Selective DNA-mediated assembly of gold nanoparticles on electroded substrates.

Motivated by the technological possibilities of electronics and sensors based on gold nanoparticles (Au NPs), we investigate the selective assembly of such NPs on electrodes via DNA hybridization. Protocols are demonstrated for maximizing selectivity and coverage using 15mers as the active binding agents. Detailed studies of the dependences on time, ionic strength, and temperature are used to understand the underlying mechanisms and their limits. Under optimized conditions, coverage of Au NPs on Au electrodes patterned on silicon dioxide (SiO2) substrates was found to be approximately 25-35%. In all cases, Au NPs functionalized with non-complementary DNA show no attachment and essentially no nonspecific adsorption is observed by any Au NPs on the SiO2 surfaces of the patterned substrates. DNA-guided assembly of multilayers of NPs was also demonstrated and, as expected, found to further increase the coverage, with three deposition cycles resulting in a surface coverage of approximately 60%.

Phage-displayed peptides as biosensor reagents.

This study investigated the potential to utilize phage-displayed peptides as reagents in sensor applications. A library of random 12-mers displayed on phage was panned against staphylococcal enterotoxin B (SEB), a causative agent of food poisoning. Nine SEB binding phage clones were isolated, all of which share the consensus sequence Trp His Lys at their amino terminus. Binding of several of these phage was shown to be inhibited when they were assayed in a competitive enzyme-linked immunosorbent assay (ELISA) format with synthesized peptide corresponding to the peptide-encoding region of one of the clones. Whole phage were labeled with the dye Cy5, and incorporated into fluoroimmunoassays. Labeled phage were able to detect SEB down to a concentration of 1.4 ng/well in a fluorescence-based immunoassay. When incorporated into an automated fluorescence-based sensing assay, Cy5-labeled phage bound to probes coated with SEB generated a robust signal of about 10,000 pA, vs a signal of 1,000 pA using a control fiber coated with streptavidin. These results demonstrate the potential for development of phage-based sensor reagents.

Anatomy of an antibody molecule: structure, kinetics, thermodynamics and mutational studies of the antilysozyme antibody D1.3.

Using site-directed mutagenesis, x-ray crystallography, microcalorimetric, equilibrium sedimentation and surface plasmon resonance detection techniques, we have examined the structure of an antibody-antigen complex and the structural and thermodynamic consequences of removing specific hydrogen bonds and van der Waals interactions in the antibody-antigen interface. These observations show that the complex is considerably tolerant, both structurally and thermodynamically, to the truncation of antibody and antigen side chains that form contacts. Alterations in interface solvent structure for two of the mutant complexes appear to compensate for the unfavorable enthalpy changes when antibody-antigen interactions are removed. These changes in solvent structure, along with the increased mobility of side chains near the mutation site, probably contribute to the observed entropy compensation. In concert, data from structural studies, reaction rates, calorimetric measurements and site directed mutations are beginning to detail the nature of antibody-protein antigen interactions.

A mutational analysis of binding interactions in an antigen-antibody protein-protein complex.

Alanine scanning mutagenesis, double mutant cycles, and X-ray crystallography were used to characterize the interface between the anti-hen egg white lysozyme (HEL) antibody D1.3 and HEL. Twelve out of the 13 nonglycine contact residues on HEL, as determined by the high-resolution crystal structure of the D1.3-HEL complex, were individually truncated to alanine. Only four positions showed a DeltaDeltaG (DeltaGmutant - DeltaGwild-type) of greater than 1.0 kcal/mol, with HEL residue Gln121 proving the most critical for binding (DeltaDeltaG = 2.9 kcal/mol). These residues form a contiguous patch at the periphery of the epitope recognized by D1.3. To understand how potentially disruptive mutations in the antigen are accommodated in the D1.3-HEL interface, we determined the crystal structure to 1.5 A resolution of the complex between D1.3 and HEL mutant Asp18 --> Ala. This mutation results in a DeltaDeltaG of only 0.3 kcal/mol, despite the loss of a hydrogen bond and seven van der Waals contacts to the Asp18 side chain. The crystal structure reveals that three additional water molecules are stably incorporated in the antigen-antibody interface at the site of the mutation. These waters help fill the cavity created by the mutation and form part of a rearranged solvent network linking the two proteins. To further dissect the energetics of specific interactions in the D1.3-HEL interface, double mutant cycles were carried out to measure the coupling of 14 amino acid pairs, 10 of which are in direct contact in the crystal structure. The highest coupling energies, 2.7 and 2.0 kcal/mol, were measured between HEL residue Gln121 and D1.3 residues VLTrp92 and VLTyr32, respectively. The interaction between Gln121 and VLTrp92 consists of three van der Waals contacts, while the interaction of Gln121 with VLTyr32 is mediated by a hydrogen bond. Surprisingly, however, most cycles between interface residues in direct contact in the crystal structure showed no significant coupling. In particular, a number of hydrogen-bonded residue pairs were found to make no net contribution to complex stabilization. We attribute these results to accessibility of the mutation sites to water, such that the mutated residues exchange their interaction with each other to interact with water. This implies that the strength of the protein-protein hydrogen bonds in these particular cases is comparable to that of the protein-water hydrogen bonds they replace. Thus, the simple fact that two residues are in direct contact in a protein-protein interface cannot be taken as evidence that there necessarily exists a productive interaction between them. Rather, the majority of such contacts may be energetically neutral, as in the D1.3-HEL complex.

