Abstract

Here we present a technology to facilitate synthetic memory in a living system via repurposing Transcriptional Programming (i.e., our decision-making technology) parts, to regulate (intercept) recombinase function post- translation. We show that interception synthetic memory can facilitate pro- grammable loss-of-function via site-specific deletion, programmable gain-of- function by way of site-specific inversion, and synthetic memory operations with nested Boolean logical operations. We can expand interception synthetic memory capacity more than 5-fold for a single recombinase, with reconfi- guration specificity for multiple sites in parallel. Interception synthetic mem- ory is ~10-times faster than previous generations of recombinase-based memory. We posit that the faster recombination speed of our next-generation memory technology is due to the post-translational regulation of recombinase function. This iteration of synthetic memory is complementary to decision- making via Transcriptional Programming – thus can be used to develop intelligent synthetic biological systems for myriad applications.

Abstract

Allosteric transcription factors (aTFs) are used in a myriad of processes throughout biology and biotech- nology. aTFs have served as the workhorses for developments in synthetic biology, fundamental research, and protein manufacturing. One of the most utilized TFs is the lactose repressor (LacI). In addition to being an exceptional tool for gene regulation, LacI has also served as an outstanding model system for understand- ing allosteric communication. In this perspective, we will use the LacI TF as the principal exemplar for engineering alternate functions related to allostery—i.e., alternate protein DNA interactions, alternate protein-ligand interactions, and alternate phenotypic mechanisms. In addition, we will summarize the design rules and heuristics for each design goal and demonstrate how the resulting design rules and heuristics can be extrapolated to engineer other aTFs with a similar topology—i.e., from the broader LacI/GalR family of TFs.

Abstract

Transcriptional programming leverages systems of engineered transcription factors to impart decision-making (e.g.,Boolean logic) in chassis cells. The number of components used to construct said decision-making systems is rapidly increasing, making an exhaustive experimental evaluation of iterations of biological circuits impractical. Accordingly, we posited that a predictive tool is needed to guide and accelerate the design of transcriptional programs. The work described here involves the development and experimental characterization of a large collection of network-capable single-INPUT logical operations i.e., engineered BUFFER (repressor) and engineered NOT (antirepressor) logical operations. Using this single-INPUT data and developed metrology, we were able to model and predict the performances of all fundamental two-INPUT compressed logical operations (i.e., compressed AND gates and compressed NOR gates). In addition, we were able to model and predict the performance of compressed mixed phenotype logical operations (A NIMPLY B gates and complementary B NIMPLY A gates). These results demonstrate that single-INPUT data is sufficient to accurately predict both the qualitative and quantitative performance of a complex circuit. Accordingly, this work has set the stage for the predictive design of transcriptional programs of greater complexity.

Abstract

Engineering synthetic tools that facilitate decision-making in mammalian cells could enable myriad biomedical applications. Researchers have now developed a new system of inducer-controlled transcription factors to facilitate synthetic decision-making (LOGIC) in human cells based on modular protein-fusion cascades.

Abstract

Bacteroides species are prominent members of the human gut microbiota. The prevalence and stability of Bacteroides in humans make them ideal candidates to engineer as programmable living therapeutics. Here we report a biotic decision-making technology in a community of Bacteroides (consortium transcriptional programming) with genetic circuit compression. Circuit compression requires systematic pairing of engineered transcription factors with cognate regulatable promoters. In turn, we demonstrate the compression workflow by designing, building, and testing all fundamental two-input logic gates dependent on the inputs isopropyl-β-D-1-thiogalactopyranoside and D-ribose. We then deploy complete sets of logical operations in five human donor Bacteroides, with which we demonstrate sequential gain-of-function control in co-culture. Finally, we couple transcriptional programs with CRISPR interference to achieve loss-of-function regulation of endogenous genes—demonstrating complex control over community composition in co-culture. This work provides a powerful toolkit to program gene expression in Bacteroides for the development of bespoke therapeutic bacteria.

Abstract

In observance of Earth Day 2022 and the looming, urgent need to fight climate change and biodiversity loss, scientists gathered in New York City and online for the Bioinspired Green Science and Technology Symposium to share the latest technological and design breakthroughs that hold promise for the environment and human health.

Abstract

Signal processing is critical to a myriad of biological phenomena (natural and engineered) that involve gene regulation. Biological signal processing can be achieved by way of allosteric transcription factors. In canonical regulatory systems (e.g., the lactose repressor), an INPUT signal results in the induction of a given transcription factor and objectively switches gene expression from an OFF state to an ON state. In such biological systems, to revert the gene expression back to the OFF state requires the aggressive dilution of the input signal, which can take 1 or more d to achieve in a typical biotic system. In this study, we present a class of engineered allosteric transcription factors capable of processing two-signal INPUTS, such that a sequence of INPUTS can rapidly transition gene expression between alternating OFF and ON states. Here, we present two fundamental biological signal processing filters, BANDPASS and BANDSTOP, that are regulated by D-fucose and isopropyl-β-D-1-thiogalactopyranoside. BANDPASS signal processing filters facilitate OFF–ON–OFF gene regulation. Whereas, BANDSTOP filters facilitate the antithetical gene regulation, ON–OFF–ON. Engineered signal processing filters can be directed to seven orthogonal promoters via adaptive modular DNA binding design. This collection of signal processing filters can be used in collaboration with our established transcriptional programming structure. Kinetic studies show that our collection of signal processing filters can switch between states of gene expression within a few minutes with minimal metabolic burden—representing a paradigm shift in general gene regulation.

