Next-Generation Synthetic Memory via Intercepting Recombinase Function
Short, A.E., Kim, D., Milner, P., and Wilson, C. J.
2023 Nature Communications. DOI: 10.1038/s41467-023-41043-w
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.
Engineering Allosteric Transcription Factors Guided by the LacI Topology
Hersey, A.N., Kay, V.E, Lee, S., Realff, M., and Wilson, C. J.
2023 Cell Systems. DOI: 10.1016/j.cels.2023.04.008
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.
Performance Prediction of Fundamental Transcriptional Programs
Milner, P., Zhang, Z., Herde, Z., Vedire, N. R., Zhang, F., Realff, M., and Wilson, C. J.
2023 ACS Synthetic Biology. 12(4):1094-1108. PMID: 36935615
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.
Synthesizing cellular LOGIC
Huang, B.D., De Pereda, A.S., and Wilson, C. J.
2023 Nature Chemical Biology. (6):671-672. PMID: 36894720
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.
Transcriptional programming in a Bacteroides consortium
Huang, B.D., Groseclose, T.M., and Wilson, C. J.
2022 Nature Communications. 13(1):3901 PMID: 35794179
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.
Bioinspired Green Science and Technology Symposium in NYC
Alcantar, N.A., Banta, S., Cak, A.D., Chen, X., DelRe, C., Deravi, L.F., Dordick, J.S., Giebel, B.M., Groffman, M.G., Holford, M., John, G., Joshi, N.S., Kotov, N.A., Montclare, J.K., Moore, B.S., Ortony, J.H., Reinmann, A.B., Son, J., Stark, R.E., Ulijn, R.V., Vörösmarty, C.J., and Wilson, C. J.
2022 Matter. Vol. 5, Iss. 7, Pg. 1980-1984. PMID:
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.
Biological Signal Processing Filters via Engineering Allosteric Transcription Factors
Groseclose, T.M., Hersey, A.N., Huang, B.D., Realff,M.J., and Wilson, C. J.
2021 Proceedings of the National Academy of Sciences of the United States of America. Vol. 118, No 46 PMID: 34772815
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.
Biomolecular Systems Engineering – Unlocking the Potential of Engineered Allostery via the Lactose Repressor (LacI) Topology
Groseclose, T.M., Rondon, R.E., Hersey, A.N., Millner,P.T., Kim,D., Zhang,F., Realff,M.J., and Wilson, C. J.
2021 Annual Review of Biophysics. 6;50:303-321 PMID: 33606944
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.
Engineering Alternate Ligand Recognition in the PurR Topology: A System of Novel Caffeine Biosensing Transcriptional Anti-Repressors
Rondon, R.E. and Wilson, C. J.
2021 ACS Synthetic Biology. 9;10(3):552-565 PMID: 33689294
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.
Engineered Systems of Inducible Anti-Repressors for the Next Generation of Biological Programming
Groseclose, T.M., Rondon, R.E., Herde, Z.D., Aldrete, C.A., and Wilson, C. J.
2020 Nature Communications. 11(1):4440 PMID: 32895374
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.
Engineered Signal-Coupled Inducible Promoters: Measuring the Endogenous RNA-polymerase Resource Budget
Davey. J.A. and Wilson, C. J.
2020 Nucleic Acids Research. 48(17):9995-10012 PMID: 32890400
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.
Biomimetic Desalination Membranes with Sub-nanometer Channels: Where Are We Now?
Porter, C.J., Werber,J.R., Zhong, M., Wilson, C. J., and Elimelech, M.
2020 ACS Nano. 14(9):10894-10916 PMID: 32886487
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.
A Son of Redlines
Wilson, C. J.
2020 Cell Systems. 11(1):5-8 PMID: 32702319
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.
Engineering Allosteric Communication
Herde, Z.D., Short, A.E., Kay, V.E, Huang, B.D., Realff, M.J., and Wilson, C. J.
2020 Current Opinion in Structural Biology. 63:115-122 PMID: 32575020
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.
Transcriptional Programming using Engineered Systems of Transcription Factors and Genetic Architectures
Rondon, R.E., Groseclose, T.M., Short, A.E. and Wilson, C. J.
2019 Nature Communications. 10(1):4784. PMID 31636266
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.
Engineering a New Class of Anti-LacI Transcription Factors with Alternate DNA Recognition
Rondon, R.E. and Wilson, C. J.
2019 ACS Synthetic Biology. 8(2):307-317. PMID: 30601657
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).
