Protein Nanocontainers from Nonviral Origin: Testing the Mechanics of Artificial and Natural Protein Cages by AFM.

Self-assembling protein nanocontainers are promising candidates for an increasingly wide scope of purposes. Their applications range from drug delivery vehicles and imaging agents to nanocompartments for controlled enzymatic activity. In order to exploit their full potential in these different fields, characterization of their properties is vital. For example, their mechanical properties give insight into the stability of a particle as a function of their internal content. The mechanics can be probed by atomic force microscopy nanoindentation, and while this single particle method is increasingly used to probe material properties of viral nanocages, it has hardly been used to characterize nonviral nanocages. Here we report nanoindentation studies on two types of nonviral nanocontainers: (i) lumazine synthase from Aquifex aeolicus (AaLS), which naturally self-assembles into icosahedral cages, and (ii) the artificial protein cage O3-33 originating from a computational design approach. In addition, we tested particles that had been engineered toward improved cargo loading capacity and compared these nanocages in empty and loaded states. We found that the thermostable AaLS cages are stiffer and resist higher forces before breaking than the O3-33 particles, but that mutations affecting the size of AaLS particles have a dramatic effect on their structural stability. Furthermore, we show that cargo packaging can occur while maintaining the cage's mechanical properties.

Searching sequence space for protein catalysts.

Genetic selection was used to explore the probability of finding enzymes in protein sequence space. Large degenerate libraries were prepared by replacing all secondary structure units in a dimeric, helical bundle chorismate mutase with simple binary-patterned modules based on a limited set of four polar and four nonpolar residues. Two-stage in vivo selection yielded catalytically active variants possessing biophysical and kinetic properties typical of the natural enzyme even though approximately 80% of the protein originates from the simplified modules and >90% of the protein consists of only eight different amino acids. This study provides a quantitative assessment of the number of sequences compatible with a given fold and implicates previously unidentified residues needed to form a functional active site. Given the extremely low incidence of enzymes in completely unbiased libraries, strategies that combine chemical information with genetic selection, like the one used here, may be generally useful in designing novel protein scaffolds with tailored activities.

Improved synthesis of C-terminal peptide thioesters on "safety-catch" resins using LiBr/THF.

[reaction: see text] The alkanesulfonamide "safety-catch" resin has proven useful for Fmoc-based synthesis of C-terminal peptide thioesters. We now report that the yield of isolated thioester can increase significantly when the cleavage reaction is carried out in 2 M LiBr/THF rather than DMF or THF. The largest effects are seen with problematic peptides that aggregate or form secondary structures on the resin.

Probing the role of the C-terminus of Bacillus subtilis chorismate mutase by a novel random protein-termination strategy.

A novel strategy combining random protein truncation and genetic selection has been developed to identify dispensable C-terminal segments of an enzyme. This approach, which entails the random introduction of premature termination codons, was applied to the last 17 residues of chorismate mutase from Bacillus subtilis (BsCM). Although structurally ill-defined, the C-terminus of BsCM has been proposed to cap the active site upon substrate binding and affect catalysis. However, sequence patterns of 178 selected gene variants show that the final 11 residues of the protein can be mutated and even removed without significantly impairing activity in vivo. In fact, none of the randomized residues is absolutely required, but a preference for wild-type Lys111, Ala112, Leu115, and Arg116 is apparent. These residues are part of a C-terminal 3(10)-helix and provide contacts with the rest of the protein or its ligands. The kinetic parameters of selected enzyme variants show that truncations and mutations do not significantly impair catalytic turnover (k(cat)) but substantially decrease k(cat)/K(m). Thus, while the 17 C-terminal residues of BsCM do not participate directly in the chemical rearrangement, they appear to contribute to enzymatic efficiency via uniform binding of the substrate and transition state.

Critical analysis of antibody catalysis.

Antibody molecules elicited with rationally designed transition-state analogs catalyze numerous reactions, including many that cannot be achieved by standard chemical methods. Although relatively primitive when compared with natural enzymes, these catalysts are valuable tools for probing the origins and evolution of biological catalysis. Mechanistic and structural analyses of representative antibody catalysts, generated with a variety of strategies for several different reaction types, suggest that their modest efficiency is a consequence of imperfect hapten design and indirect selection. Development of improved transition-state analogs, refinements in immunization and screening protocols, and elaboration of general strategies for augmenting the efficiency of first-generation catalytic antibodies are identified as evident, but difficult, challenges for this field. Rising to these challenges and more successfully integrating programmable design with the selective forces of biology will enhance our understanding of enzymatic catalysis. Further, it should yield useful protein catalysts for an enhanced range of practical applications in chemistry and biology.

A strategically positioned cation is crucial for efficient catalysis by chorismate mutase.

