Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a ...typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ∼2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20°C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at http://pross.weizmann.ac.il.
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•A new computational method is used to stabilize five recalcitrant proteins•Designed variants show higher expression and stability with unmodified function•A designed human acetylcholinesterase variant expresses solubly in bacteria•The method is fully automated and implemented on a webserver
Heterologous expression of proteins and their mutants often results in misfolding and aggregation. Goldenzweig et al. (2016) developed an automated algorithm for protein stabilization requiring minimal experimental testing; for instance, the five tested variants of human acetylcholinesterase showed ≥100-fold higher soluble bacterial expression and higher melting temperatures than wild-type.
Many promising vaccine candidates from pathogenic viruses, bacteria, and parasites are unstable and cannot be produced cheaply for clinical use. For instance, Plasmodium falciparum ...reticulocyte-binding protein homolog 5 (PfRH5) is essential for erythrocyte invasion, is highly conserved among field isolates, and elicits antibodies that neutralize in vitro and protect in an animal model, making it a leading malaria vaccine candidate. However, functional RH5 is only expressible in eukaryotic systems and exhibits moderate temperature tolerance, limiting its usefulness in hot and low-income countries where malaria prevails. Current approaches to immunogen stabilization involve iterative application of rational or semirational design, random mutagenesis, and biochemical characterization. Typically, each round of optimization yields minor improvement in stability, and multiple rounds are required. In contrast, we developed a one-step design strategy using phylogenetic analysis and Rosetta atomistic calculations to design PfRH5 variants with improved packing and surface polarity. To demonstrate the robustness of this approach, we tested three PfRH5 designs, all of which showed improved stability relative to wild type. The best, bearing 18 mutations relative to PfRH5, expressed in a folded form in bacteria at >1 mg of protein per L of culture, and had 10–15 °C higher thermal tolerance than wild type, while also retaining ligand binding and immunogenic properties indistinguishable from wild type, proving its value as an immunogen for a future generation of vaccines against the malaria blood stage. We envision that this efficient computational stability design methodology will also be used to enhance the biophysical properties of other recalcitrant vaccine candidates from emerging pathogens.
The GroE chaperonin system in Escherichia coli comprises GroEL and GroES and facilitates ATP-dependent protein folding in vivo and in vitro. Proteins with very similar sequences and structures can ...differ in their dependence on GroEL for efficient folding. One potential but unverified source for GroEL dependence is frustration, wherein not all interactions in the native state are optimized energetically, thereby potentiating slow folding and misfolding. Here, we chose enhanced green fluorescent protein as a model system and subjected it to random mutagenesis, followed by screening for variants whose in vivo folding displays increased or decreased GroEL dependence. We confirmed the altered GroEL dependence of these variants with in vitro folding assays. Strikingly, mutations at positions predicted to be highly frustrated were found to correlate with decreased GroEL dependence. Conversely, mutations at positions with low frustration were found to correlate with increased GroEL dependence. Further support for this finding was obtained by showing that folding of an enhanced green fluorescent protein variant designed computationally to have reduced frustration is indeed less GroEL-dependent. Our results indicate that changes in local frustration also affect partitioning in vivo between spontaneous and chaperonin-mediated folding. Hence, the design of minimally frustrated sequences can reduce chaperonin dependence and improve protein expression levels.
Abstract
Improving an enzyme's initially low catalytic efficiency with a new target substrate by an order of magnitude or two may require only a few rounds of mutagenesis and screening or selection. ...However, subsequent rounds of optimization tend to yield decreasing degrees of improvement (diminishing returns) eventually leading to an optimization plateau. We aimed to optimize the catalytic efficiency of bacterial phosphotriesterase (PTE) toward V-type nerve agents. Previously, we improved the catalytic efficiency of wild-type PTE toward the nerve agent VX by 500-fold, to a catalytic efficiency (kcat/KM) of 5 × 106 M−1 min−1. However, effective in vivo detoxification demands an enzyme with a catalytic efficiency of >107 M−1 min−1. Here, following eight additional rounds of directed evolution and the computational design of a stabilized variant, we evolved PTE variants that detoxify VX with a kcat/KM ≥ 5 × 107 M−1 min−1 and Russian VX (RVX) with a kcat/KM ≥ 107 M−1 min−1. These final 10-fold improvements were the most time consuming and laborious, as most libraries yielded either minor or no improvements. Stabilizing the evolving enzyme, and avoiding tradeoffs in activity with different substrates, enabled us to obtain further improvements beyond the optimization plateau and evolve PTE variants that were overall improved by >5000-fold with VX and by >17 000-fold with RVX. The resulting variants also hydrolyze G-type nerve agents with high efficiency (GA, GB at kcat/KM > 5 × 107 M−1 min−1) and can thus serve as candidates for broad-spectrum nerve-agent prophylaxis and post-exposure therapy using low enzyme doses.
