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At least 775 records · Page 43Linked to original sources
PubMed2004

Orthogonal site-specific protein modification by engineering reversible thiol protection mechanisms.

Covalent modification is an important strategy for introducing new functions into proteins. As engineered proteins become more sophisticated, it is often desirable to introduce multiple, modifications involving several different functionalities in a site-specific manner. Such orthogonal labeling schemes require independent labeling of differentially reactive nucleophilic amino acid side chains. We have developed two protein-mediated protection schemes that permit independent labeling of multiple thiols. These schemes exploit metal coordination or disulfide bond formation to reversibly protect cysteines in a Cys(2)His(2) zinc finger domain. We constructed a variety of N- and C-terminal fusions of these domains with maltose-binding protein, which were labeled with two or three different fluorophores. Multiple modifications were made by reacting an unprotected cysteine in MBP first, deprotecting the zinc finger, and then reacting the zinc finger cysteines. The fusion proteins were orthogonally labeled with two different fluorophores, which exhibited intramolecular fluorescene resonance energy transfer (FRET). These conjugates showed up to a threefold ratiometric change in emission intensities in response to maltose binding. We also demonstrated that the metal- and redox-mediated protection methods can be combined to produce triple independent modifications, and prepared a protein labeled with three different fluorophores that exhibited a FRET relay. Finally, labeled glucose-binding protein was covalently patterned on glass slides using thiol-mediated immobilization chemistries. Together, these experiments demonstrated that reversible thiol protection schemes provide a rapid, straightforward method for producing multiple, site-specific modifications.

Binding Sites↗
PubMed1998

Genetic engineering of a single-chain antibody fragment for surface immobilization in an optical biosensor.

The development of a biosensor based on a genetically engineered biomolecule offers many potential advantages to sensors that rely on natural proteins only. Here we present how protein engineering techniques can be used to introduce a functional unit for surface immobilization into a single-chain antibody fragment (scFv). A peptide known to mimic the binding properties of biotin was fused to the carboxyterminus of the phosphorylcholine-binding scFv fragment of IgA McPC603. This fusion protein could be immobilized on a streptavidin monolayer. The resulting scFv monolayer was capable of binding a fluorescently labeled phosphorylcholine analog, as detected by total internal reflection fluorescence. In contrast, an scFv monolayer formed by introducing biotin through chemical modification was not capable of binding phosphorylcholine. These results demonstrate the utility of site-specific, oriented attachment strategies in the formation of protein monolayers in optical sensors, made possible by the use of protein engineering techniques.

Adhesiveness↗
PubMed2005

Design and folding of a multidomain protein.

To test whether the folding process of a large protein can be understood on the basis of the folding behavior of the domains that constitute it, we coupled two well-studied small -helical proteins, the B-domain of protein A (60 amino acids) and Rd-apocytochrome b562 (Rd-apocyt b562, 106 amino acids), by fusing the C-terminal helix of the B-domain of protein A with the N-terminal helix of Rd-apocyt b562 without changing their hydrophobic core residues. The success of the design was confirmed by determining the structure of the engineered protein with multidimensional NMR methods. Kinetic studies showed that the logarithms of the folding/unfolding rate constants of the engineered protein are linearly dependent on concentrations of guanidinium chloride in the measurable range from 1.7 to 4 M. Their slopes (m-values) are close to those of Rd-apocyt b562. In addition, the 1H-15N HSQC spectrum taken at 1.5 M guanidinium chloride reveals that only the Rd-apocyt b562 domain in the designed protein remained folded. These results suggest that the two domains have weak energetic coupling. Interestingly, the redesigned protein folds faster than Rd-apocyt b562, suggesting that the fused helix stabilizes the rate-limiting transition state.

Cytochrome b Group↗
PubMed2006

Engineering controllable protein degradation.

Complex biological networks are regulated via alterations in protein expression, degradation, and function. Synthetic control of these processes allows dissection of natural systems and the design of new networks. In E. coli, the adaptor SspB tethers ssrA-tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. To engineer controlled degradation, we have designed a series of modified ssrA tags that have weakened interactions with ClpXP. When SspB is present, ClpXP degrades purified substrates bearing these engineered peptide tags 100-fold more efficiently. Importantly, substrates bearing these tags are stable in the absence of SspB in vivo but are rapidly degraded upon SspB induction. Our studies supply a conceptual foundation and working components for controllable degradation, improve mechanistic understanding of adaptor-mediated proteolysis, and demonstrate that the relative importance of adaptor proteins in degradation is correlated with the strength of protease-substrate contacts.