Abstract

Allosteric function is a critical component of many of the parts used to construct gene networks throughout synthetic biology. In this review, we discuss an emerging field of research and education, biomolecular systems engineering, that expands on the synthetic biology edifice—integrating workflows and strategies from protein engineering, chemical engineering, electrical engineering, and computer science principles. We focus on the role of engineered allosteric communication as it relates to transcriptional gene regulators—i.e., transcription factors and corresponding unit opera- tions. In this review, we (a) explore allosteric communication in the lactose repressor LacI topology, (b) demonstrate how to leverage this understanding of allostery in the LacI system to engineer non-natural BUFFER and NOT logical operations, (c) illustrate how engineering workflows can be used to confer alternate allosteric functions in disparate systems that share the LacI topology, and (d) demonstrate how fundamental unit operations can be directed to form combinational logical operations.

Abstract

Recent advances in synthetic biology and protein engineering have increased the number of allosteric transcription factors used to regulate independent promoters. These developments represent an important increase in our biological computing capacity, which will enable us to construct more sophisticated genetic programs for a broad range of biological technologies. However, the majority of these transcription factors are represented by the repressor phenotype (BUFFER), and require layered inversion to confer the antithetical logical function (NOT), requiring additional biological resources. Moreover, these engineered transcription factors typically utilize native ligand binding functions paired with alternate DNA binding functions. In this study, we have advanced the state-of-the-art by engineering and redesigning the PurR topology (a native antirepressor) to be responsive to ca!eine, while mitigating responsiveness to the native ligand hypoxanthine!i.e., a deamination product of the input molecule adenine. Importantly, the resulting ca!eine responsive transcription factors are not antagonized by the native ligand hypoxanthine. In addition, we conferred alternate DNA binding to the ca!eine antirepressors, and to the PurR sca!old, creating 38 new transcription factors that are congruent with our current transcriptional programming structure. Finally, we leveraged this system of transcription factors to create integrated NOR logic and related feedback operations. This study represents the "rst example of a system of transcription factors (antirepressors) in which both the ligand binding site and the DNA binding functions were successfully engineered in tandem.

Abstract

Traditionally engineered genetic circuits have almost exclusively used naturally occurring transcriptional repressors. Recently, non-natural transcription factors (repressors) have been engineered and employed in synthetic biology with great success. However, transcriptional anti-repressors have largely been absent with regard to the regulation of genes in engineered genetic circuits. Here, we present a work!ow for engineering systems of non-natural anti- repressors. In this study, we create 41 inducible anti-repressors. This collection of tran- scription factors respond to two distinct ligands, fructose (anti-FruR) or D-ribose (anti-RbsR); and were complemented by 14 additional engineered anti-repressors that respond to the ligand isopropyl à-d-1-thiogalactopyranoside (anti-LacI). In turn, we use this collection of anti-repressors and complementary genetic architectures to confer logical control over gene expression. Here, we achieved all NOT oriented logical controls (i.e., NOT, NOR, NAND, and XNOR). The engineered transcription factors and corresponding series, parallel, and series- parallel genetic architectures represent a nascent anti-repressor based transcriptional pro- gramming structure.

Abstract

Inducible promoters are a central regulatory com- ponent in synthetic biology, metabolic engineering, and protein production for laboratory and commer- cial uses. Many of these applications utilize two or more exogenous promoters, imposing a currently un- quantifiable metabolic burden on the living system. Here, we engineered a collection of inducible pro- moters (regulated by LacI-based transcription fac- tors) that maximize the free-state of endogenous RNA polymerase (RNAP). We leveraged this collec- tion of inducible promotors to construct simple two- channel logical controls that enabled us to measure metabolic burden – as it relates to RNAP resource partitioning. The two-channel genetic circuits utilized sets of signal-coupled transcription factors that reg- ulate cognate inducible promoters in a coordinated logical fashion. With this fundamental genetic ar- chitecture, we evaluated the performance of each inducible promoter as discrete operations, and as coupled systems to evaluate and quantify the ef- fects of resource partitioning. Obtaining the ability to systematically and accurately measure the ap- parent RNA-polymerase resource budget will enable researchers to design more robust genetic circuits, with significantly higher fidelity. Moreover, this study presents a workflow that can be used to better under- stand how living systems adapt RNAP resources, via the complementary pairing of constitutive and regu- lated promoters that vary in strength.

Abstract

Transmembrane protein channels, including ion channels and aquaporins that are responsible for fast and selective transport of water, have inspired membrane scientists to exploit and mimic their performance in membrane technologies. These biomimetic membranes comprise discrete nanochannels aligned within amphiphilic matrices on a robust support. While biological components have been used directly, extensive work has also been conducted to produce stable synthetic mimics of protein channels and lipid bilayers. However, the experimental performance of biomimetic membranes remains far below that of biological membranes. In this review, we critically assess the status and potential of biomimetic desalination membranes. We !rst review channel chemistries and their transport behavior, identifying key characteristics to optimize water permeability and salt rejection. We compare various channel types within an industrial context, considering transport performance, processability, and stability. Through a re-examination of previous vesicular stopped-"ow studies, we demonstrate that incorrect permeability equations result in an overestimation of the water permeability of nanochannels. We find in particular that the most optimized aquaporin-bearing bilayer had a pure water permeability of 2.1 L m-2 h-1 bar-1, which is comparable to that of current state- of-the-art polymeric desalination membranes. Through a quantitative assessment of biomimetic membrane formats, we analytically show that formats incorporating intact vesicles o#er minimal bene!t, whereas planar biomimetic selective layers could allow for dramatically improved salt rejections. We then show that the persistence of nanoscale defects explains observed subpar performance. We conclude with a discussion on optimal strategies for minimizing these defects, which could enable breakthrough performance.