Analysis of binding interactions in an idiotope-antiidiotope protein-protein complex by double mutant cycles.

The idiotope-antiidiotope complex between the anti-hen egg white lysozyme antibody D1.3 and the anti-D1.3 antibody E5.2 provides a useful model for studying protein-protein interactions. A high-resolution crystal structure of the complex is available [Fields, B. A., Goldbaum, F. A., Ysern, X., Poljak, R.J., & Mariuzza, R. A. (1995) Nature 374, 739-742], and both components are easily produced and manipulated in Escherichia coli. We previously analyzed the relative contributions of individual residues of D1.3 to complex stabilization by site-directed mutagenesis [Dall'Acqua, W., Goldman, E. R., Eisenstein, E., & Mariuzza, R. A. (1996) Biochemistry 35, 9667-9676]. In the current work, we introduced single alanine substitutions in 9 out of 21 positions in the combining site of E5.2 involved in contacts with D1.3 and found that 8 of them play a significant role in ligand binding (delta Gmutant-delta Gwild type > 1.5 kcal/mol). Furthermore, energetically important E5.2 and D1.3 residues tend to be juxtaposed in the crystal structure of the complex. In order to further dissect the energetics of specific interactions in the D1.3-E5.2 interface, double mutant cycles were carried out to measure the coupling of 13 amino acid pairs, 9 of which are in direct contact in the crystal structure. The highest coupling energy (4.3 kcal/mol) was measured for a charged-neutral pair which forms a buried hydrogen bond, while side chains which interact through solvated hydrogen bonds have lower coupling energies (1.3-1.7 kcal/mol), irrespective of whether they involve charged-neutral or neutral-neutral pairs. Interaction energies of similar magnitude (1.3-1.6 kcal/mol) were measured for residues forming only van der Waals contacts. Cycles between distant residues not involved in direct contacts in the crystal structure also showed significant coupling (0.5-1.0 kcal/mol). These weak long-range interactions could be due to rearrangements in solvent or protein structure or to secondary interactions involving other residues.

A mutational analysis of the binding of two different proteins to the same antibody.

The crystal structures of the complexes between the anti-hen egg white lysozyme (HEL) antibody D1.3 and HEL and between D1.3 and the anti-D1.3 antibody E5.2 have shown that D1.3 contacts these two proteins through essentially the same set of combining site residues [Fields, B. A., Goldbaum, F. A., Ysern, X., Poljak, R. J., & Mariuzza, R. A. (1995) Nature 374, 739-742]. To probe the relative contribution of individual residues to complex stabilization, single alanine substitutions were introduced in the combining site of D1.3, and their effects on affinity for HEL and for E5.2 were measured using surface plasmon resonance detection, fluorescence quench titration, or sedimentation equilibrium. The energetics of the binding to HEL are dominated by only 3 of the 13 contact residues tested (delta Gmutant-delta Gwild type > 2.5 kcal/mol): VLW92, VHD100, and VHY101. These form a patch at the center of the interface and are surrounded by residues whose apparent contributions are much less pronounced ( < 1.5 kcal/mol). This contrasts with the interaction of D1.3 with E5.2 in which most the contact residues (11 of 15) were found to play a significant role in ligand binding ( > 1.5 kcal/mol). Furthermore, even though D1.3 contacts HEL and E5.2 in very similar ways, the functionally important residues of D1.3 are different for the two interactions, with only substitutions at D1.3 positions VH100 and VH101 greatly affecting binding to both ligands. Thus, the same protein may recognize different ligands in ways that are structurally similar yet energetically distinct.

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.

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.

Applications of imaging spectroscopy in molecular biology. II. Colony screening based on absorption spectra.

Digital imaging spectroscopy has been used to obtain the grayscale spectrum of colored bacterial colonies directly from petri dishes. Up to 500 individual colony spectra can be simultaneously recorded and processed from a single plate. Spectra can be obtained in the visible to near infrared region (400nm-900nm) with 10nm resolution. Instrument response is normalized through run-time radiometric calibration such that each grayscale spectrum can be converted to the ground-state absorption spectrum of the colony. In this study, mutants of the photosynthetic bacterium Rhodobacter capsulatus have been differentiated by the absorption spectra of their pigment-protein complexes. This imaging technique is applicable to chromogenic systems in which colony and/or media color (e.g. indicator plates) provides a quantitative indicator of gene expression.