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PubMed2004

Molecular engineering of myosin.

Protein engineering and design provide excellent tools to investigate the principles by which particular structural features relate to the mechanisms that underlie the biological function of a protein. In addition to studies aimed at dissecting the communication pathways within enzymes, recent advances in protein engineering approaches make it possible to generate enzymes with increased catalytic efficiency and specifically altered or newly introduced functions. Here, two approaches using state-of-the-art protein design and engineering are described in detail to demonstrate how key features of the myosin motor can be changed in a specific and predictable manner. First, it is shown how replacement of an actin-binding surface loop with synthetic sequences, whose flexibility and charge density is varied, can be employed to manipulate the actin affinity, the catalytic activity and the efficiency of coupling between actin- and nucleotide-binding sites of myosin motor constructs. Then the use of pre-existing molecular building blocks, which are derived from unrelated proteins, is described for manipulating the velocity and even the direction of movement of recombinant myosins.

Actins↗
PubMed2007

Enhanced protein production by engineered zinc finger proteins.

Increasing the yield of therapeutic proteins from mammalian production cell lines reduces costs and decreases the time to market. To this end, we engineered a zinc finger protein transcription factor (ZFP TF) that binds a DNA sequence within the promoter driving transgene expression. This ZFP TF enabled >100% increase in protein yield from CHO cells in transient, stable, and fermentor production run settings. Expression vectors engineered to carry up to 10 ZFP binding sites further enhanced ZFP-mediated increases in protein production up to approximately 500%. The multimerized ZFP binding sites function independently of the promoter, and therefore across vector platforms. CHO cell lines stably expressing ZFP TFs demonstrated growth characteristics similar to parental cell lines. ZFP TF expression and gains in protein production were stable over >30 generations in the absence of antibiotic selection. Our results demonstrate that ZFP TFs can rapidly and stably increase protein production in mammalian cells.

Animals↗
PubMed1997

Adenosylcobalamin-dependent glutamate mutase: examination of substrate and coenzyme binding in an engineered fusion protein possessing simplified subunit structure and kinetic properties.

Glutamate mutase is comprised of two weakly associating subunits; E and S, that combine to form the coenzyme binding site. The active holoenzyme assembles in a kinetically complex process in which both the stoichiometry and apparent Kd for adenosylcobalamin (AdoCbl) are dependent upon the relative concentrations of the two subunits, as is the enzyme's specific activity. To facilitate mechanistic and structural studies on this enzyme we have genetically fused the S subunit to the C-terminus of the E subunit through an 11 amino acid (Gly-Gln)5-Gly linker segment. This protein, GlmES, binds AdoCbl stoichiometrically and neither the affinity for AdoCbl nor the turnover number depends upon protein concentration. The kcat and Km for both substrate and coenzyme, together with the deuterium isotope effects on Vmax and Vmax/Km, have been determined for the GlmES-catalyzed reaction proceeding in both directions. Compared with wild type, the affinity for AdoCbl is unchanged, but for the conversion of L-glutamate to (2S,3S)-3-methylaspartate both kcat and Km for L-glutamate are decreased by about a third and the isotope effects are reduced, suggesting product release to be more rate-limiting. To test hypotheses concerning the activation of the coenzyme, we examined the binding of adenosylcobalamin, methylcobalamin, and cob(II)alamin to the enzyme. Each of these is bound with essentially the same affinity (2 microM), suggesting that, contrary to expectations, interactions between the protein and the adenosyl moiety do not serve to weaken the cobalt-carbon bond in the ground state.

Apoenzymes↗
PubMed2006

Structure of anti-FLAG M2 Fab domain and its use in the stabilization of engineered membrane proteins.