Abstract

Three brothers, black males, one murdered, one incarcerated—one unscathed (me). Three cousins, black males, two incarcerated—one with his liberties intact (me). Six siblings, only two completed high school—one being me. I remember, when I was 4 years old, walking to the store with my sister and being scolded for picking up a hypodermic needle someone used to inject heroin… and we lived in the better part of my neighborhood. This is the legacy of redlining, compounded by mass incarceration. If you do not know what redlining is, look it up. Purportedly, redlining ended in 1968; however, it is still the birthright of many black Americans. My early life experience is the product of this legacy. So, when people ask me "what was the hardest part of my academic journey?", I tell them "getting here to begin with." Studies suggest that success in STEM correlates with a student’s experience and exposure in middle school—my first positive experience with STEM was in col- lege, in the form of a calculus course. From that moment, academia (STEM) was a sanctuary for me—once I realized it was an option. After college, I finished graduate school (PhD) in 3 years with 10 published papers and started my position as faculty 2 years later. Ending the legacy of redlining is synonymous to fixing the "pipeline" for increasing the number of black scientist and engineers.

Abstract

Protein allostery is a vitally important protein function that has proven to be a vexing problem to understand at the molecular level. Allosteric communication is a hallmark of many protein functions. However, despite more than four decades of study the details regarding allosteric communication in protein systems are still being developed. Engineering of LacI and related homologues to confer alternate allosteric communication has shed light on the pre-requisites for the de novo design of allosteric communication. While the de novo design of an allosteric pathway and complementary functional surfaces has not been realized, this review highlights recent advances that set the stage for true predictive design for a given protein topology.

Abstract

The control of gene expression is an important tool for metabolic engineering, the design of synthetic gene networks, and protein manufacturing. The most successful approaches to date are based on modulating mRNA synthesis via an inducible coupling to transcriptional effectors. Here we present a biological programming structure that leverages a system of engineered transcription factors and complementary genetic architectures. We use a modular design strategy to create 27 non-natural and non-synonymous transcription factors using the lactose repressor topology as a guide. To direct systems of engineered transcription factors we employ parallel and series genetic (DNA) architectures and confer fundamental and combinatorial logical control over gene expression. Here we achieve AND, OR, NOT, and NOR logical controls in addition to two non-canonical half-AND operations. The basic logical operations and corresponding parallel and series genetic architectures represent the building blocks for subsequent combinatorial programs, which display both digital and analog performance.

Abstract

The lactose repressor, LacI (I+YQR), is an archetypal transcription factor that has been a workhorse in many synthetic genetic networks. LacI represses gene expression (apo ligand) and is induced upon binding of the ligand isopropyl β-d-1-thiogalactopyranoside (IPTG). Recently, laboratory evolution was used to confer inverted function in the native LacI topology resulting in anti-LacI (antilac) function (IAYQR), where IPTG binding results in gene suppression. Here we engineered 46 antilacs with alternate DNA binding function (IAADR). Phenotypically, IAADR transcription factors are the inverse of wild-type I+YQR function and possess alternate DNA recognition (ADR). This collection of bespoke IAADR bind orthogonally to disparate non-natural operator DNA sequences and suppress gene expression in the presence of IPTG. This new class of IAADR gene regulators were designed modularly via the systematic pairing of nine alternate allosteric regulatory cores with six alternate DNA binding domains that interact with complementary synthetic operator DNA sequences. The 46 IAADR identified in this study are also orthogonal to the naturally occurring operator O1. Finally, a demonstration of full orthogonality was achieved via the construction of synthetic genetic toggle switches composed of two nonsynonymous unit pair operations that control two distinct fluorescent outputs. This new class of IAADR transcription factors will facilitate the expansion of the computational capacity of engineered gene circuits, via the scalable increase in the control over the number of gene outputs by way of the expansion of the number of unique transcription factors (or systems of transcription factors) that can simultaneously regulate one or more promoter(s).

Abstract

Biomolecular assembly is a key driving force in nearly all life processes, providing structure, information storage, and communication within cells and at the whole organism level. These assembly processes rely on precise interactions between functional groups on nucleic acids, proteins, carbohydrates, and small molecules, and can be fine-tuned to span a range of time, length, and complexity scales. Recognizing the power of these motifs, researchers have sought to emulate and engineer biomolecular assemblies in the laboratory, with goals ranging from modulating cellular function to the creation of new polymeric materials. In most cases, engineering efforts are inspired or informed by understanding the structure and properties of naturally occurring assemblies, which has in turn fueled the development of predictive models that enable computational design of novel assemblies. This Review will focus on selected examples of protein assemblies, highlighting the story arc from initial discovery of an assembly, through initial engineering attempts, toward the ultimate goal of predictive design. The aim of this Review is to highlight areas where significant progress has been made, as well as to outline remaining challenges, as solving these challenges will be the key that unlocks the full power of biomolecules for advances in technology and medicine.

Abstract

The control of gene expression is an important tool for metabolic engineering, the design of synthetic gene networks, gene-function analysis, and protein man- ufacturing. The most successful approaches to date are based on modulating messenger RNA (mRNA) synthesis via their inducible coupling to transcriptional effectors, which requires biosensing functionality. A hallmark of biological sen- sing is the conversion of an exogenous signal, usually a small molecule or envi- ronmental cue such as a protein–ligand interaction, into a useful output or response. One of the most utilized regulatory proteins is the lactose repressor (LacI). In this review we will (1) explore the mechanochemical structure–function relationship of LacI; (2) discuss how the physical attributes of LacI can be lever- aged to identify and understand other regulatory proteins; (3) investigate the designability (tunability) of LacI; (4) discuss the potential of the modular design of novel regulatory proteins, fashioned after the topology and mechanochemical properties of LacI.

Abstract

The lactose repressor (LacI) is a classic genetic switch that has been used as a fundamental component in a host of synthetic genetic networks. To expand the function of LacI for use in the development of novel networks and other biotechnological applications, we engineered alternate communication in the LacI scaffold via laboratory evolution. Here we produced 14 new regulatory elements based on the LacI topology that are responsive to isopropyl β-d-1-thiogalactopyranoside (IPTG) with variation in repression strengths and ligand sensitivities-on solid media. The new variants exhibit repressive as well as antilac (i.e., inverse-repression + IPTG) functions and variations in the control of gene output upon exposure to different concentrations of IPTG. In addition, examination of this collection of variants in solution results in the controlled output of a canonical florescent reporter, demonstrating the utility of this collection of new regulatory proteins under standard conditions.

