animal feeding have not been found in naturethus far. We decided to de novo construct consensus phytasesusing primary protein sequence comparisons. A consensus enzymebased on 13 fungal phytase sequences had normal catalytic properties,but showed an unexpected 15–22°C increase in unfoldingtemperature compared with each of its parents. As a first steptowards understanding the molecular basis of increased heatresistance, the crystal structure of consensus phytase was determinedand compared with that of Aspergillus niger phytase. Aspergillusniger phytase unfolds at much lower temperatures. In most cases,consensus residues were indeed expected, based on comparisonsof both three-dimensional structures, to contribute more tophytase stabilization than non-consensus amino acids. For someconsensus amino acids, predicted by structural comparisons todestabilize the protein, mutational analysis was performed.Interestingly, these consensus residues in fact increased theunfolding temperature of the consensus phytase. In summary,for fungal phytases apparently an unexpected direct link betweenprotein sequence conservation and protein stability exists.