Combinatorial mutagenesis and in vivo selection experiments previously afforded functional variants of the AroH class Bacillus subtilis chorismate mutase lacking the otherwise highly conserved active site residue Arg(90). Here, we present a detailed kinetic and crystallographic study of several such variants. Removing the arginine side chain (R90G and R90A) reduced catalytic efficiency by more than 5 orders of magnitude. Reintroducing a positive charge to the active site through lysine substitutions restored more than a factor of a thousand in k(cat). Remarkably, the lysine could be placed at position 90 or at the more remote position 88 provided a sterically suitable residue was present at the partner site. Crystal structures of the double mutants C88S/R90K and C88K/R90S show that the lysine adopts an extended conformation that would place its epsilon-ammonium group within hydrogen-bonding distance of the ether oxygen of bound chorismate in the transition state. These results provide support for the hypothesis that developing negative charge in the highly polarized transition state is stabilized electrostatically by a strategically placed cation. The implications of this finding for the mechanism of all natural chorismate mutases and for the design of artificial catalysts are discussed.

Facile, fmoc-compatible solid-phase synthesis of peptide C-terminal thioesters.

A short route to peptide C-terminal thioesters was developed that does not require the use of special linkers or resins and is compatible with standard Fmoc chemistry. Following conventional solid-phase peptide synthesis, an excess of Me(2)AlCl and EtSH in dichloromethane cleaves peptides from Wang or Pam resins to give the corresponding thioesters directly in good yield and purity.

Molecular dynamics simulations highlight mobile regions in proteins: A novel suggestion for converting a murine V(H) domain into a more tractable species.

The V(H) region of the murine antibody 1F7 has been identified as a single-domain chorismate mutase, but a tendency to denature and aggregate has hampered its biochemical characterization. Standard mutagenesis approaches targeting antibody chain dimerization areas have been exhausted. We describe a new approach to the problem, where we use molecular dynamics (MD) simulations to find the differences between the untractable protein and the known soluble V(H) domain from a llama antibody. MD simulations of proteins yield information on the relative stability and fluctuations of parts of the proteins. By comparing simulation results of two related proteins their differences in stability and fluctuations can be analyzed and may suggest mutations aimed at (de)stabilization of one of the two proteins. For the mouse versus llama simulations, this approach highlights an untried area in the protein which shows increased fluctuations. The replacement of this eight-residue segment with the corresponding llama sequence gave a chimeric mutant which shows significantly decreased fluctuations. We see this as a general scheme to generate suggestions for mutagenesis experiments, not only obviously generalizable to other immunoglobulin domains, but to other protein systems as well.

Catalysis of decarboxylation by a preorganized heterogeneous microenvironment: crystal structures of abzyme 21D8.

Antibody 21D8 catalyzes the solvent-sensitive decarboxylation of 3-carboxybenzisoxazoles. The crystal structure of chimeric Fab 21D8 with and without hapten at 1.61 A and 2.10 A, respectively, together with computational analysis, shows how a melange of polar and non-polar sites are exploited to achieve both substrate binding and acceleration of a reaction normally facilitated by purely aprotic dipolar media. The striking similarity of the decarboxylase and a series of unrelated esterase antibodies also highlights the chemical versatility of structurally conserved anion binding sites and the relatively subtle changes involved in fine-tuning the immunoglobulin pocket for recognition of different ligands and catalysis of different reactions.

Shape complementarity, binding-site dynamics, and transition state stabilization: a theoretical study of Diels-Alder catalysis by antibody 1E9.

Antibody 1E9 is a protein catalyst for the Diels-Alder reaction between tetrachlorothiophene dioxide and N-ethylmaleimide. Quantum mechanical calculations have been employed to study the 1E9-catalyzed Diels-Alder reaction in the gas phase. The transition states and intermediates were all determined at the B3LYP/6-31G*//HF/6-31G* level. The cycloaddition step is predicted to be rate-determining, and the endo reaction pathway is strongly favored. Binding of the reactants and the transition states to antibody 1E9 was investigated by docking and molecular dynamics simulations. The linear interaction energy (LIE) method was adopted to estimate the free energy barrier of the 1E9-catalyzed Diels-Alder reaction. The catalytic efficiency of antibody 1E9 is achieved by enthalpic stabilization of the transition state, near-perfect shape complementarity of the hydrophobic binding site for the transition state, and a strategically placed hydrogen-bonding interaction.

Evolution of shape complementarity and catalytic efficiency from a primordial antibody template.