Abstract

Characterization of the mechanisms underlying hypohalous acid (i.e., hypochlorous acid or hypobromous acid) degradation of proteins is important for understanding how the immune system deactivates pathogens during infections and damages human tissues during inflammatory diseases. Proteins are particularly important hypohalous acid reaction targets in pathogens and in host tissues, as evidenced by the detection of chlorinated and brominated oxidizable residues. While a significant amount of work has been conducted for reactions of hypohalous acids with a range of individual amino acids and small peptides, the assessment of oxidative decay in full-length proteins has lagged in comparison. The most rigorous test of our understanding of oxidative decay of proteins is the rational redesign of proteins with conferred resistances to the decay of structure and function. Toward this end, in this study, we experimentally determined a putative mechanism of oxidative decay using adenylate kinase as the model system. In turn, we leveraged this mechanism to rationally design new proteins and experimentally test each system for oxidative resistance to loss of structure and function. From our extensive assessment of secondary structure, protein hydrodynamics, and enzyme activity upon hypochlorous acid or hypobromous acid challenge, we have identified two key strategies for conferring structural and functional resistance, namely, the design of proteins (adenylate kinase enzymes) that are resistant to oxidation requires complementary consideration of protein stability and the modification (elimination) of certain oxidizable residues proximal to catalytic sites.

Abstract

Proteins are important targets of chemical disinfectants. To improve the understanding of disinfectant-protein reactions, this study characterized the disinfectant:protein molar ratios at which 50% degradation of oxidizable amino acids (i.e., Met, Tyr, Trp, His, Lys) and structure were observed during HOCl, HOBr, and O3 treatment of three well-characterized model proteins and bacteriophage MS2. A critical question is the extent to which the targeting of amino acids is driven by their disinfectant rate constants rather than their geometrical arrangement. Across the model proteins and bacteriophage MS2 (coat protein), differing widely in structure, methionine was preferentially targeted, forming predominantly methionine sulfoxide. This targeting concurs with its high disinfectant rate constants and supports its hypothesized role as a sacrificial antioxidant. Despite higher HOCl and HOBr rate constants with histidine and lysine than for tyrosine, tyrosine generally was degraded in preference to histidine, and to a lesser extent, lysine. These results concur with the prevalence of geometrical motifs featuring histidines or lysines near tyrosines, facilitating histidine and lysine regeneration upon Cl[+1] transfer from their chloramines to tyrosines. Lysine nitrile formation occurred at or above oxidant doses where 3,5-dihalotyrosine products began to degrade. For O3, which lacks a similar oxidant transfer pathway, histidine, tyrosine, and lysine degradation followed their relative O3 rate constants. Except for its low reactivity with lysine, the O3 doses required to degrade amino acids were as low as or lower than for HOCl or HOBr, indicating its oxidative efficiency. Loss of structure did not correlate with loss of particular amino acids, suggesting the need to characterize the oxidation of specific geometric motifs to understand structural degradation.

Abstract

Proteins are the most functionally diverse macromolecules observed in nature, participating in a broad array of catalytic, biosensing, transport, scaffolding, and regulatory functions. Fittingly, proteins have become one of the most promising nanobiotechnological tools to date, and through the use of recombinant DNA and other laboratory methods we have produced a vast number of biological therapeutics derived from human genes. Our emerging ability to rationally design proteins (e.g., via computational methods) holds the promise of significantly expanding the number and diversity of protein therapies and has opened the gateway to realizing true and uncompromised personalized medicine. In the last decade computational protein design has been transformed from a set of fundamental strategies to stringently test our understanding of the protein structure-function relationship, to practical tools for developing useful biological processes, nano-devices, and novel therapeutics. As protein design strategies improve (i.e., in terms of accuracy and efficiency) clinicians will be able to leverage individual genetic data and biological metrics to develop and deliver personalized protein therapeutics with minimal delay.

Abstract

Pseudomonas aeruginosa azurin has been an important model system for investigating fundamental electron transfer (EleT) in proteins. Early pioneering studies used ruthenium photosensitizers to induce EleT in azurin and this experimental data continues to be used to develop theories for EleT mediated through a protein matrix. In this study we show that putative EleT rates in the P. aeruginosa azurin model system, measured via photoinduced methods, can also be explained by an alternate energy transfer (EngT) mechanism. Investigation of EngT in azurin, conducted in this study, isolates and resolves confounding phenomena--i.e., zinc contamination and excited state emission--that can lead to erroneous kinetic assignments. Here we employ two azurin photosensitizer systems, the previously reported Ru(2,2'-bipyridine)2(imidazole) and an unreported phototrigger, Ru(bpy)2(phen-IA), Ru(2,2'-bipyridine)2(5-iodoacetamido-1,10-phenanthroline), that has a longer lifetime, to better resolve convoluted kinetic observations and allow us to draw clear distinctions between photoinduced EngT and EleT. Extensive metal analysis, in addition to electrochemical and photochemical (photoinduced transfer) measurements, suggests Zn-metalated azurin contamination can result in a biexponential reaction, which can be mistaken for EleT. Namely, upon photoinduction, the observed slow phase is exclusively the contribution from Zn-metalated azurin, not EleT, whereas the fast phase is the result of EngT between the photosensitizer and the Cu-site, rather than simple excited-state decay of the phototrigger.

