Engineering protein assemblies with allosteric control via monomer fold-switching

The macromolecular machines of life use allosteric control to self-assemble, dissociate and change shape in response to signals. Despite enormous interest, the design of nanoscale allosteric assemblies has proven tremendously challenging. Here we present a proof of concept of allosteric assembly in which an engineered fold switch on the protein monomer triggers or blocks assembly. Our design is based on the hyper-stable, naturally monomeric protein CI2, a paradigm of simple two-state folding, and the toroidal arrangement with 6-fold symmetry that it only adopts in crystalline form. We engineer CI2 to enable a switch between the native and an alternate, latent fold that self-assembles onto hexagonal toroidal particles by exposing a favorable inter-monomer interface. The assembly is controlled on demand via the competing effects of temperature and a designed short peptide. These findings unveil a remarkable potential for structural metamorphosis in proteins and demonstrate key principles for engineering protein-based nanomachinery.This work was supported by the European Research Council (grant ERC-2012-ADG- 323059 to V.M.) and by the PRODESTECH network funded through the CONSOLIDER program from the Spanish Government (grant CSD2009-00088). L.A.C. acknowledges support from Ministry of Economy and Competitiveness through grants BIO2016- 78768-P and RYC-2013-13197. V.M. acknowledges additional support from the W.M. Keck Foundation and from the CREST Center for Cellular and Biomomolecular Machines (grant NSF-CREST-1547848). J.M.V. acknowledges additional support from Ministry of Economy and Competitiveness through grant BFU2016-75984. F.M.R. and A.R. thank the staff from the ALBA synchrotron (Spain) for assistance with the XALOC beamline. Structural data are deposited in the Protein Data Bank with accession codes 6QIY (X-ray CI2 classical geometry) and 6QIZ (X-ray CI2 domain swapped) and EMD- 4568 (cryo-EM CI2eng assembly)

Engineering protein assemblies with allosteric control via monomer fold-switching

The macromolecular machines of life use allosteric control to self-assemble, dissociate and change shape in response to signals. Despite enormous interest, the design of nanoscale allosteric assemblies has proven tremendously challenging. Here we present a proof of concept of allosteric assembly in which an engineered fold switch on the protein monomer triggers or blocks assembly. Our design is based on the hyper-stable, naturally monomeric protein CI2, a paradigm of simple two-state folding, and the toroidal arrangement with 6-fold symmetry that it only adopts in crystalline form. We engineer CI2 to enable a switch between the native and an alternate, latent fold that self-assembles onto hexagonal toroidal particles by exposing a favorable inter-monomer interface. The assembly is controlled on demand via the competing effects of temperature and a designed short peptide. These findings unveil a remarkable potential for structural metamorphosis in proteins and demonstrate key principles for engineering protein-based nanomachinery.This work was supported by the European Research Council (grant ERC-2012-ADG-323059 to V.M.) and by the PRODESTECH network funded through the CONSOLIDER program from the Spanish Government (grant CSD2009-00088). L.A.C. acknowledges support from Ministry of Economy and Competitiveness through grants BIO2016-78768-P and RYC-2013-13197. V.M. acknowledges additional support from the W.M.Keck Foundation and from the CREST Center for Cellular and Biomomolecular Machines (grant NSF-CREST-1547848). J.M.V. acknowledges additional support from Ministry of Economy and Competitiveness through grant BFU2016-75984.Peer reviewe

Atom-by-atom analysis of global downhill protein folding

5 pages, 4 figures.-- PMID: 16799571 [PubMed].-- Supplementary information available at: http://www.nature.com/nature/journal/v442/n7100/suppinfo/nature04859.htmlThe atomic coordinates of Naf-BBL have been deposited in the Protein Data Bank with the accession number 2QYU.Protein folding is an inherently complex process involving coordination of the intricate networks of weak interactions that stabilize native three-dimensional structures. In the conventional paradigm, simple protein structures are assumed to fold in an all-or-none process that is inaccessible to experiment. Existing experimental methods therefore probe folding mechanisms indirectly. A widely used approach interprets changes in protein stability and/or folding kinetics, induced by engineered mutations, in terms of the structure of the native protein. In addition to limitations in connecting energetics with structure, mutational methods have significant experimental uncertainties and are unable to map complex networks of interactions. In contrast, analytical theory predicts small barriers to folding and the possibility of downhill folding. These theoretical predictions have been confirmed experimentally in recent years, including the observation of global downhill folding. However, a key remaining question is whether downhill folding can indeed lead to the high-resolution analysis of protein folding processes. Here we show, with the use of nuclear magnetic resonance (NMR), that the downhill protein BBL from Escherichia coli unfolds atom by atom starting from a defined three-dimensional structure. Thermal unfolding data on 158 backbone and side-chain protons out of a total of 204 provide a detailed view of the structural events during folding. This view confirms the statistical nature of folding, and exposes the interplay between hydrogen bonding, hydrophobic forces, backbone conformation and side-chain entropy. From the data we also obtain a map of the interaction network in this protein, which reveals the source of folding cooperativity. Our approach can be extended to other proteins with marginal barriers (less than 3RT), providing a new tool for the study of protein folding.The research described in this article was supported by the NIH and the NSF.Peer reviewe