The crystal structure of an efficient Diels-Alder antibody catalyst at 1.9 angstrom resolution reveals almost perfect shape complementarity with its transition state analog. Comparison with highly related progesterone and Diels-Alderase antibodies that arose from the same primordial germ line template shows the relatively subtle mutational steps that were able to evolve both structural complementarity and catalytic efficiency.

alpha-Functionalized phosphonylphosphinates: synthesis and evaluation as transcarbamoylase inhibitors.

Diverse alpha-methyl-substituted phosphonylphosphinates (P-C-P-C-X) are accessible from a protected, pentafluorophenylsulfonated phosphonylphosphinate via nucleophilic displacement. The utility of this route is demonstrated with several nitrogen nucleophiles. The resulting amine and amino acid phosphonylphosphinate derivatives were evaluated as inhibitors of Streptococcus faecalis ornithine transcarbamoylase (OTC). Compared with the structurally related phosphonoacetyl-L-ornithine (L-PALO), a known inhibitor of OTCs from various sources, the phosphonylphosphinates are surprisingly poor inhibitors, binding several orders of magnitude less tightly to the enzyme. These results suggest that the tetrahedral intermediate formed in the normal transcarbamoylase reaction is poorly mimicked by a tetrahedral and anionic phosphonate, either because of directly unfavorable interactions with a hydrogen-bond acceptor within the active site or because transition-state analogues are unable to induce the protein conformation changes that normally accompany reaction.

Electric fields in active sites: substrate switching from null to strong fields in thiol- and selenol-subtilisins.

Although known to be important factors in promoting catalysis, electric field effects in enzyme active sites are difficult to characterize from an experimental standpoint. Among optical probes of electric fields, Raman spectroscopy has the advantage of being able to distinguish electronic ground-state and excited-state effects. Earlier Raman studies on acyl derivatives of cysteine proteases [Doran, J. D., and Carey, P. R. (1996) Biochemistry 35, 12495-502], where the acyl group has extensive pi-electron conjugation, showed that electric field effects in the active site manifest themselves by polarizing the pi-electrons of the acyl group. Polarization gives rise to large shifts in certain Raman bands, e.g. , the C=C stretching band of the alpha,beta-unsaturated acyl group, and a large red shift in the absorption maximum. It was postulated that a major source of polarization is the alpha-helix dipole that originates from the alpha-helix terminating at the active-site cysteine of the cysteine protease family. In contrast, using the acyl group 5-methylthiophene acryloyl (5-MTA) as an active-site Raman probe, acyl enzymes of thiol- or selenol-subtilisin exhibit no polarization even though the acylating amino acid is at the terminus of an alpha-helix. Quantum mechanical calculations on 5-MTA ethyl thiol and selenol ethyl esters allowed us to identify the conformational states of these molecules along with their corresponding vibrational signatures. The Raman spectra of 5-MTA thiol and selenol subtilisins both showed that the acyl group binds in a single conformation in the active site that is s-trans about the =C-C=O single bond. Moreover, the positions of the C=C stretching bands show that the acyl group is not experiencing polarization. However, the release of steric constraints in the active site by mutagenesis, by creating the N155G form of selenol-subtilisin and the P225A form of thiol-subtilisin, results in the appearance of a second conformer in the active sites that is s-cis about the =C-C=O bond. The Raman signature of this second conformer indicates that it is strongly polarized with a permanent dipole being set up through the acyl group's pi-electron chain. Molecular modeling for 5-MTA in the active sites of selenol-subtilisin and N155G selenol-subtilisin confirms the findings from Raman spectroscopic studies and identifies the active-site features that give rise to polarization. The determinants of polarization appear to be strong electron pull at the acyl carbonyl group by a combination of hydrogen bonds and the field at the N-terminus of the alpha-helix and electron push from a negatively charged group placed at the opposite end of the chromophore.

Bacillus subtilis chorismate mutase is partially diffusion-controlled.

The effect of viscosogens on the enzyme-catalyzed rearrangement of chorismate to prephenate has been studied. The steady-state parameters kcat and kcat/Km for the monofunctional chorismate mutase from Bacillus subtilis (BsCM) decreased significantly with increasing concentrations of glycerol, whereas the 'sluggish' BsCM mutants C75A and C75S were insensitive to changes in microviscosity. The latter results rule out extraneous interactions of the viscosogen as an explanation for the effects observed with the wild-type enzyme. Additional control experiments show that neither viscosogen-induced shifts in the pH-dependence of the enzyme-catalyzed reaction nor small perturbations of the conformational equilibrium of chorismate can account for the observed effects. Instead, BsCM appears to be limited by substrate binding and product release at low and high substrate concentrations, respectively. Analysis of the kinetic data indicates that diffusive transition states are between 30 and 40% rate-determining in these concentration regimes; the chemical step must contribute to the remaining kinetic barrier. The relatively low value of the 'on' rates for chorismate and prephenate (approximately 2 x 106 m-1.s-1) probably reflects the need for a rare conformation of the enzyme, the ligand, or both for successful binding. Interestingly, the chorismate mutase domain of the bifunctional chorismate mutase-prephenate dehydratase from Escherichia coli, which has steady-state kinetic parameters comparable to those of BsCM but has a much less accessible active site, is insensitive to changes in viscosity and the reaction it catalyses is not diffusion-controlled.

