Application of a very high-throughput digital imaging screen to evolve the enzyme galactose oxidase.

Directed evolution has become an important enabling technology for the development of new enzymes in the chemical and pharmaceutical industries. Some of the most interesting substrates for these enzymes, such as polymers, have poor solubility or form highly viscous solutions and are therefore refractory to traditional high-throughput screens used in directed evolution. We combined digital imaging spectroscopy and a new solid-phase screening method to screen enzyme variants on problematic substrates highly efficiently and show here that the specific activity of the enzyme galactose oxidase can be improved using this technology. One of the variants we isolated, containing the mutation C383S, showed a 16-fold increase in activity, due in part to a 3-fold improvement in K(m). The present methodology should be applicable to the evolution of numerous other enzymes, including polysaccharide-modifying enzymes that could be used for the large-scale synthesis of modified polymers with novel chemical properties.

Molecular phylogenetic evidence for noninvasive zoonotic transmission of Staphylococcus intermedius from a canine pet to a human.

rRNA-based molecular phylogenetic techniques were used to identify the bacterial species present in the ear fluid from a female patient with otitis externa. We report the identification of Staphylococcus intermedius from the patient and a possible route of transmission. Analysis of 16S ribosomal DNA restriction fragment length polymorphisms indicated that the dominant species present was S. intermedius. A pet dog owned by the patient also was tested and found to harbor S. intermedius. In humans, the disease is rare and considered a zoonosis. Previously, S. intermedius has been associated with dog bite wounds, catheter-related injuries, and surgery. This study represents the first reported case of a noninvasive infection with S. intermedius.

Characterization of a symmetrized mutant RC with 42 residues from the QA site replacing residues in the Q(B) site.

The electron transfer reactions involving Q(A) and Q(B) were investigated in Rb. capsulatus RCs where the Q(B) site was mutated to contain 42 residues from the Q(A) site. The RCs have M220-M261 in the Q(A) site substituted for L193-L227 in the Q(B) site plus the M subunit second-site mutations, M144MI and M145AS, which had been found to restore the ability of the bacteria to grow photosynthetically. These mutants lack L210D, L212E, L213D, and L223S which have been previously shown to affect the electron transfer from Q(A) (-) to Q(B). Despite the large change in the Q(B) pocket, secondary quinone function still can be reconstituted. The UQ(4) dissociation constant for the Q(B) site in the mutant is only three times as large as in the wild type RCs. The rate of charge recombination (P(+)Q(A)Q(B) (-) --> PQ(A)Q(B)) (k (BP)) is reduced from 8.9 s(-1) in wild type RCs to 0.05s(-1) in the mutant, This indicates that Q(A)Q(B) (-) is stabilized relative to Q(A) (-)Q(B) by at least 60 meV more than in wild type protein. k (BP) is pH independent in the mutant RCs, while in wild type RCs k (BP) increases at alkaline pHs as reduction of Q(B) becomes energetically less favorable. Similar pH independent, slow k (BP) has been found in the L212EA/L213DA double mutant. The largest change found in the mutant is that the electron transfer from Q(A) (-) to Q(B) (k (AB) ((1)) approximately 14 s(-1)) is 3 orders of magnitude slower than in wild type RCs (10(4) s(-1)).

Hydrogen bonding and circular dichroism of bacteriochlorophylls in the Rhodobacter capsulatus light-harvesting 2 complex altered by combinatorial mutagenesis.