Abstract

Careful balance between structural stability and flexibility is a hallmark of enzymatic function, and temperature can affect both properties. Canonical (fixed-backbone) enzyme design strategies currently do not consider the role of these properties. Herein, we describe the rational design of 100 temperature-adapted adenylate kinase enzymes using a multistate design strategy that incorporates the impact of conformational changes to backbone structure and stability, in addition to experimental analysis of thermostability and function. Comparison of the experimental temperature of maximum activity to the melting temperature across all 100 variants reveals a strong correlation between these two parameters. In turn, experimental stability data were used to produce accurate predictions of thermostability, providing the requisite complement for de novo temperature-adapted enzyme design. In principle, this level of design-based analysis can be applied to any protein, paving the way toward identifying and understanding the hallmarks of the thermodynamic and structural limits of function.

Abstract

The application of chemical oxidants may alter the sorption properties of dissolved organic matter (DOM), such as humic and fulvic acids, proteins, polysaccharides, and lipids, affecting their fate in water treatment processes, including attachment to other organic components, activated carbon, and membranes (e.g., organic fouling). Similar reactions with chlorine (HOCl) and bromine (HOBr) produced at inflammatory sites in vivo affect the fate of biomolecules (e.g., protein aggregation). In this study, quartz crystal microbalance with dissipation monitoring (QCM-D) was used to evaluate changes in the noncovalent interactions of proteins, polysaccharides, fatty acids, and humic and fulvic acids with a model hydrophobic surface as a function of increasing doses of HOCl, HOBr, and ozone (O3). All three oxidants enhanced the sorption tendency of proteins to the hydrophobic surface at low doses but reduced their sorption tendency at high doses. All three oxidants reduced the sorption tendency of polysaccharides and fatty acids to the hydrophobic surface. HOCl and HOBr increased the sorption tendency of humic and fulvic acids to the hydrophobic surface with maxima at moderate doses, while O3 decreased their sorption tendency. The behavior observed with two water samples was similar to that observed with humic and fulvic acids, pointing to the importance of these constituents. For chlorination, the highest sorption tendency to the hydrophobic surface was observed within the range of doses typically applied during water treatment. These results suggest that ozone pretreatment would minimize membrane fouling by DOM, while chlorine pretreatment would promote DOM removal by activated carbon.

Abstract

Protein engineering holds the potential to transform the metabolic drug landscape through the development of smart, stimulusresponsive drug systems. Protein therapeutics are a rapidly expanding segment of Food and Drug Administration approved drugs that will improve clinical outcomes over the long run. Engineering of protein therapeutics is still in its infancy, but recent general advances in protein engineering capabilities are being leveraged to yield improved control over both pharmacokinetics and pharmacodynamics. Stimulus- responsive protein therapeutics are drugs which have been designed to be metabolized under targeted conditions. Protein engineering is being utilized to develop tailored smart therapeutics with biochemical logic. This review focuses on applications of targeted drug neutralization, stimulus-responsive engineered protein prodrugs, and emerging multicomponent smart drug systems (e.g., antibody-drug conjugates, responsive engineered zymogens, prospective biochemical logic smart drug systems, drug buffers, and network medicine applications).

Abstract

To expand our understanding of the hallmarks of allosteric control we used directed-evolution to engineer alternate cooperative communication in the lactose repressor protein (LacI) scaffold. Starting with an I(s) type LacI mutant D88A (i.e. a LacI variant that is insensitive to the exogenous ligand isopropyl-β-d-thiogalactoside (IPTG) and remains bound to operator DNA, + or -IPTG) we used error-prone polymerase chain reaction to introduce compensatory mutations to restore modulated DNA binding function to the allosterically 'dead' I(s)(D88A) background. Five variants were generated, three variants (C4, C32 and C80) with wild-type like function and two co-repressor variants (C101 and C140) that are functionally inverted. To better resolve the residues that define new allosteric networks in the LacI variants, we conducted mutational tolerance analysis via saturation mutagenesis at each of the evolved positions to assess sensitivity to mutation--a hallmark of allosteric residues. To better understand the physicochemical bases of alternate allosteric function, variant LacI(C80) was characterized to assess IPTG ligand binding at equilibrium, kinetically using stopped-flow, and via isothermal titration calorimetry. These data suggest that the conferred modulated DNA binding function observed for LacI(C80), while thermodynamically similar to wild-type LacI, is mechanistically different from the wild-type repressor, suggesting a new allosteric network and communication route.

Abstract

Although protein degradation by neutrophil-derived hypochlorous acid (HOCl) and eosinophil-derived hypobromous acid (HOBr) can contribute to the inactivation of pathogens, collateral damage to host proteins can also occur and has been associated with inflammatory diseases ranging from arthritis to atherosclerosis. Though previous research suggested halotyrosines as biomarkers of protein damage and lysine as a mediator of the transfer of a halogen to tyrosine, these reactions within whole proteins are poorly understood. Herein, reactions of HOCl and HOBr with three well-characterized proteins [adenylate kinase (ADK), ribose binding protein, and bovine serum albumin] were characterized. Three assessments of oxidative modifications were evaluated for each of the proteins: (1) covalent modification of electron-rich amino acids (assessed via liquid chromatography and tandem mass spectrometry), (2) attenuation of secondary structure (via circular dichroism), and (3) fragmentation of protein backbones (via sodium dodecyl sulfate-polyacrylamide gel electrophoresis). In addition to forming halotyrosines, HOCl and HOBr converted lysine into lysine nitrile (2-amino-5-cyanopentanoic acid), a relatively stable and largely overlooked product, in yields of up to 80%. At uniform oxidant levels, fragmentation and loss of secondary structure correlated with protein size. To further examine the role of lysine, a lysine-free ADK variant was rationally designed. The absence of lysine increased yields of chlorinated tyrosines and decreased yields of brominated tyrosines following treatments with HOCl and HOBr, respectively, without influencing the susceptibility of ADK to HOX-mediated losses of secondary structure. These findings suggest that lysine serves predominantly as a sacrificial antioxidant (via formation of lysine nitrile) toward HOCl and as a halogen-transfer mediator [via reactions involving ε-N-(di)haloamines] with HOBr.