A small, thermostable, and monofunctional chorismate mutase from the archaeon Methanococcus jannaschii.

The gene for chorismate mutase (CM) from the archaeon Methanococcus jannaschii, an extreme thermophile, was subcloned and expressed in Escherichia coli. This gene, which belongs to the aroQ class of CMs, encodes a monofunctional enzyme (AroQf) able to complement the CM deficiency of an E. coli mutant strain. The purified protein follows Michaelis-Menten kinetics (kcat = 5.7 s-1 and Km = 41 microM at 30 degreesC) and displays pH-independent activity in the range of pH 5-9. Its activation parameters [Delta H = 16.2 kcal/mol, Delta S = -1. 7 cal/(mol.K)] are similar to those of another well characterized AroQ class CM, the mesophilic AroQp domain from E. coli. Like AroQp, the thermophilic CM is an alpha-helical dimer, but approximately 5 kcal/mol more stable than its mesophilic counterpart as judged from equilibrium denaturation studies. The possible origins of the thermostability of M. jannaschii AroQf, the smallest natural CM characterized to date, are discussed in light of available sequence and tertiary structural information.

Exploring sequence constraints on an interhelical turn using in vivo selection for catalytic activity.

The role of interhelical turns in determining protein structure has been investigated previously in relatively simple four-helix-bundle proteins using combinatorial mutagenesis coupled with screening for functional variants. To assess the tolerance to sequence substitution of a short, interhelical turn in a larger, more complicated protein, we have exploited a more sensitive in vivo selection for catalytic activity. Randomization of three solvent-exposed turn residues in Escherichia coli chorismate mutase (Ala65, His66, and His67), followed by selection, indicated that >63% of tripeptides, including some with significantly altered backbone conformations, can functionally replace the native sequence. The increased sensitivity of the catalytic assay allowed optimal sequences to be distinguished from less appropriate ones, revealing a statistically significant preference for hydrophilic residues in solvent-exposed positions. It also enabled investigation of the extent to which either secondary structure or tertiary interactions influence substitution patterns. Randomization of an alpha-helical residue (Lys64), together with the adjacent solvent-exposed tripeptide, Ala65-His66-His67, showed that the secondary structure at position 64 does not limit the range of side chains allowed at this site. In contrast, randomization of a buried turn residue (Leu68), together with the same tripeptide, revealed an extremely strict requirement for hydrophobic aliphatic amino acids at this position. The strong constraint imposed by the tertiary interaction, in contrast to the weak influence of secondary structure, has important implications for protein design.

Redesigning enzyme topology by directed evolution.

Genetic selection was exploited in combination with structure-based design to transform an intimately entwined, dimeric chorismate mutase into a monomeric, four-helix-bundle protein with near native activity. Successful reengineering depended on choosing a thermostable starting protein, introducing point mutations that preferentially destabilize the wild-type dimer, and using directed evolution to optimize an inserted interhelical turn. Contrary to expectations based on studies of other four-helix-bundle proteins, only a small fraction of possible turn sequences (fewer than 0.05 percent) yielded well-behaved, monomeric, and highly active enzymes. Selection for catalytic function thus provides an efficient yet stringent method for rapidly assessing correctly folded polypeptides and may prove generally useful for protein design.

Probing enzyme quaternary structure by combinatorial mutagenesis and selection.

Genetic selection provides an effective way to obtain active catalysts from a diverse population of protein variants. We have used this tool to investigate the role of loop sequences in determining the quaternary structure of a domain-swapped enzyme. By inserting random loops of four to seven residues into a dimeric chorismate mutase and selecting for functional variants by genetic complementation, we have obtained and characterized both monomeric and hexameric enzymes that retain considerable catalytic activity. The low percentage of active proteins recovered from these selection experiments indicates that relatively few loop sequences permit a change in quaternary structure without affecting active site structure. The results of our experiments suggest further that protein stability can be an important driving force in the evolution of oligomeric proteins.

3D structural information as a guide to protein engineering using genetic selection.

A great variety of protein systems have been investigated in the past year using structure-guided evolutionary strategies. On the basis of available 3D structural information, critical regions of proteins have been targeted for randomizing mutagenesis and active variants of the corresponding genes have been selected. These approaches help characterize structural and mechanistic features of proteins and have important implications for design.