The inherent difficulties of stabilizing detergent-solubilized integral membrane proteins for biophysical or structural analysis demand the development of new methodologies to improve success rates. One proven strategy is the use of antibody fragments to increase the ;soluble' portion of any membrane protein, but this approach is limited by the difficulties and expense associated with producing monoclonal antibodies to an appropriate exposed epitope on the target protein. Here, the stabilization of a detergent-solubilized K(+) channel protein, KvPae, by engineering a FLAG-binding epitope into a known loop region of the protein and creating a complex with Fab fragments from commercially available anti-FLAG M2 monoclonal antibodies is reported. Although well diffracting crystals of the complex have not yet been obtained, during the course of crystallization trials the structure of the anti-FLAG M2 Fab domain was solved to 1.86 A resolution. This structure, which should aid future structure-determination efforts using this approach by facilitating molecular-replacement phasing, reveals that the binding pocket appears to be specific only for the first four amino acids of the traditional FLAG epitope, namely DYKD. Thus, the use of antibody fragments for improving the stability of target proteins can be rapidly applied to the study of membrane-protein structure by placing the short DKYD motif within a predicted peripheral loop of that protein and utilizing commercially available anti-FLAG M2 antibody fragments.

Amino Acid Sequence↗
PubMed1992

A genetically engineered purpurin/retinol-binding protein hybrid that binds to transthyretin.

A mini-gene encoding rat retinol-binding protein (RBP) and a cDNA encoding chicken purpurin were separately transfected into HeLa cells. In contrast to RBP, expressed purpurin did not bind to transthyretin (TTR). A purpurin/RBP hybrid protein was constructed by substituting the cDNA sequence encoding the N-terminal 29 amino acids of purpurin for the corresponding part of RBP. The expressed hybrid molecule bound to the TTR-Sepharose. These results demonstrate that purpurin does not bind to TTR, that a functional purpurin/RBP hybrid can be constructed, and that the N-terminal coil of RBP is not required for TTR binding.

Amino Acid Sequence↗
PubMed2006

Conformational stabilization of an engineered binding protein.

We analyzed the thermodynamic basis for improvement of a binding protein by disulfide engineering. The Z(SPA)(-)(1) affibody binds to its Z domain binding partner with a dissociation constant K(d) = 1.6 microM, and previous analyses suggested that the moderate affinity is due to the conformational heterogeneity of free Z(SPA)(-)(1) rather than to a suboptimal binding interface. Studies of five stabilized Z(SPA)(-)(1) double cystein mutants show that it is possible to improve the affinity by an order of magnitude to K(d) = 130 nM, which is close to the range (20 to 70 nM) observed with natural Z domain binders, without altering the protein-protein interface obtained by phage display. Analysis of the binding thermodynamics reveals a balance between conformational entropy and desolvation entropy: the expected and favorable reduction of conformational entropy in the best-binding Z(SPA)(-)(1) mutant is completely compensated by an unfavorable loss of desolvation entropy. This is consistent with a restriction of possible conformations in the disulfide-containing mutant and a reduction of average water-exposed nonpolar surface area in the free state, resulting in a smaller conformational entropy penalty, but also a smaller change in surface area, for binding of mutant compared to wild-type Z(SPA)(-)(1). Instead, higher Z domain binding affinity in a group of eight Z(SPA)(-)(1) variants correlates with more favorable binding enthalpy and enthalpy-entropy compensation. These results suggest that protein-protein binding affinity can be improved by stabilizing conformations in which enthalpic effects can be fully explored.

Algorithms↗
PubMed2002

Recombinantly engineered human proteins: transforming the treatment of psoriasis.

Psoriasis is a chronic, inflammatory disease with lesions that produce considerable physical discomfort and often lead to substantial disruption in patients' daily activities. The use of currently available, nonspecific, systemic immunosuppressive therapies for patients with moderate to severe psoriasis is limited by an inability to maintain disease remission safely. Advances in recombinant DNA technology paralleled with increased understanding of the immunopathology of psoriasis have led to the development of numerous biologic agents for the treatment of this disease. These new biologic therapies target specific steps in psoriasis pathology, including direct effects on T cells, T cell activation, T cell migration, and cellular production and secretion of cytokines. By selectively targeting the activities of T cells that are directly involved in psoriasis pathogenesis, these novel agents offer improved safety profiles and enhanced efficacy. In this article, the mechanisms of T cell pathogenicity that guided the development of these new biologic therapies are reviewed along with clinical data on the progress of these agents.

Alefacept↗
PubMed2004

Engineering a thermostable protein with two DNA-binding domains using the hyperthermophile protein Sac7d.