Abstract

Antigen-specific activation of cytotoxic T cells can be enhanced up to three-fold more than soluble controls when using functionalized bundled carbon nanotube substrates ((b) CNTs). To overcome the denaturing effects of direct adsorption on (b) CNTs, a simple but robust method is demonstrated to stabilize the T cell stimulus on carbon nanotube substrates through non-covalent attachment of the linker neutravidin.

Abstract

We demonstrate the self-organization of quasi-one-dimensional nanostructures with periodic features using nature's primary three building blocks: lipids, DNA, and proteins. The periodicity of these "BioNanoStacks" is controllable through selection of the length of the DNA spacers. We show that BioNanoStacks can be reversibly assembled and disassembled through thermal melting of the DNA duplex, where the melting transition temperature is controllable not just by the DNA sequence and salt concentration, but also by the lipid composition within these superstructures. These novel materials may find applications in fields such as templated nanomaterial assembly, tissue-engineering scaffolds, or therapeutic delivery systems. Well-established techniques for chemical modification of biomolecules will also provide a broad platform for adaption and remodeling of these structures to provide optimal features for the required application.

Abstract

The fundamental principles that govern monomer folding are believed to be congruent with those of protein oligomers. However, the effects of protein assembly during the folding reaction can result in a series of complex transitions that are considerably more challenging to deconvolute. Here we developed the experimental protein folding mechanism for the lactose repressor (LacI), for both the dimeric and the tetrameric states, using equilibrium unfolding and kinetic experiments, and by leveraging the previously reported monomer folding landscape. Reaction details for LacI oligomers were observed by way of circular dichroism, intrinsic fluorescence, and Förster resonance energy transfer (FRET) and as a function of protein concentration. In general, the dimer and tetramer are four-phase folding reactions in which the first three transitions are tantamount to the folding of constituent monomers. The final reaction phase of the LacI dimer can be attributed to protein assembly, based on the concentration dependence of the observed folding rates and intermolecular FRET measurements. Unlike the dimer, the latter reaction phase of the LacI tetramer is not dependent on protein concentration, likely because of a strong tethering of the monomers, which simplifies the folding reaction by eliminating an explicit protein assembly phase. Finally, folding of the LacI dimer and tetramer was assessed in the presence of polyethylene glycol to rule out inert molecular crowding as the driving force for the protein folding reaction; in addition, these data provide insight into the folding mechanism in vivo.

Abstract

Success in evolution depends critically upon the ability of organisms to adapt, a property that is also true for the proteins that contribute to the fitness of an organism. Successful protein evolution is enhanced by mutational pathways that generate a wide range of physicochemical mechanisms to adaptation. In an earlier study, we used a weak-link method to favor changes to an essential but maladapted protein, adenylate kinase (AK), within a microbial population. Six AK mutants (a single mutant followed by five double mutants) had success within the population, revealing a diverse range of adaptive strategies that included changes in nonpolar packing, protein folding dynamics, and formation of new hydrogen bonds and electrostatic networks. The first mutation, AK(BSUB) Q199R, was essential in defining the structural context that facilitated subsequent mutations as revealed by a considerable mutational epistasis and, in one case, a very strong dependence upon the order of mutations. Namely, whereas the single mutation AK(BSUB) G213E decreases protein stability by >25 degrees C, the same mutation in the background of AK(BSUB) Q199R increases stability by 3.4 degrees C, demonstrating that the order of mutations can play a critical role in favoring particular molecular pathways to adaptation. In turn, protein folding kinetics shows that four of the five AK(BSUB) double mutants utilize a strategy in which an increase in the folding rate accompanied by a decrease in the unfolding rate results in additional stability. However, one mutant exhibited a dramatic increase in the folding relative to a modest increase in the unfolding rate, suggesting a different adaptive strategy for thermostability. In all cases, an increase in the folding rates for the double mutants appears to be the preferred mechanism in conferring additional stability and may be an important aspect of protein evolution. The range of overlapping as well as contrasting strategies for success illustrates both the power and subtlety of adaptation at even the smallest unit of change, a single amino acid.

Abstract

Protein function is a balance between activity and stability. However, the relevance of stability-activity trade-offs for protein evolution and their impact on organismal fitness have been difficult to determine. Previously, we have linked organismal survival at increasing temperatures to adaptive changes to a single protein sequence through allelic replacement of an essential gene, adenylate kinase (adk), in a thermophile. In vivo continuous evolution of the temperature-sensitive thermophile has shown that the first step toward increased organismal fitness is mutation of glutamine-199 to arginine in the mesophilic enzyme (AKsub Q199R). Here, we show that although substitution of Arg-199 did confer a modest increase in stability (0.6 kcal mol(-1)at 20 degrees C; DeltaT(m) = 3.0 degrees C), it is a large change in the activity profile of the enzyme that is responsible for its exceptional robustness during the earlier experimental evolution study. Kinetic studies of AKsub Q199R show that it has a strong loss of enzymatic activity (>50%) at lower temperatures (20-45 degrees C) and a subsequent increase at elevated temperatures. The stability-activity trade-off observed for AKsub Q199R was linked to the rigidification of the overall structure through stabilization of a polypeptide loop containing Arg-199 that is part of the ATP-binding site of the enzyme. Structural analysis revealed the formation of new ionic interactions facilitated by Arg-199. Our results suggest that stability-activity trade-offs are employed readily as an evolutionary strategy during natural selection to increase organismal fitness.