We have investigated the spectroscopic properties of two classes of light-harvesting 2 (LH2, B800-850) mutants of Rhodobacter capsulatus obtained by combinatorial mutagenesis to the C-terminal half of the beta-apoprotein: a pseudoLH2 (pLH2) class, in which the 800-nm absorption was normal but the 850-nm peak was blue-shifted by up to 14 nm, and the other a pseudoLH1 (pLH1) class, which lacked the 800-nm absorption band and showed 850-nm absorption red-shifts of up to 30 nm. In several of the pLH1 antennae, carotenoid depletion contributed to the phenotype, while in the pLH2 complexes there was some carotenoid enrichment. A number of mutants from each class have also been characterized by low-temperature absorption and fluorescence spectroscopy, resonance Raman spectroscopy, and circular dichroism. In all of the mutants investigated, the B850 bacteriochlorophyll a binding site remained intact, conserving both the hydrogen bonding environment of the chromophores and their conformation and liganding. In contrast, the intensity of the CD spectra of pLH1 complexes was considerably reduced, relative to that of wild-type or pLH2 complexes, consistent with alterations in the interactions between pigments and in their relative orientation. Elevated fluorescence polarization over the red wing of the B850 band in the pLH2 complexes indicated a reduction of exciton mobility within the ring of BChl molecules. Possible structural alterations governing the spectral properties of the different mutants are discussed.

Site-directed mutations near the L-subunit D-helix of the purple bacterial reaction center: a partial model for the primary donor of photosystem II.

We have engineered a photosynthetically competent mutant of the purple non-sulfur bacterium Rhodobacter capsulatus which seeks to mimic the behavior of the primary electron donor (P) of the plant photosystem II (PS II) reaction center (RC). To construct this mutant (denoted D1-ILMH), four residues in the bacterial L subunit were mutagenized, such that an 11-residue segment was made identical to the analogous segment from the D1 subunit of PS II. The electronic properties of the bacteriochlorophyll (Bchl) dimer which constitutes the primary donor are substantially altered by these modifications, to the degree that the dimer becomes functionally much more "monomeric". The changes include (1) an increase in the values of the zero-field splitting (ZFS) parameters, as measured by electron paramagnetic resonance (EPR), for the spin-polarized triplet state, 3P, from /D/ = 185 x 10(-4) cm(-1) and /E/ = 31 x 10(-4) cm(-1) in wild-type (WT) chromatophore membranes to /D/ = 200 x 10(-4) cm(-1) and /E/ = 44 x 10(-4) cm(-1) in the mutant and (2) an increase in the EPR line width of the oxidized state, P+, from 0.97 mT in WT to 1.09 mT in D1-ILMH RCs. However, unlike the PS II primary donor (P680), the orientation of 3P in the D1-ILMH mutant is the same as in WT bacteria and does not display the unusual orientation found for PS II. And whereas the redox couple P/P+ has a very high midpoint potential in PS II, P/P+ in the D1-ILMH mutant has a lower midpoint (90 mV more negative) than in WT Rb. capsulatus. In addition, Raman measurements indicate that the hydrogen bond between HisL168 and the C2 acetyl carbonyl oxygen of the Bchl on the active electron transfer pathway (P(A)) is absent in the mutant, due to the fact that HisL168 in the WT sequence has been replaced by a leucine in D1-ILMH. However, the Raman data also reveal the presence of a new hydrogen bond in the D1-ILMH RCs, between the C9 keto carbonyl oxygen of P(A) and an unknown hydrogen-bond donor. Thus, although the protein environment around one of the Bchls of the special pair is significantly changed in D1-ILMH, the chimeric RC does not, as a result of these changes, have a primary donor that is oriented like the one in PS II.

Fluorescence resonance energy transfer between blue-emitting and red-shifted excitation derivatives of the green fluorescent protein.

We report fluorescent resonance energy transfer (FRET) between two linked variants of the green fluorescent protein (GFP). The C terminus of a red-shifted variant of GFP (RSGFP4) is fused to a flexible polypeptide linker containing a Factor X a protease cleavage site. The C terminus of this linker is in turn fused to the N terminus of a blue variant of GFP (BFP5). The gene product has spectral properties that suggest energy transfer is occurring from BFP5 to RSGFP4. Upon incubation with Factor X(a), the protein is cleaved, and the two fluorescent proteins dissociate. This is accompanied by a marked decrease in energy transfer. The RSGFP4::BFP5 fusion protein demonstrates the feasibility of using FRET between two GFP derivatives as a tool to monitor protein-protein interactions; in addition, this construct may find applications as an intracellular screen for protease inhibitors.