The acid- and thermostable Sac7d is a small, non-specific DNA-binding protein of the hyperthermophile archaea Sulfolobus acidocaldarius. In this study, Sac7d was employed as a structural unit in the design of a thermostable protein containing two putative DNA-binding domains. By linking two Sac7d proteins together and comparing the DNA interaction of dimer to that of monomer, this study may provide structural insights into other dimeric DNA-binding proteins. The engineered protein, Sac7dK66C, was over-expressed and purified. Dimeric Sac7d was obtained by cross-linking two mutant Sac7d molecules through the C-terminal disulfide bond. Thermal stability and DNA-binding ability of dimeric Sac7d were assessed and compared to those of wild type Sac7d by gel retardation assay, circular dichroism spectroscopy, and crystallization experiments. Dimeric Sac7d was shown to be equally thermostable as wild type, and its ability to stabilize DNA duplex is the same as wild type. However, the interaction of dimeric Sac7d with DNA diverged from that of wild type, suggesting different DNA-binding modes for dimeric Sac7d. In addition, a large difference in extinction coefficient was observed in all dimer/DNA CD spectra, which was reminiscent of the spectrum of Psi-DNA. Conjugation of various chemical groups to mutant Sac7d is possible through the C-terminal thiol group. This offers a possible approach in the design of a thermostable biomolecule with novel functions.

Amino Acid Sequence↗
PubMed1991

Heterologous protein production by filamentous fungi.

There are clearly many facets to successful production of heterologous proteins from filamentous fungi. The objectives are to exploit the natural ability of some species to secrete high levels of protein. The heterologous target proteins produced in a fungal host must be acceptable to the public and be economic to produce, i.e. the targets must be authentic (in structure and activity) and be produced in high yield to necessary levels of purity. The appearance of heterologous products from fungi on the market is testament to some success but, equally, there are considerable limitations in our ability to produce desired yields of many target proteins. We endorse the view of van den Hondel, Punt and van Gorcom (1991) that for the commercial production of heterologous proteins from filamentous fungi more information is required on transcriptional control, introns, mRNA stability and processing, translational efficiency, protein secretion, glycosylation and proteolysis. In addition, there is scope for yield improvement based on a better understanding of the physiology of growth/product secretion coupled to appropriate bioreactor operation. The authenticity of product is an aspect which will assume increasing importance, particularly for therapeutic proteins. The level at which the structures and functional activity of heterologous proteins are assessed will ultimately be determined by legislation. The analytical methods currently available are not always sufficient, for example, to reveal folded structures, and most proteins are not amenable to analysis by two-dimensional NMR. The authenticity of target heterologous proteins will also need to be assessed in relation to the glycosylation level and pattern. This is not easily done and explains the paucity of detailed information published to date on glycosylation of fungal proteins. Novel engineered proteins are already being produced from filamentous fungi where expression is an aid to investigation of structure-function relationships. Commercial production of such engineered proteins will require approval subject to a range of stringently applied tests and analyses. This imposes an even greater need to be able to specify and control, in a rational manner, the structures of recombinant proteins. The research needs for realization of improved yields are equally important in assuring authenticity of product. It is encouraging that progress is being made on all fronts, primarily with Aspergillus spp. and T. reesei, but also with other species, such as N. crassa.

Animals↗
PubMed1995

Advances in the use of Bacillus subtilis for the expression and secretion of heterologous proteins.

During the past year, significant progress has been made using Bacillus subtilis to produce a wide range of foreign proteins. Through strain improvement and co-expression of molecular chaperones, secretory proteins can be produced at a higher level. Through protein engineering, target proteins can be redesigned to have better stability and solubility. A combination of these two strategies would be a useful approach to produce heterologous proteins from B. subtilis at high quality and with a high yield.

Bacillus subtilis↗
PubMed2000

Reverse engineering a protein: the mechanochemistry of ATP synthase.

ATP synthase comprises two rotary motors in one. The F(1) motor can generate a mechanical torque using the hydrolysis energy of ATP. The F(o) motor generates a rotary torque in the opposite direction, but it employs a transmembrane proton motive force. Each motor can be reversed: The F(o) motor can drive the F(1) motor in reverse to synthesize ATP, and the F(1) motor can drive the F(o) motor in reverse to pump protons. Thus ATP synthase exhibits two of the major energy transduction pathways employed by the cell to convert chemical energy into mechanical force. Here we show how a physical analysis of the F(1) and F(o) motors can provide a unified view of the mechanochemical principles underlying these energy transducers.