Dynamics, energetics, and structure in protein folding

10 pages, 7 figures.-- PMID: 16834320 [PubMed].-- PMCID: PMC2546509.-- Author manuscript available in PMC: http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=16834320Printed version published on Jul 18, 2006.For many decades, protein folding experimentalists have worked with no information about the time scales of relevant protein folding motions and without methods for estimating the height of folding barriers. Protein folding experiments have been interpreted using chemical models in which the folding process is characterized as a series of equilibria between two or more distinct states that interconvert with activated kinetics. Accordingly, the information to be extracted from experiments was circumscribed to apparent equilibrium constants and relative folding rates. Recent developments are changing this situation dramatically. The combination of fast-folding experiments with the development of analytical methods more closely connected to physical theory reveals that folding barriers in native conditions range from minimally high (~14RT for the very slow folder AcP) to nonexistent. While slow-folding (i.e., ≥ 1 ms) single-domain proteins are expected to fold in a two-state fashion, microsecond-folding proteins should exhibit complex behavior arising from crossing marginal or negligible folding barriers. This realization opens a realm of exciting opportunities for experimentalists. The free energy surface of a protein with a marginal (or no) barrier can be mapped using equilibrium experiments, which could resolve energetic factors from structural factors in folding. Kinetic experiments on these proteins provide the unique opportunity to measure folding dynamics directly. Furthermore, the complex distributions of time-dependent folding behaviors expected for these proteins might be accessible to single-molecule measurements. Here, we discuss some of these recent developments in protein folding, emphasizing aspects that can serve as a guide for experimentalists interested in exploiting this new avenue of research.The research described here has been supported by NIH grant GM-066800 and NSF grant MCB-0317294.Peer reviewe

Lessons about Protein Folding and Binding from Archetypal Folds

The function of proteins as biological nanomachines relies on their ability to fold into complex 3D structures, bind selectively to partners, and undergo conformational changes on cue. The native functional structures, and the rates of interconversion between conformational states (folded-unfolded, bound-free), are all encoded in the physical chemistry of their amino acid sequence. However, despite extensive research over decades, this code has proven difficult to fully crack, in terms of both prediction and understanding the molecular mechanisms at play. Earlier work on single-domain proteins reported a commonality of slow rates (10–2–102 s–1) and simple behavior in both kinetic and thermodynamic unfolding experiments, which suggested the process was all-or-none and thereby analogous to a chemical reaction (e.g., A ⇄ B). In the absence of a first-principles pre-exponential factor for protein (un)folding dynamics, the rates could only be interpreted in relative terms, e.g., the changes induced by mutation, and hence, neither the height of nor the entropic contribution to the free energy barriers was known. The rates were also many orders of magnitude too slow for direct atomistic simulations, and the computational focus was on predicting rate changes induced by mutation via coarse grained simulations. However, even the effects of mutation proved to be strikingly homogeneous with all experimental data clustering at ∼1/3 of the free energy perturbation recovered on folding and ∼2/3 on unfolding. The implementation of ultrafast kinetic methods turned the field upside down because they allowed researchers to measure the time scales of elementary (un)folding motions, which set the pre-exponential factor for protein conformational transitions at ∼1 μs. In parallel, we and others set out to investigate the simplest possible protein structures capable of autonomous folding, which we defined as archetypal folds. The rationale was to recapitulate the hierarchical organization of protein structure, starting from the bottom up. The study of fold archetypes ended up opening new research avenues in protein (un)folding, but also making unexpected connections with the folding upon binding of intrinsically disordered proteins and suggesting their functioning as conformational rheostats. This Account describes our work on the kinetic, thermodynamic, mechanistic, and functional analysis of fold archetypes. We first discuss the kinetic studies, emphasizing their impact on our understanding of (un)folding rates, of barrierless (downhill) folding, and as benchmarks for atomistic simulations. We continue with the thermodynamic analysis, introducing the differential scanning calorimetry, multiprobe, and NMR approaches that we developed to dissect their gradual, minimally cooperative (un)folding transitions and to probe the underlying mechanisms with unprecedented detail. The last two sections cover single-molecule analyses and some recent, mostly computational, results on the exploration of possible biological and technological roles for the gradual conformational transitions of fold archetypes.V.M. acknowledges support from the European Research Council (ERC-2012-ADG- 323059), the National Science foundation (NSF-MCB-1616759), the CREST Center for Cellular and Biomolecular Machines (NSF-CREST-1547848), and the W.M. Keck Foundation