The isolation of nanobodies from pre-immune libraries by means of biopanning is a straightforward process. Nevertheless, the recovered candidates often require optimization to improve some of their ...biophysical characteristics. In principle, CDRs are not mutated because they are likely to be part of the antibody paratope, but in this work, we describe a mutagenesis strategy that specifically addresses CDR1. Its sequence was identified as an instability hot spot by the PROSS program, and the available structural information indicated that four CDR1 residues bound directly to the antigen. We therefore modified the loop flexibility with the addition of an extra glycine rather than by mutating single amino acids. This approach significantly increased the nanobody yields but traded-off with moderate affinity loss. Accurate modeling coupled with atomistic molecular dynamics simulations enabled the modifications induced by the glycine insertion and the rationale behind the engineering design to be described in detail.
Proteins are increasingly used in basic and applied biomedical research. Many proteins, however, are only marginally stable and can be expressed in limited amounts, thus hampering research and ...applications. Research has revealed the thermodynamic, cellular, and evolutionary principles and mechanisms that underlie marginal stability. With this growing understanding, computational stability design methods have advanced over the past two decades starting from methods that selectively addressed only some aspects of marginal stability. Current methods are more general and, by combining phylogenetic analysis with atomistic design, have shown drastic improvements in solubility, thermal stability, and aggregation resistance while maintaining the protein's primary molecular activity. Stability design is opening the way to rational engineering of improved enzymes, therapeutics, and vaccines and to the application of protein design methodology to large proteins and molecular activities that have proven challenging in the past.
Although it is well-known
that limited local mutations of enzymes,
such as matrix metalloproteinases (MMPs), may change enzyme activity
by orders of magnitude as well as its stability, the completely ...rational
design of proteins is still challenging. These local changes alter
the electrostatic potential and thus local electrostatic fields, which
impacts the dynamics of water molecules close the protein surface.
Here we show by a combined computational design, experimental, and
molecular dynamics (MD) study that local mutations have not only a
local but also a global effect on the solvent: In the specific case
of the matrix metalloprotease MMP14, we found that the nature of local
mutations, coupled with surface morphology, have the ability to influence
large patches of the water hydrogen-bonding network at the protein
surface, which is correlated with stability. The solvent contribution
can be experimentally probed via terahertz (THz) spectroscopy, thus
opening the door to the exciting perspective of rational protein design
in which a systematic tuning of hydration water properties allows
manipulation of protein stability and enzymatic activity.
Stabilization of the metastable envelope glycoprotein (Env) of HIV-1 is hypothesized to improve induction of broadly neutralizing antibodies. We improved the expression yield and stability of the ...HIV-1 envelope glycoprotein BG505SOSIP.664 gp140 by means of a previously described automated sequence and structure-guided computational thermostabilization approach, PROSS. This combines sequence conservation information with computational assessment of mutant stabilization, thus taking advantage of the extensive natural sequence variation present in HIV-1 Env. PROSS is used to design three gp140 variants with 17–45 mutations relative to the parental construct. One of the designs is experimentally observed to have a fourfold improvement in yield and a 4 °C increment in thermostability. In addition, the designed immunogens have similar antigenicity profiles to the native flexible linker version of wild type, BG505SOSIP.664 gp140 (NFL Wt) to major epitopes targeted by broadly neutralizing antibodies. PROSS eliminates the laborious process of screening many variants for stability and functionality, providing a proof of principle of the method for stabilization and improvement of yield without compromising antigenicity for next generation complex, highly glycosylated vaccine candidates.
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•One-step stabilization of HIV-1 Env gp140.•One-step yield improvement of HIV-1 Env gp140.•Native-like oligomeric conformation of designed vaccine candidates.•Unaltered antigenicity of designed vaccine candidates.
ABSTRACT
Summary
Many natural and designed proteins are only marginally stable limiting their usefulness in research and applications. Recently, we described an automated structure and sequence-based ...design method, called PROSS, for optimizing protein stability and heterologous expression levels that has since been validated on dozens of proteins. Here, we introduce improvements to the method, workflow and presentation, including more accurate sequence analysis, error handling and automated analysis of the quality of the sequence alignment that is used in design calculations.
Availability and implementation
PROSS2 is freely available for academic use at https://pross.weizmann.ac.il.
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