Amino Acid Sequence↗
PubMed2001

Properties of exogenously added GPI-anchored proteins following their incorporation into cells.

Isolated glycosylphosphatidylinositol (GPI)-anchored proteins, when added to cells in vitro, incorporate into their surface membranes and, once incorporated, exert their native functions. Virtually any protein of interest, if expressed as a GPI-reanchored derivative, can be modified to acquire this capacity. Such transfer of proteins directly to cells, termed "protein engineering" or "painting" constitutes an alternative to conventional gene transfer for manipulating cell surface composition that has many potential applications. Previous studies with incorporated GPI-anchored proteins have focused almost entirely on their extracellular functions. In this study, biotinylated human erythrocyte (E(hu)) decay accelerating factor, E(hu) acetylcholinesterase, and GPI-reanchored murine B7-1 and B7-2 were used as GPI-anchored reporters to characterize their plasma membrane organization and cell signalling properties following addition to Hela or Chinese hamster ovary cells. For each reporter, three types of cell-association were documented; (1) nonphysiological attachment and/or incomplete insertion, (2) uncomplexed membrane integration, and (3) organization into TX-100-resistant microdomains. Transit from the first two compartments into the third, i.e., microdomains, progressed slowly, continuing even after 24 to 36 h and was associated with the acquisition of cell signalling capacity. All four reporters, incorporated in two different detergents, behaved similarly. When organized in microdomains, caveolin and other GPI proteins co-isolated with the incorporated reporter. These results have implications for protein engineering of cells in general, and in particular, for cells such as modified tumor cell immunogens administered to patients for therapeutic purposes.

Acetylcholinesterase↗
PubMed2002

Screening and selection methods for large-scale analysis of protein function.

High-throughput assays hold tremendous promise for protein engineering and proteomics. With powerful assays it should be possible to evolve, for example, a stereoselective esterase for the chemical synthesis or a site-specific endonuclease for biomedical research. Entire cDNA libraries, which encode all of the proteins expressed in a given organism or cell line, should simply be passed through a battery of biochemical assays to determine the function of each individual protein. Herein we look at the types of assays that have been developed and how close we are to our goals of engineering proteins with new activities as well as rapidly assigning function to the thousands of proteins that make up each genome.

Amino Acid Sequence↗
PubMed2003

An affibody in complex with a target protein: structure and coupled folding.

Combinatorial protein engineering provides powerful means for functional selection of novel binding proteins. One class of engineered binding proteins, denoted affibodies, is based on the three-helix scaffold of the Z domain derived from staphylococcal protein A. The Z(SPA-1) affibody has been selected from a phage-displayed library as a binder to protein A. Z(SPA-1) also binds with micromolar affinity to its own ancestor, the Z domain. We have characterized the Z(SPA-1) affibody in its uncomplexed state and determined the solution structure of a Z:Z(SPA-1) protein-protein complex. Uncomplexed Z(SPA-1) behaves as an aggregation-prone molten globule, but folding occurs on binding, and the original (Z) three-helix bundle scaffold is fully formed in the complex. The structural basis for selection and strong binding is a large interaction interface with tight steric and polar/nonpolar complementarity that directly involves 10 of 13 mutated amino acid residues on Z(SPA-1). We also note similarities in how the surface of the Z domain responds by induced fit to binding of Z(SPA-1) and Ig Fc, respectively, suggesting that the Z(SPA-1) affibody is capable of mimicking the morphology of the natural binding partner for the Z domain.

Antibodies↗
PubMed2006

Selection of novel vesicular stomatitis virus glycoprotein variants from a peptide insertion library for enhanced purification of retroviral and lentiviral vectors.