Dual color microscopic imagery of cells expressing the green fluorescent protein and a red-shifted variant.

The green fluorescent protein (GFP) from the jellyfish, Aequorea victoria, has become a versatile reporter for monitoring gene expression and protein localization in a variety of cells and organisms. GFP emits bright green light (lambda max = 510 nm) when excited with ultraviolet (UV) or blue light (lambda max = 395 nm, minor peak at 470 nm). The chromophore in GFP is intrinsic to the primary structure of the protein, and fluorescence from GFP does not require additional gene products, substrates or other factors. GFP fluorescence is stable, species-independent and can be monitored noninvasively using the techniques of fluorescence microscopy and flow cytometry [Chalfie et al., Science 263 (1994) 802-805; Stearns, Curr. Biol. 5 (1995) 262-264]. The protein appears to undergo an autocatalytic reaction to create the fluorophore [Heim et al., Proc. Natl. Acad. Sci. USA 91 (1994) 12501-12504] in a process involving cyclization of a Tyr66 aa residue. Recently [Delagrave et al., Bio/Technology 13 (1995) 151-154], a combinatorial mutagenic strategy was targeted at aa 64 through 69, which spans the chromophore of A. victoria GFP, yielding a number of different mutants with red-shifted fluorescence excitation spectra. One of these, RSGFP4, retains the characteristic green emission spectra (lambda max = 505 nm), but has a single excitation peak (lambda max = 490 nm). The fluorescence properties of RSGFP4 are similar to those of another naturally occurring GFP from the sea pansy, Renilla reniformis [Ward and Cormier, Photobiochem. Photobiol. 27 (1978) 389-396]. In the present study, we demonstrate by fluorescence microscopy that selective excitation of A. victoria GFP and RSGFP4 allows for spectral separation of each fluorescent signal, and provides the means to image these signals independently in a mixed population of bacteria or mammalian cells.

Green fluorescent protein: untapped potential in immunotechnology.

Many invertebrates produce bioluminescence using green-fluorescent proteins (GFPs) as energy-transfer acceptors. GFPs fluoresce in vivo upon receiving energy from either a luciferase-oxyluciferin excited-state complex or a Ca(2+)-activated photoprotein depending upon the organism. These highly fluorescent proteins are unique due to the chemical nature of their chromophore, which is comprised of modified amino acid residues within the polypeptide chain. Recently GFP was sequenced and cloned. GFP, GFP mutants or related proteins with altered spectra will have widespread use as a markers of gene expression and as a protein tags in cell culture and in multicellular organisms. Many of the uses of fluorescent-labeled proteins or antibodies in immunotechnology will be improved by the use of GFP. Many new applications were discussed at a recent international symposium [1].

Context dependence of phenotype prediction and diversity in combinatorial mutagenesis.

Two different combinatorial mutagenesis experiments on the light-harvesting II (LH2) protein of Rhodobacter capsulatus indicate that heuristic rules relating sequence directly to phenotype are dependent on which sets or groups of residues are mutated simultaneously. Previously reported combinatorial mutagenesis of this chromogenic protein (based on both phylogenetic and structural models) showed that substituting amino acids with large molar volumes at Gly beta 31 caused the mutated protein to have a spectrum characteristic of light-harvesting I (LH1). The six residues that underwent combinatorial mutagenesis were modeled to lie on one side of a transmembrane alpha-helix that binds bacteriochlorophyll. In a second experiment described here, we have not used structural models or phylogeny in choosing mutagenesis sites. Instead, a set of six contiguous residues was selected for combinatorial mutagenesis. In this latter experiment, the residue substituted at Gly beta 31 was not a determining factor in whether LH2 or LH1 spectra were obtained; therefore, we conclude that the heuristic rules for phenotype prediction are context dependent. While phenotype prediction is context dependent, the ability to identify elements of primary structure causing phenotype diversity appears not to be. This strengthens the argument for performing combinatorial mutagenesis with an arbitrary grouping of residues if structural models are unavailable.