Adenosine Triphosphate↗
PubMed2003

Engineering a protein scaffold from a PHD finger.

The design of proteins with tailored functions remains a relatively elusive goal. Small size, a well-defined structure, and the ability to maintain structural integrity despite multiple mutations are all desirable properties for such designer proteins. Many zinc binding domains fit this description. We determined the structure of a PHD finger from the transcriptional cofactor Mi2beta and investigated the suitability of this domain as a scaffold for presenting selected binding functions. The two flexible loops in the structure were mutated extensively by either substitution or expansion, without affecting the overall fold of the domain. A binding site for the corepressor CtBP2 was also grafted onto the domain, creating a new PHD domain that can specifically bind CtBP2 both in vitro and in the context of a eukaryotic cell nucleus. These results represent a step toward designing new regulatory proteins for modulating aberrant gene expression in vivo.

Amino Acid Sequence↗
PubMed1998

Genetic engineering of proteins with cell membrane permeability.

The discovery of methods for generating proteins with inherent cell membrane-translocating activity will expand our ability to study and manipulate various intracellular processes in living systems. We report a method to engineer proteins with cell-membrane permeability. After a 12-amino acid residue membrane-translocating sequence (MTS) was fused to the C-terminus of glutathione S-transferase (GST), the resultant GST-MTS fusion proteins were efficiently imported into NIH 3T3 fibroblasts and other cells. To explore the applicability of this nondestructive import method to the study of intracellular processes, a 41-kDa GST-Grb2SH2-MTS fusion protein containing the Grb2 SH2 domain was tested for its effect on the epidermal growth factor (EGF)-stimulated signaling pathway. This fusion protein entered cells, formed a complex with phosphorylated EGF receptor (EGFR), and inhibited EGF-induced EGFR-Grb2 association and mitogen-activated protein kinase activation.

3T3 Cells↗
PubMed1996

Mutational modulation of substrate bond-type specificity and thermostability of glucoamylase from Aspergillus awamori by replacement with short homologue active site sequences and thiol/disulfide engineering.

Rational protein engineering based on three-dimensional structure, sequence alignment, and previous mutational analysis served to increase thermostability and modulate bond-type specificity in glucoamylase from Aspergillus awamori. The single free cysteine, Cys320, became disulfide bonded in the Ala246 --> Cys mutant, thus enhancing T50 by 4 degrees C to 73 degrees C. Compared to wild-type, Ala246 --> Cys was roughly twice as active at 66 degrees C, but half as active at 45 degrees C. The alternative, elimination of the thiol group in Cys320 --> Ala, barely improved thermostability or altered activity. Secondly, to acquire exceptionally high specificity toward alpha-1,6 glucosidic linkages, characteristic of Hormoconis resinae glucoamylase, two short sequential mutants, Val181 --> Thr/Asn182 --> Tyr/Gly183 --> Ala(L3 glucoamylase) and Pro307 --> Ala/Thr310 --> Val/Tyr312 --> Met/Asn313 --> Gly (L5 glucoamylase), were made. These homologue mutants are located in the (alpha/alpha)6-fold of the catalytic domain in segments that connect alpha-helices 5 and 6 and alpha-helices 9 and 10, respectively. The kinetics of malto- and isomaltooligosaccharides hydrolysis clearly demonstrated that combination of the mutations in L3L5 compensated adverse effects of the single replacements in L3 or L5 glucoamylases to yield wild-type or higher activity. On alpha-1,4-linked substrates, typically Km increased 2-fold for L3, and Kcat decreased up to 15-fold for L5 glucoamylase. In contrast, on alpha-1,6-linked substrates L3 showed both a 2-fold increase in Km and a 3-fold decrease in kcat, while L5 GA caused a similar kcat reduction, but up to 9-fold increase in Km. L3L5 glucoamylase had remarkably low Km for isomaltotriose through isomaltoheptaose and elevated kcat on isomaltose, resulting in an approximately 2-fold improved catalytic efficiency (kcat/Km). Rational loop replacement thus proved powerful in achieving variants with enhanced properties of a highly evolved enzyme.