Biomolecular Assemblies: Moving from Observation to Predictive Design
Wilson, C. J., Bommarius, A. S., Champion, J. A., Chernoff, Y. O., Lynn, D. G., Paravastu, A. K., Liang, C., Hsieh M. C. and Heemstra, J. M.
2018 ACS Chemical Reviews. 118(24): 11519–11574.
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.
Deconstruction of Complex Protein Signaling Switches - A Roadmap Toward Engineering Higher-Order Gene Regulators
Davey, J.A. and Wilson, C. J.
2017 WIREs Nanomedicine & Nanobiotechnology. 9(6):1939-5116. PMID: 28185424
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.
Fourteen Ways to Reroute Cooperative Communication in the Lactose Repressor: Engineering Regulatory Proteins with Alternate Repressive Functions
Richards, D.H., Meyer, S. and Wilson, C. J.
2017 ACS Synthetic Biology. 6(1):6-12. PMID: 27598336
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.
Leveraging Rational Design and Detailed Structural Analysis to Elucidate the Mechanism of Oxidative Decay in Adenylate Kinase
Howell, S. C., Mitch, W. A., Richards, D.H. and Wilson, C. J.
2015 ACS Chemical Biology. 10(10):2393-404. PMID: 26266833
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.
Degradation of Amino Acids and Structure in Model Proteins and Bacteriophage MS2 by Chlorine, Bromine, and Ozone
Choe, J. K., Richards, D.H., Wilson, C. J. and Mitch, W.A.
2015 Environmental Science & Technology. 49(22): 13331-9. PMID: 26488608
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.
Rational Protein Design: Developing Next Generation Biological Therapeutics and Nanobiotechnological Tools
Wilson, C.J.
2015 WIREs Nanomedicine & Nanobiotechnology. 7(3):330-41. PMID: 25348497
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.
Examining Photoinduced Energy Transfer in Pseudomonas aeruginosa Azurin
Tobin, P. and Wilson, C.J.
2014 Journal of American Chemical Society. 136(5):1793-802. PMID: 24467236
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.
Understanding Thermal Adaptation of Enzymes through the Multistate Rational Design and Stability Prediction of 100 Adenylate Kinases
Howell, S.C., Inampudi, K.K., Bean, D.P. and Wilson, C.J.
2014 Structure. 4;22(2):218-29. PMID: 24361272
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.
Effect of Chemical Oxidation on the Hydrophobicity of Dissolved Organic Matter
Zeng, T, Wilson, C. J., and Mitch, W. A.
2014 Environmental Science & Technology. 48(9):5118-26. PMID: 24697505
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.
Protein Engineering: A New Frontier for Drug Metabolism
Tobin, P., Richards, D.H., Randolph, R.A. and Wilson, C.J.
2014 Current Drug Metabolism. 15(7):743-56. PMID: 25495737
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).
Engineering Alternate Cooperative-Communications in the Lactose Repressor Protein Scaffold, Protein Engineering, Design & Selection
Meyer, S., Ramot, R., Kishore Inampudi, K., Luo, B., Lin, C., Amere, S., and Wilson, C. J.
2013 Protein Engineering Design & Selection 26, 433-443. PMID: 23587523
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.
Role of Lysine during Protein Modification by HOCl and HOBr: Halogen-Transfer Agent or Sacrificial Antioxidant?
Sivey, J. D., Howell, S. C., Bean, D. J., McCurry, D. L., Mitch, W. A., and Wilson, C.J.
2013 Biochemistry 52, 1260-1271. PMID: 23327477
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.
Adsorption of Multimeric T Cell Antigens on Carbon Nanotubes: Effect on Protein Structure and Antigen - Specific T Cell Stimulation
Fadel, T.R., Li, N., Shah, S., Look, M., Pfefferle, L. D., Haller, G.L., Justesen, S., Wilson, C.J. and Fahmy, T.M.
2013 Small 9, 666-672. PMID: 23090793
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.
Reversible Assembly of Stacked Membrane Nanodiscs with Reduced Dimensionality and Variable Periodicity
Beales, P. A., Geerts, N., Inampudi, K. K., Shigematsu, H., Wilson, C. J., and Vanderlick, T. K
2013 Journal of the American Chemical Society 135, 3335-38. PMID: 23405911
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.
Lactose Repressor Experimental Folding Landscape: Fundamental Functional Unit and Tetramer Folding Mechanisms
Ramot, R., Kishore Inampudi, K., and Wilson, C. J.
2012 Biochemistry 51, 7569-79. PMID: 22931511
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.