Red-shifted excitation mutants of the green fluorescent protein.

Using optimized combinatorial mutagenesis techniques and Digital Imaging Spectroscopy (DIS), we have isolated mutants of the cloned Aequorea victoria green fluorescent protein (GFP) that show red-shifted excitation spectra similar to that of Renilla reniformis GFP. Selective excitation of wild-type versus Red-Shifted GFP (RSGFP) enables spectral separation of these proteins. Six contiguous codons spanning the tyrosine chromophore region were randomized and sequence analysis of the mutants revealed a tyrosineglycine consensus. These mutants will enable the simultaneous analysis of two promoters or proteins per cell or organism. In consideration of the multitude of applications which are developing for GFP alone, we envisage that spectrally shifted fluorescent proteins will be of value to a diversity of research programs, including developmental and cell biology, drug-screening, and diagnostic assays.

Imaging sequence space.

Digital imaging spectrophotometers can simultaneously measure the spectra of hundreds of features in a two-dimensional scene. While a variety of applications can be anticipated, a colorimetric analysis of mutants expressing pigmented proteins has already led to the development of efficient algorithms for optimizing combinatorial mutagenesis.

Searching sequence space to engineer proteins: exponential ensemble mutagenesis.

We describe an efficient method for generating combinatorial libraries with a high percentage of unique and functional mutants. Combinatorial libraries have been successfully used in the past to express ensembles of mutant proteins in which all possible amino acids are encoded at a few positions in the sequence. However, as more positions are mutagenized the proportion of functional mutants is expected to decrease exponentially. Small groups of residues were randomized in parallel to identify, at each altered position, amino acids which lead to functional proteins. By using optimized nucleotide mixtures deduced from the sequences selected from the random libraries, we have simultaneously altered 16 sites in a model pigment binding protein: approximately one percent of the observed mutants were functional. Mathematical formalization and extrapolation of our experimental data suggests that a 10(7)-fold increase in the throughput of functional mutants has been obtained relative to the expected frequency from a random combinatorial library. Exponential ensemble mutagenesis should be advantageous in cases where many residues must be changed simultaneously to achieve a specific engineering goal, as in the combinatorial mutagenesis of phage displayed antibodies. With the enhanced functional mutant frequencies obtained by this method, entire proteins could be mutagenized combinatorially.

Hydropathy and molar volume constraints on combinatorial mutants of the photosynthetic reaction center.

Combinatorial cassette mutagenesis was used to substitute randomly nine amino acid residues in the vicinity of the active branch monomeric bacteriochlorophyll in the photosynthetic reaction center of Rhodobacter capsulatus. The bacteriochlorophyll environment was targeted because of the potential role of this cofactor in the initial charge separation event of photosynthesis. Mutants with perturbed binding and which have altered energy levels of this chromophore, would be useful for electron transfer studies. Four sites in the M-subunit D-helix and five sites in the L-subunit cd-helix, including residue L153, the axial histidine ligand to the bacteriochlorophyll, were randomly substituted. The cd and D-helix regions were mutagenized independently of each other and simultaneously, resulting in three libraries of mutants. Digital imaging spectroscopy was used to screen photosynthetically selected mutants for ground state absorption spectra in the visible and near-infrared. The functional mutants of each library have distinct spectroscopic characteristics. One unusual spectral phenotype with an absorbance band at 825 nm occurred only in the nine-site library, with a frequency of about one out of 10(6) mutants. A mutant with an 825 nm band also has an unusual light-minus-dark difference spectra, which does not show a blue shift of the 800 nm absorption band. The mean molar volume and hydropathy of the substitutions occurring in functional mutants are biased towards the mean values of a random dope to various extents, indicating different stringencies at each site for these physicochemical properties.