Abstract

Understanding how the folding of proteins establishes their functional characteristics at the molecular level challenges both theorists and experimentalists. The simplest test beds for confronting this issue are provided by electron transfer proteins. The environment provided by the folded protein to the cofactor tunes the metal's electron transport capabilities as envisioned in the entatic hypothesis. To see how the entatic state is achieved one must study how the folding landscape affects and in turn is affected by the metal. Here, we develop a coarse-grained functional to explicitly model how the coordination of the metal (which results in a so-called entatic or rack-induced state) modifies the folding of the metallated Pseudomonas aeruginosa azurin. Our free-energy functional-based approach directly yields the proper nonlinear extra-thermodynamic free energy relationships for the kinetics of folding the wild type and several point-mutated variants of the metallated protein. The results agree quite well with corresponding laboratory experiments. Moreover, our modified free-energy functional provides a sufficient level of detail to explicitly model how the geometric entatic state of the metal modifies the dynamic folding nucleus of azurin.

Abstract

Specific interactions between proteins and ligands that modify their functions are crucial in biology. Here, we examine sugars that bind the lactose repressor protein (LacI) and modify repressor affinity for operator DNA using isothermal titration calorimetry and equilibrium DNA binding experiments. High affinity binding of the commonly-used inducer isopropyl-beta,D-thiogalactoside is strongly driven by enthalpic forces, whereas inducer 2-phenylethyl-beta,D-galactoside has weaker affinity with low enthalpic contributions. Perturbing the dimer interface with either pH or oligomeric state shows that weak inducer binding is sensitive to changes in this distant region. Effects of the neutral compound o-nitrophenyl-beta,D-galactoside are sensitive to oligomerization, and at elevated pH this compound converts to an anti-inducer ligand with slightly enhanced enthalpic contributions to the binding energy. Anti-inducer o-nitrophenyl-beta,D-fucoside exhibits slightly enhanced affinity and increased enthalpic contributions at elevated pH. Collectively, these results both demonstrate the range of energetic consequences that occur with LacI binding to structurally-similar ligands and expand our insight into the link between effector binding and structural changes at the subunit interface.

Abstract

In 1961, Jacob and Monod proposed the operon model for gene regulation based on metabolism of lactose in Escherichia coli. This proposal was followed by an explication of allosteric behavior by Monod and colleagues. The operon model rationally depicted how genetic mechanisms can control metabolic events in response to environmental stimuli via coordinated transcription of a set of genes with related function (e.g. metabolism of lactose). The allosteric response found in the lactose repressor and many other proteins has been extended to a variety of cellular signaling pathways in all organisms. These two models have shaped our view of modern molecular biology and captivated the attention of a surprisingly broad range of scientists. More recently, the lactose repressor monomer was used as a model system for experimental and theoretical explorations of protein folding mechanisms. Thus, the lac system continues to advance our molecular understanding of genetic control and the relationship between sequence, structure and function.

Abstract

Pseudomonas aeruginosa azurin is a 128-residue beta-sandwich metalloprotein; in vitro kinetic experiments have shown that it folds in a two-state reaction. Here, we used a variational free energy functional to calculate the characteristics of the transition state ensemble (TSE) for folding of the apo-form of P. aeruginosa azurin and investigate how it responds to thermal and mutational changes. The variational method directly yields predicted chevron plots for wild-type and mutant apo-forms of azurin. In parallel, we performed in vitro kinetic-folding experiments on the same set of azurin variants using chemical perturbation. Like the wild-type protein, all apo-variants fold in apparent two-state reactions both in calculations and in stopped-flow mixing experiments. Comparisons of phi (phi) values determined from the experimental and theoretical chevron parameters reveal an excellent agreement for most positions, indicating a polarized, highly structured TSE for folding of P. aeruginosa apo-azurin. We also demonstrate that careful analysis of side-chain interactions is necessary for appropriate theoretical description of core mutants.

Abstract

The role of water in protein folding, specifically its presence or not in the transition-state structure, is an unsolved question. There are two common classes of folding-transition states: diffuse transition states, in which almost all side chains have similar, rather low phi (phi) values, and polarized transition states, which instead display distinct substructures with very high phi-values. Apo-and zinc-forms of Pseudomonas aeruginosa azurin both fold in two-state equilibrium and kinetic reactions; while the apo-form exhibits a polarized transition state, the zinc form entails a diffuse, moving transition state. To examine the presence of water in these two types of folding-transition states, we probed the equilibrium and kinetic consequences of replacing core valines with isosteric threonines at six positions in azurin. In contrast to regular hydrophobic-to-alanine phi-value analysis, valine-to-threonine mutations do not disrupt the core packing but stabilize the unfolded state and can be used to assess the degree of solvation in the folding-transition state upon combination with regular phi-values. We find that the transition state for folding of apo-azurin appears completely dry, while that for zinc-azurin involves partially formed interactions that engage water molecules. This distinct difference between the apo-and holo-folding nuclei can be rationalized in terms of the shape of the free-energy barrier.

Abstract

This paper reports a combined computational and experimental study of the correlation between protein stability cores and folding kinetics. An empirical potential function was developed, and it was used for analyzing interaction energies among secondary structure elements. Studies on a beta sandwich protein, Pseudomonas aeruginosa azurin, showed that the computationally identified substructure with the strongest interactions in the native state is identical to the "interlocked pair" of beta strands, an invariant motif found in most sandwich-like proteins. Moreover, previous and new in vitro folding results revealed that the identified substructure harbors most residues that form native-like interactions in the folding transition state. These observations demonstrate that the potential function is effective in revealing the relative strength of interactions among various protein parts; they also strengthen the suggestion that the most stable regions in native proteins favor stable interactions early during folding.

Abstract

An invariant substructure that forms two interlocked pairs of neighboring beta-strands occurs in essentially all known sandwich-like proteins. Eight conserved positions in these strands were recently shown to act as structural determinants. To test whether the residues at these invariant positions are conserved for mechanistic (i.e., part of folding nucleus) or energetic (i.e., governing native-state stability) reasons, we characterized the folding behavior of eight point-mutated variants of the sandwich-like protein Pseudomonas aeruginosa apo-azurin. We find a simple relationship among the conserved positions: half of the residues form native-like interactions in the folding transition state, whereas the others do not participate in the folding nucleus but govern high native-state stability. Thus, evolutionary preservation of these specific positions gives both mechanistic and energetic advantages to members of the sandwich-like protein family.