Engrailed homeodomain uses an electrostatic spring-loaded mechanism to change conformation upon binding to DNA

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A modular approach to map out the conformational landscapes of unbound intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) fold upon binding to select/recruit multiple partners, morph around the partners structure, and exhibit allostery. However, we do not know whether these properties emerge passively from disorder, or rather are encoded into the IDPs folding mechanisms. A main reason for this gap is the lack of suitable methods to dissect the energetics of IDP conformational landscapes without partners. Here we introduce such an approach that we term molecular LEGO, and apply it to NCBD, a helical, molten globule–like IDP, as proof of concept. The approach entails the experimental and computational characterization of the protein, its separate secondary structure elements (LEGO building blocks), and their supersecondary combinations. Comparative analysis uncovers specific, yet inconspicuous, energetic biases in the conformational/folding landscape of NCBD, including 1) strong local signals that define the three native helices, 2) stabilization of helix–helix interfaces via soft pairwise tertiary interactions, 3) cooperative stabilization of a heterogeneous three-helix bundle fold, and 4) a dynamic exchange between sets of tertiary interactions (native and nonnative) that recapitulate the different structures NCBD adopts in complex with various partners. Crucially, a tug of war between sets of interactions makes NCBD gradually shift between structural subensembles as a conformational rheostat. Such conformational rheostatic behavior provides a built-in mechanism to modulate binding and switch/recruit partners that is likely at the core of NCBDs function as transcriptional coactivator. Hence, the molecular LEGO approach emerges as a powerful tool to dissect the conformational landscapes of unbound IDPs and rationalize their functional mechanisms

Engrailed homeodomain uses an electrostatic spring-loaded mechanism to change conformation upon binding to DNA

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Inhibition by Aplidine of the aggregation of the prion peptide PrP 106–126 into β-sheet fibrils

Aplidine, a cyclic peptide, from the tunicate Aplidium albican, prevents the in vitro aggregation into β-sheet containing fibrils of the prion peptide 106–126 when co-incubated in a 1:1 molar ratio. The blocking of fibril formation induced by Aplidine has clear sequence specificity, being much stronger for the 106–126 prion peptide than for the β-amyloid 25–35 peptide. In addition to the known ability of Aplidine to cross the plasmatic membrane, these results indicate that Aplidine is a potential leading compound for the development of therapeutic blockers of prion aggregation.This work was supported in part by grants from the Spanish CICYT, Comunidad de Madrid, Neuropharma, Fundación Lilly and an institutional grant from Fundación Ramón Areces. PharmaMar supplied Aplidine. Victor Muñoz is a recipient of a Dreyfus New Faculty Award, a Packard Fellowship for Science and Engineering and a Searle Scholar Award.Peer reviewe

The Effect of Electrostatics on the Marginal Cooperativity of an Ultrafast Folding Protein*

Proteins fold up by coordinating the different segments of their polypeptide chain through a network of weak cooperative interactions. Such cooperativity results in unfolding curves that are typically sigmoidal. However, we still do not know what factors modulate folding cooperativity or the minimal amount that ensures folding into specific three-dimensional structures. Here, we address these issues on BBL, a small helical protein that folds in microseconds via a marginally cooperative downhill process (Li, P., Oliva, F. Y., Naganathan, A. N., and Muñoz, V. (2009) Proc. Natl. Acad. Sci. USA. 106, 103–108). Particularly, we explore the effects of salt-induced screening of the electrostatic interactions in BBL at neutral pH and in acid-denatured BBL. Our results show that electrostatic screening stabilizes the native state of the neutral and protonated forms, inducing complete refolding of acid-denatured BBL. Furthermore, without net electrostatic interactions, the unfolding process becomes much less cooperative, as judged by the broadness of the equilibrium unfolding curve and the relaxation rate. Our experiments show that the marginally cooperative unfolding of BBL can still be made twice as broad while the protein retains its ability to fold into the native three-dimensional structure in microseconds. This result demonstrates experimentally that efficient folding does not require cooperativity, confirming predictions from theory and computer simulations and challenging the conventional biochemical paradigm. Furthermore, we conclude that electrostatic interactions are an important factor in determining folding cooperativity. Thus, electrostatic modulation by pH-salt and/or mutagenesis of charged residues emerges as an attractive tool for tuning folding cooperativity