Recursive ensemble mutagenesis.

We have developed a generally applicable experimental procedure to find functional proteins that are many mutational steps from wild type. Optimization algorithms, which are typically used to search for solutions to certain combinatorial problems, have been adapted to the problem of searching the 'sequence space' of proteins. Many of the steps normally performed by a digital computer are embodied in this new molecular genetics technique, termed recursive ensemble mutagenesis (REM). REM uses information gained from previous iterations of combinatorial cassette mutagenesis (CCM) to search sequence space more efficiently. We have used REM to simultaneously mutate six amino acid residues in a model protein. As compared to conventional CCM, one iteration of REM yielded a 30-fold increase in the frequency of 'positive' mutants. Since a multiplicative factor of similar magnitude is expected for the mutagenesis of additional sets of six residues, performing REM on 18 sites is expected to yield an exponential (30,000-fold) increase in the throughput of positive mutants as compared to random [NN(G,C)]18 mutagenesis.

An algorithmically optimized combinatorial library screened by digital imaging spectroscopy.

Combinatorial cassettes based on a phylogenetic "target set" were used to simultaneously mutagenize seven amino acid residues on one face of a transmembrane alpha helix comprising a bacteriochlorophyll binding site in the light harvesting II antenna of Rhodobacter capsulatus. This pigmented protein provides a model system for developing complex mutagenesis schemes, because simple absorption spectroscopy can be used to assay protein expression, structure, and function. Colony screening by Digital Imaging Spectroscopy showed that 6% of the optimized library bound bacteriochlorophyll in two distinct spectroscopic classes. This is approximately 200 times the throughput (ca. 0.03%) of conventional combinatorial cassette mutagenesis using [NN(G/C)]. "Doping" algorithms evaluated in this model system are generally applicable and should enable simultaneous mutagenesis at more positions in a protein than currently possible, or alternatively, decrease the screening size of combinatorial libraries.

Probing the primary donor environment in the histidineM200-->leucine and histidineL173-->leucine heterodimer mutants of Rhodobacter capsulatus by light-induced Fourier transform infrared difference spectroscopy.

Light-induced P+QB-/PQB FTIR difference spectra of reaction centers (RCs) have been obtained from chromatophores lacking light-harvesting B800-850 antenna for Rhodobacter capsulatus wild type (WT) and for the two mutants HisM200-->Leu and HisL173-->Leu. The primary donor (P) in both mutants consists of a bacteriochlorophyll-bacteriopheophytin heterodimer. The most prominent difference between the WT and the mutant spectra is in the 1600-1200-cm-1 region. The WT spectrum displays large positive bands at approximately 1290, 1500-1430, and 1580-1530 cm-1. These three bands are either small or altogether absent in the heterodimer spectra. In addition, both heterodimer spectra compare well with the electrochemically generated BChla+/BChla spectrum [Mäntele, W.G., Wollenweber, A. M., Nabedryk, E., & Breton, J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8468-8472]. These observations indicate that the positive charge is localized on the monomeric BChl in the heterodimers. The overall shape of the ester and keto C = O signals in the BChla+/BChla spectrum is maintained in the in situ spectra although significant differences are observed in the frequency, width, and splitting of the bands. The shape of the signal at 1757/1744 cm-1 in HisL173-->Leu is comparable to the 1751/1737-cm-1 signal of BChla+/BChla in tetrahydrofuran, indicating a free 10a ester C = O of PM in HisL173-->Leu. The reduced amplitude of the negative 1740-cm-1 feature in both HisM200-->Leu and WT spectra suggests a hydrogen-bonded 10a ester C = O for PL.(ABSTRACT TRUNCATED AT 250 WORDS)