The introduction of new features or functions that are not present in an original protein is a significant challenge in protein engineering. For example, modifications to vesicular stomatitis virus glycoprotein (VSV-G), which is commonly used to pseudotype retroviral and lentiviral vectors for gene delivery, have been hindered by a lack of structural knowledge of the protein. We have developed a transposon-based approach that randomly incorporates designed polypeptides throughout a protein to generate saturated insertion libraries and a subsequent high-throughput selection process in mammalian cells that enables the identification of optimal insertion sites for a novel designed functionality. This method was applied to VSV-G in order to construct a comprehensive library of mutants whose combined members have a His6 tag inserted at likely every site in the original protein sequence. Selecting the library via iterative retroviral infections of mammalian cells led to the identification of several VSV-G-His6 variants that were able to package high-titer viral vectors and could be purified by Ni-nitrilotriacetic acid affinity chromatography. Column purification of vectors reduced protein and DNA impurities more than 5,000-fold and 14,000-fold, respectively, from the viral supernatant. This substantially improved purity elicited a weaker immune response in the brain, without altering the infectivity or tropism from wild-type VSV-G-pseudotyped vectors. This work applies a powerful new tool for protein engineering to construct novel viral envelope variants that can greatly improve the safety and use of retroviral and lentiviral vectors for clinical gene therapy. Furthermore, this approach of library generation and selection can readily be extended to other challenges in protein engineering.

Animals↗
PubMed2006

Immobilization of histidine-tagged proteins by magnetic nanoparticles encapsulated with nitrilotriacetic acid (NTA)-phospholipids micelle.

We described the development of functionalized magnetic nanoparticles (MNPs) with PEG-modification, a phospholipids micelle coating, and their use in manipulating histidine-tagged proteins. Highly monodisperse MNPs were synthesized in an organic solvent and could be phase-transferred into an aqueous solution by encapsulating the nanoparticles with a phospholipids micelle. The phospholipids micelle coating rendered the nanoparticles highly water-soluble, and the functional groups of the phospholipids coating allowed for the bioconjugation of various moieties, such as fluorescent molecules and engineered proteins. Functionalized phospholipids, such as nitrilotriacetic acid (NTA)-phospholipids, caused the MNPs to bind and allowed for manipulation of histidine-tagged proteins. Due to their high surface/volume ratio, the MNPs showed better performance (about 100 times higher) in immobilizing engineered proteins than conventional micrometer-sized beads. This demonstrates that MNPs coated with phospholipids micelle can be a versatile platform for the effective manipulation of various kinds of engineered proteins, which is very important in the field of proteomics. It is expected that a combination of MNPs with optical fluorescent molecules can find applications in bimodal (magnetic and optical) molecular imaging nanoprobes.

Coated Materials, Biocompatible↗
PubMed2006

The folding pathway of T4 lysozyme: an on-pathway hidden folding intermediate.

T4 lysozyme has two easily distinguishable but energetically coupled domains: the N and C-terminal domains. In earlier studies, an amide hydrogen/deuterium exchange pulse-labeling experiment detected a stable submillisecond intermediate that accumulates before the rate-limiting transition state. It involves the formation of structures in both the N and C-terminal regions. However, a native-state hydrogen exchange experiment subsequently detected an equilibrium intermediate that only involves the formation of the C-terminal domain. Here, using stopped-flow circular dichroism and fluorescence, amide hydrogen exchange-folding competition, and protein engineering methods, we re-examined the folding pathway of T4-lysozyme. We found no evidence for the existence of a stable folding intermediate before the rate-limiting transition state at neutral pH. In addition, using native-state hydrogen exchange-directed protein engineering, we created a mimic of the equilibrium intermediate. We found that the intermediate mimic folds with the same rate as the wild-type protein, suggesting that the equilibrium intermediate is an on-pathway intermediate that exists after the rate-limiting transition state.

Bacteriophage T4↗
PubMed2004

Modulation of S6 fibrillation by unfolding rates and gatekeeper residues.

We present a protein engineering analysis of the fibrillation of a protein from a thermophilic organism, the 101 residue S6 from Thermus thermophilus. When agitated, S6 fibrillates at pH 2.0 in 0.4 M NaCl. Under these solvent conditions, S6 has native-like secondary structure and also unfolds and refolds cooperatively. However, its tertiary structure appears to be more plastic than at neutral pH, and some regions of the protein may be partially unstructured. At 42 degrees C, there is a lag phase of several days after which fibrillation takes place over several hours. Data from the fibrillation behaviour of a comprehensive series of single and double mutants of S6 suggests that several factors control the onset of fibrillation. Firstly, there appears to be a contiguous region of "gatekeeper" residues that inhibit fibrillation, since their truncation significantly reduces the duration of the lag phase. This region overlaps extensively with the partially unstructured region of the protein, suggesting that residues with enhanced flexibility and solvent-accessibility are important for the initiation of fibrillation. Secondly, longer lag phases correlate with faster rates of unfolding. We interpret this to mean that kinetic stability also controls fibrillation but in the sense that the quasi-native state, rather than the denatured state, is the species that participates in nucleation. This implies that fibrillation can also occur from a quasi-native state as opposed to an ensemble of highly fluctuating structures, and highlights the delicate balance between flexibility and structure required to form organized assemblies of polypeptide chains.