Experimental Evolution of Adenylate Kinase Reveals Contrasting Strategies towards Protein Thermostability
Miller, C., Davlieva, M., Wilson, C.J., White, K., Couñago, R., Wu, G., Myers, J.C., Wittung-Stafshede, P., and Shamoo, Y.
2010 Biophysical Journal 99, 887-896. PMID: 20682267
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.
An Adaptive Mutation in Adenylate Kinase that Increases Organismal Fitness is Linked to Stability-Activity Trade-offs
Counago, R., Wilson, C. J., Pena, M. I., Wittung-Stafshede, P., and Shamoo, Y.
2008 Protein Engineering Design & Selection 21, 19-27. PMID: 18093993
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.
Establishing the Entatic State in Folding Metallated Pseudomonas Aeruginosa Azurin
Zong, C. H., Wilson, C. J., Shen, T. Y., Wittung-Stafshede, P., Mayo, S. L., and Wolynes, P. G.
2007 Proceedings of the National Academy of Sciences of the United States of America 104, 3159-64 (corresponding author). PMID: 17301232
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.
Ligand Interactions with Lactose Repressor Protein and the Repressor-Operator Complex: The Effects of Ionization and Oligomerization on Binding
Wilson, C. J., Zhan, H. L., Swint-Kruse, L., and Matthews, K. S.
2007 Biophysical Chemistry 126, 94-105. PMID: 16860458
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.
The Lactose Repressor System: Paradigms for Regulation, Allosteric Behavior and Protein Folding
Wilson, C. J., Zhan, H., Swint-Kruse, L., and Matthews, K. S.
2007 Cellular and Molecular Life Sciences 64, 3-16. PMID: 17103112
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.
ϕ-Value Analysis of Apo-Azurin Folding: Comparison between Experiment and Theory
Zong, C. H., Wilson, C. J., Shen, T. Y., Wolynes, P. G., and Wittung-Stafshede, P.
2006 Biochemistry 45, 6458-66 (authors made equal contributions). PMID: 16700556
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.
Solvation of the Folding-Transition State in Pseudomonas Aeruginosa Azurin is Modulated by Metal
Wilson, C. J., Apiyo, D., and Wittung-Stafshede, P.
2006 Protein Science 15, 843-852. PMID: 16522792
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.
Correlation between Protein Stability Cores and Protein Folding Kinetics: A Case Study on Pseudomonas Aeruginosa Apo-Azurin
Chen, M. Z., Wilson, C. J., Wu, Y. H., Wittung-Stafshede, P., and Ma, J. P.
2006 Structure 14, 1401-1410. PMID: 16962971
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.
Role of Structural Determinants in Folding of the Sandwich-like Protein Pseudomonas Aeruginosa Azurin
Wilson, C. J., and Wittung-Stafshede, P.
2005 Proceedings of the National Academy of Sciences of the United States of America 102, 3984-3987. PMID: 15753320
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.
Snapshots of a Dynamic Folding Nucleus in Zinc-Substituted Pseudomonas Aeruginosa Azurin
Wilson, C. J., and Wittung-Stafshede, P.
2005 Biochemistry 44, 10054-10062. PMID: 16042382
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.
The Experimental Folding Landscape of Monomeric Lactose Repressor, a Large Two-Domain Protein, Involves Two Kinetic Intermediates
Wilson, C. J., Das, P., Clementi, C., Matthews, K. S., and Wittung-Stafshede, P.
2005 Proceedings of the National Academy of Sciences of the United States of America 102, 14563-14568. PMID: 16203983
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.
Characterization of the Folding Landscape of Monomeric Lactose Repressor: Quantitative Comparison of Theory and Experiment
Das, P., Wilson, C. J., Fossati, G., Wittung-Stafshede, P., Matthews, K. S., and Clementi, C.
2005 Proceedings of the National Academy of Sciences of the United States of America 102, 14569-14574. PMID: 16203982
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.
Role of Cofactors in Metalloprotein Folding
Wilson, C. J., Apiyo, D., and Wittung-Stafshede, P.
2004 Quarterly Reviews of Biophysics 37, 285-314. PMID: 16194296
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.
Streptococcus Pneumoniae PstS Production is Phosphate Responsive and Enhanced during Growth in the Murine Peritoneal Cavity
Orihuela, C. J., Mills, J., Robb, C. W., Wilson, C. J., Watson, D. A., and Niesel, D. W.
2001 Infection and Immunity 69, 7565-7571. PMID: 11705934
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.