Amino Acid Sequence↗
PubMed2006

The role of protein dynamics in increasing binding affinity for an engineered protein-protein interaction established by H/D exchange mass spectrometry.

It is generally accepted that protein and solvation dynamics play fundamental roles in the mechanisms of protein-protein binding; however, assessing their contribution meaningfully has not been straightforward. Here, hydrogen/deuterium exchange mass spectrometry (H/D-Ex) was employed to assess the role of dynamics for a high-affinity human growth hormone variant (hGHv) and the wild-type growth hormone (wt-hGH) each binding to the extracellular domain of their receptor (hGHbp). Comparative analysis of the transient fluctuations in the bound and unbound states revealed that helix-1 of hGHv undergoes significant transient unfolding in its unbound state, a characteristic that was not found in wt-hGH or apparent in the temperature factor data from the X-ray analysis of the unbound hGHv structure. In addition, upon hormone binding, an overall increase in stability was observed for the beta-sheet structure of hGHbp which included sites distant from the binding interface. On the basis of the stability, binding kinetics, and thermodynamic data presented, the increase in the binding free energy of hGHv is primarily generated by factors that appear to increase the energy of the unbound state relative to the free energy of the bound complex. This implies that an alternate route to engineer new interactions aiming to increase protein-protein association energies may be achieved by introducing certain mutations that destabilize one of the interacting molecules without destabilizing the resulting bound complex. Importantly, although the hGHv molecule is less stable than its wt-hGH counterpart, its resulting active ternary complex with two copies of hGHbp has comparable stability to the wt complex.

Binding Sites↗
PubMed2007

Fluorescent proteins and engineered cell lines.

Green fluorescent protein and other fluorescent proteins provide powerful tools for high content analysis of cellular processes. Engineering fluorescent protein sensors for expression in cellular assays requires consideration of a wide range of design factors to produce fusion proteins capable of generating informative and biologically relevant data while meeting the rigorous demands of high content screening. The target protein, fluorescent protein, host cell line, construct components and orientation, expression level, and other factors all contribute to the performance of the sensor. This chapter reviews the process of selecting an optimum combination of these design elements for engineering cells for high content analysis.

Animals↗
PubMed2002

Reverse engineering of the giant muscle protein titin.

Through the study of single molecules it has become possible to explain the function of many of the complex molecular assemblies found in cells. The protein titin provides muscle with its passive elasticity. Each titin molecule extends over half a sarcomere, and its extensibility has been studied both in situ and at the level of single molecules. These studies suggested that titin is not a simple entropic spring but has a complex structure-dependent elasticity. Here we use protein engineering and single-molecule atomic force microscopy to examine the mechanical components that form the elastic region of human cardiac titin. We show that when these mechanical elements are combined, they explain the macroscopic behaviour of titin in intact muscle. Our studies show the functional reconstitution of a protein from the sum of its parts.

Connectin↗
PubMed1990

Estimating the contribution of engineered surface electrostatic interactions to protein stability by using double-mutant cycles.

Coulombic interactions between charges on the surface of proteins contribute to stability. It is difficult, however, to estimate their importance by protein engineering methods because mutation of one residue in an ion pair alters the energetics of many interactions in addition to the coulombic energy between the two components. We have estimated the interaction energy between two charged residues, Asp-12 and Arg-16, in an alpha-helix on the surface of a barnase mutant by invoking a double-mutant cycle involving wild-type enzyme (Asp-12, Thr-16), the single mutants Thr----Arg-16 and Asp----Ala-12, and the double mutant Asp----Ala-12, Thr----Arg-16. The changes in free energy of unfolding of the single mutants are not additive because of the coulombic interaction energy. Additivity is restored at high concentrations of salt that shield electrostatic interactions. The geometry of the ion pair in the mutant was assumed to be the same as that in the highly homologous ribonuclease from Bacillus intermedius, binase, which has Asp-12 and Arg-16 in the native enzyme. The ion pair does not form a hydrogen-bonded salt bridge, but the charges are separated by 5-6 A. The mutant barnase containing the ion pair Asp-12/Arg-16 is more stable than wild type by 0.5 kcal/mol, but only a part of the increased stability is attributable to the electrostatic interaction. We present a formal analysis of how double-mutant cycles can be used to measure the energetics of pairwise interactions.

Amino Acid Sequence↗