Amino Acids↗
PubMed2005

Design and construction of highly stable, protease-resistant chimeric avidins.

The chicken avidin gene family consists of avidin and seven separate avidin-related genes (AVRs) 1-7. Avidin protein is a widely used biochemical tool, whereas the other family members have only recently been produced as recombinant proteins and characterized. In our previous study, AVR4 was found to be the most stable biotin binding protein thus far characterized (T(m) = 106.4 degrees C). In this study, we studied further the biotin-binding properties of AVR4. A decrease in the energy barrier between the biotin-bound and unbound state of AVR4 was observed when compared with that of avidin. The high resolution structure of AVR4 facilitated comparison of the structural details of avidin and AVR4. In the present study, we used the information obtained from these comparative studies to transfer the stability and functional properties of AVR4 to avidin. A chimeric avidin protein, ChiAVD, containing a 21-amino acid segment of AVR4 was found to be significantly more stable (T(m) = 96.5 degrees C) than native avidin (T(m) = 83.5 degrees C), and its biotin-binding properties resembled those of AVR4. Optimization of a crucial subunit interface of avidin by an AVR4-inspired point mutation, I117Y, significantly increased the thermostability of the avidin mutant (T(m) = 97.5 degrees C) without compromising its high biotin-binding properties. By combining these two modifications, a hyperthermostable ChiAVD(I117Y) was constructed (T(m) = 111.1 degrees C). This study provides an example of rational protein engineering in which another member of the protein family has been utilized as a source in the optimization of selected properties.

Amino Acid Sequence↗
PubMed1991

Pathway and stability of protein folding.

We describe an experimental approach to the problem of protein folding and stability which measures interaction energies and maps structures of intermediates and transition states during the folding pathway. The strategy is based on two steps. First, protein engineering is used to remove interactions that stabilize defined positions in barnase, the RNAse from Bacillus amyloliquefaciens. The consequent changes in stability are measured from the changes in free energy of unfolding of the protein. Second, each mutation is used as a probe of the structure around the wild-type side chain during the folding process. Kinetic measurements are made on the folding and unfolding of wild-type and mutant proteins. The kinetic and thermodynamic data are combined and analysed to show the role of individual side chains in the stabilization of the folded, transition and intermediate states of the protein. The protein engineering experiments are corroborated by nuclear magnetic resonance studies of hydrogen exchange during the folding process. Folding is a multiphasic process in which alpha-helices and beta-sheet are formed relatively early. Formation of the hydrophobic core by docking helix and sheet is (partly) rate determining. The final steps involve the forming of loops and the capping of the N-termini of helices.

Amino Acid Sequence↗
PubMed2003

Genetic engineering of an allosterically based glucose indicator protein for continuous glucose monitoring by fluorescence resonance energy transfer.

Real-time monitoring of blood glucose could vastly reduce a number of the long-term complications associated with diabetes. In this article, we present a novel approach that relies on a glucose-binding protein engineered such that a 20% reduction in fluorescence due to the fluorescence resonance energy transfer occurs as a result of glucose binding. This change in fluorescence provides a signal for the optical detection of glucose. The novel glucose indicator protein (GIP) was created by fusing two fluorescent reporter proteins (green fluorescent proteins) to each end of an Escherichia coli glucose-binding protein in such a manner that the spatial separation between the fluorescent moieties changes when glucose binds, thus generating a distinct optical signal that can be used for glucose detection. By placing the GIP within a dialysis hollow fiber sensor, a microsensor has been developed for continuous monitoring of glucose. The sensor had a response time to sudden glucose changes within 100 s and was reversible. The sensor was shown to have an optional range on the order of 10 microM of glucose.

Amino Acid Sequence↗
PubMed2007

Protein library design and screening: working out the probabilities.

In designing protein libraries for selection, we must coordinate our capacity to create a large diversity of protein variants with the physical limitations of what we can actually screen. This chapter aims to bring the language of probabilities into the protein engineer's laboratory to answer some of our common questions: How can we most efficiently design a library? What fraction of the theoretical library diversity have we actually sampled at the end of the day? What is the probability of missing an individual of the library? Are the mutations present in the variants we have selected statistically meaningful or the product of random variation? The computation of these criteria throughout the process of experimental protein engineering will enable us to better design and evaluate the products of our libraries of protein variants.