Abstract

Zinc-substituted Pseudomonas aeruginosa azurin folds in two-state equilibrium and kinetic reactions. In the unfolded state, the zinc ion remains bound to the unfolded polypeptide via two native-state ligands (His117 and Cys112). The significantly curved Chevron plot for zinc-substituted azurin was earlier ascribed to movement of the folding-transition state. At low concentrations of denaturant, the transition state occurs early in the folding reaction (low Tanford beta-value), whereas at high-denaturant concentration, it moves closer to the native structure (high Tanford beta-value). Here, we use this movement to track the formation and growth of zinc-substituted azurin's folding nucleus with atomic resolution using protein engineering. The average phi (phi) value for 17 positions (covering all secondary-structure elements) goes from 0.25 in 0 M GuHCl (beta approximately 0.46) to 0.76 in 4 M GuHCl (beta approximately 0.86); a phi-value of 1 or 0 indicates native-like or unfolded-like interactions, respectively. Analysis of individual phi-values reveals a delocalized nucleus where structure condenses around a leading density centered on Leu50 in the core. The diffuse moving transition state for zinc-substituted azurin is in sharp contrast to the fixed polarized folding nucleus observed for apo-azurin. The dramatic difference in apparent kinetic behavior for the two forms of azurin can be rationalized as a minor alteration on a common free-energy profile that exhibits a broad activation barrier.

Abstract

To probe the experimental folding behavior of a large protein with complex topology, we created a monomeric variant of the lactose repressor protein (MLAc), a well characterized tetrameric protein that regulates transcription of the lac operon. Purified MLAc is folded, fully functional, and binds the inducer isopropyl beta-d-thiogalactoside with the same affinity as wild-type LacI. Equilibrium unfolding of MLAc induced by the chemical denaturant urea is a reversible, apparent two-state process (pH 7.5, 20 degrees C). However, time-resolved experiments demonstrate that unfolding is single-exponential, whereas refolding data indicate two transient intermediates. The data reveal the initial formation of a burst-phase (tau < ms) intermediate that corresponds to approximately 50% of the total secondary-structure content. This step is followed by a rearrangement reaction that is rate-limited by an unfolding process (tau approximately 3 s; pH 7.5, 20 degrees C) and results in a second intermediate. This MLAc intermediate converts to the native structure (tau approximately 30 s; pH 7.5, 20 degrees C). Remarkably, the experimental folding-energy landscape for MLAc is in excellent agreement with theoretical predictions using a simple topology-based C(alpha)-model as presented in a companion article in this issue.

Abstract

Recent theoretical/computational studies based on simplified protein models and experimental investigation have suggested that the native structure of a protein plays a primary role in determining the folding rate and mechanism of relatively small single-domain proteins. Here, we extend the study of the relationship between protein topology and folding mechanism to a larger protein with complex topology, by analyzing the folding process of monomeric lactose repressor (MLAc) computationally by using a Gō-like C(alpha) model. Next, we combine simulation and experimental results (see companion article in this issue) to achieve a comprehensive assessment of the folding landscape of this protein. Remarkably, simulated kinetic and equilibrium analyses show an excellent quantitative agreement with the experimental folding data of this study. The results of this comparison show that a simplified, completely unfrustrated C(alpha) model correctly reproduces the complex folding features of a large multidomain protein with complex topology. The success of this effort underlines the importance of synergistic experimental/theoretical approaches to achieve a broader understanding of the folding landscape.

Abstract

Metals are commonly found as natural constituents of proteins. Since many such metals can interact specifically with their corresponding unfolded proteins in vitro, cofactor-binding prior to polypeptide folding may be a biological path to active metalloproteins. By interacting with the unfolded polypeptide, the metal may create local structure that initiates and directs the polypeptide-folding process. Here, we review recent literature that addresses the involvement of metals in protein-folding reactions in vitro . To date, the best characterized systems are simple one such as blue-copper proteins, heme-binding proteins, iron-sulfur-cluster proteins and synthetic metallopeptides. Taken together, the available data demonstrates that metals can play diverse roles: it is clear that many cofactors bind before polypeptide folding and influence the reaction; yet, some do not bind until a well-structured active site is formed. The significance of characterizing the effects of metals on protein conformational changes is underscored by the many human diseases that are directly linked to anomalous protein-metal interactions.

Abstract

Differential display-PCR (DDPCR) was used to identify a Streptococcus pneumoniae gene with enhanced transcription during growth in the murine peritoneal cavity. Northern dot blot analysis and comparative densitometry confirmed a 1.8-fold increase in expression of the encoded sequence following murine peritoneal culture (MPC) versus laboratory culture or control culture (CC). Sequencing and basic local alignment search tool analysis identified the DDPCR fragment as pstS, the phosphate-binding protein of a high-affinity phosphate uptake system. PCR amplification of the complete pstS gene followed by restriction analysis and sequencing suggests a high level of conservation between strains and serotypes. Quantitative immunodot blotting using antiserum to recombinant PstS (rPstS) demonstrated an approximately twofold increase in PstS production during MPC from that during CCs, a finding consistent with the low levels of phosphate observed in the peritoneum. Moreover, immunodot blot and Northern analysis demonstrated phosphate-dependent production of PstS in six of seven strains examined. These results identify pstS expression as responsive to the MPC environment and extracellular phosphate concentrations. Presently, it remains unclear if phosphate concentrations in vivo contribute to the regulation of pstS. Finally, polyclonal antiserum to rPstS did not inhibit growth of the pneumococcus in vitro, suggesting that antibodies do not block phosphate uptake; moreover, vaccination of mice with rPstS did not protect against intraperitoneal challenge as assessed by the 50% lethal dose.