Combinatorial Chemistry Techniques↗
PubMed2005

Engineering high affinity superantigens by phage display.

Protein L (PpL) is a B-cell superantigen from Peptostreptococcus magnus known to bind to mammalian Vkappa light chains. PpL from P.magnus strain 312 comprises five homologous immunoglobulin (Ig) binding domains. We first analysed the binding of the individual domains (B1-B5) of PpL(312) to human Vkappa light chains (huVkappa) subtypes 1 (huVkappaI) and 3 (huVkappaIII). Using a combination of rational design and phage selection we isolated mutants of the N-terminal B1 domain with a 14-fold increased affinity for huVkappa1 (B1kappa1) and >tenfold increased affinity for huVkappaIII (B1kappa3). We investigated the potential of the selected domains, in particular the B1kappa1 domain, as reagents in immunochemistry and immunotherapy. B1kappa1 proved a superior reagent than the wild-type domain, allowing up to tenfold more sensitive detection of human Vkappa antibody fragments in ELISA. A fusion protein of B1kappa1 with a human Vlambda antibody scFv fragment promoted the efficient recruitment of antibody encoded effector functions including complement, mononuclear phagocyte respiratory burst and phagocytosis through retargeting of IgGkappa and IgMkappa. Our results suggest that superantigens with improved affinity and/or specificity are easily accessible through protein engineering. Such engineered superantigens should prove useful as reagents in immunochemistry and may have potential as agents in immunotherapy.

Amino Acid Sequence↗
PubMed2004

Removal of N-terminal methionine from recombinant proteins by engineered E. coli methionine aminopeptidase.

The removal of N-terminal translation initiator Met by methionine aminopeptidase (MetAP) is often crucial for the function and stability of proteins. On the basis of crystal structure and sequence alignment of MetAPs, we have engineered Escherichia coli MetAP by the mutation of three residues, Y168G, M206T, Q233G, in the substrate-binding pocket. Our engineered MetAPs are able to remove the Met from bulky or acidic penultimate residues, such as Met, His, Asp, Asn, Glu, Gln, Leu, Ile, Tyr, and Trp, as well as from small residues. The penultimate residue, the second residue after Met, was further removed if the antepenultimate residue, the third residue after Met, was small. By the coexpression of engineered MetAP in E. coli through the same or a separate vector, we have successfully produced recombinant proteins possessing an innate N terminus, such as onconase, an antitumor ribonuclease from the frog Rana pipiens. The N-terminal pyroglutamate of recombinant onconase is critical for its structural integrity, catalytic activity, and cyto-toxicity. On the basis of N-terminal sequence information in the protein database, 85%-90% of recombinant proteins should be produced in authentic form by our engineered MetAPs.

Amino Acid Substitution↗
PubMed1998

Structure of the transition state in the folding process of human procarboxypeptidase A2 activation domain.

The transition state for the folding pathway of the activation domain of human procarboxypeptidase A2 (ADA2h) has been analyzed by the protein engineering approach. Recombinant ADA2h is an 81-residue globular domain with no disulfide bridges or cis-prolyl bonds, which follows a two-state folding transition. Its native fold is arranged in two alpha-helices packing against a four-stranded beta-sheet. Application of the protein engineering analysis for 20 single-point mutants spread throughout the whole sequence indicates that the transition state for this molecule is quite compact, possessing some secondary structure and a hydrophobic core in the process of being consolidated. The core (folding nucleus) is made by the packing of alpha-helix 2 and the two central beta-strands. The other two strands, at the edges of the beta-sheet, and alpha-helix 1 seem to be completely unfolded. These results, together with previous analysis of ADA2h with either of its two alpha-helices stabilized through improved local interactions, suggest that alpha-helix 1 does not contribute to the folding nucleus, even though it is partially folded in the denatured state under native conditions. On the other hand, alpha-helix 2 folds partly in the transition state and is part of the folding nucleus. It is suggested that a good strategy to improve folding speed in proteins would be to stabilize the helices that are not folded in the denatured state but are partly present in the transition state. Comparison with other proteins shows that there is no clear relationship between fold and/or size with folding speed and level of structure in the transition state of proteins.

Amino Acid Sequence↗