Core-Directed Protein Design. II. Rescue of a Multiply Mutated and Destabilized Variant of Ubiquitin

Finucane, Michael D.; Woolfson, Derek N.

doi:10.1021/bi990766f

Cited by 47 publications

(59 citation statements)

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“…11 As temperature is increased, however, the spectrum shifts to one that strongly resembles Ub. 12,13 The 231 nm minimum disappears, and the position and molar ellipticity of the new minimum are consistent with native Ub. The transition has a midpoint of ~30 ºC (Figure 2(b)) and is fully reversible upon cooling (not shown).…”

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confidence: 58%

Allosteric Switching by Mutually Exclusive Folding of Protein Domains

Radley

Markowska

Bettinger

et al. 2003

Journal of Molecular Biology

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Many proteins are built from structurally and functionally distinct and domains. A major goal is to understand how conformational change transmits information between domains in order to achieve biological activity. A two-domain, bi-functional fusion protein has been designed so that the mechanical stress imposed by the folded structure of one subunit causes the other subunit to unfold, and vice versa. The construct consists of ubiquitin inserted into a surface loop of barnase. The distance between the amino and carboxyl ends of ubiquitin is much greater than the distance between the termini of the barnase loop. This topological constraint causes the two domains to engage in a thermodynamic tug-of-war in which only one can exist in its folded state at any given time. This conformational equilibrium, which is cooperative, reversible, and controllable by ligand binding, serves as a model for the coupled binding and folding mechanism widely used to mediate protein-protein interactions and cellular signaling processes. The position of the equilibrium can be adjusted by temperature or ligand binding and is monitored in vivo by cell death. This design forms the basis for a new class of cytotoxic proteins that can be activated by cell-specific effector molecules, and can thus target particular cell types for destruction. Keywords molecular switch; unfolding; natively unfolded; allostery Proteins often display modular architecture that combines protein or small molecule interaction domains with catalytic domains. In such cases, the domains must be coupled, both functionally and structurally, for the protein to attain overall biological activity. For example, ligand binding or phosphorylation can induce structural changes within a regulatory domain that then trigger activity in a catalytic domain. A related type of switching mechanism is illustrated by the recent discovery of proteins that are unstructured in physiological conditions but fold upon binding to their cellular targets. 1,2 Examples include elongin C 3,4 and the GTPase-binding domain of the Wiskott-Aldrich syndrome protein. 5 In these instances, the folding/unfolding of a regulatory domain modulates function of the intact protein via propagation of structural changes. Protein folding makes a particularly effective functional switch because it is reversible and inherently cooperative. Understanding the molecular basis for this type of mechanism is important because it is widely used to regulate protein-protein interactions and in signaling pathways that control cellular behavior. The system consists of a fusion protein in which human ubiquitin (Ub) is inserted into a surface loop of the ribonuclease barnase (Bn) from Bacillus amyloliquefaciens. These proteins were chosen for the following reasons. First, Bn is extremely lethal to both prokaryotic and eukaryotic cells. It is able to be synthesized in B. amyloliquefaciens only because it is co-expressed with its intracellular inhibitor barstar (Bs). 7 This cytotoxic property allows the enzymatic activity...

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confidence: 58%

Allosteric Switching by Mutually Exclusive Folding of Protein Domains

Radley

Markowska

Bettinger

et al. 2003

Journal of Molecular Biology

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“…First, the proteins in our system are potentially more exposed to proteolysis as they only have one fusion partner instead of two, and hence the selection may be more efficient; second, after selection, proteins can be expressed free from pIII and ready for characterization or large-scale production simply by changing the bacterial strain that is used (39).…”

Section: Discussionmentioning

confidence: 99%

“…low levels compared with WT-UBQ and was also totally lost in the protease selection studies (39). These properties provide good evidence that the AL 7 mutant was destabilized, although no direct stability measurement was possible.…”

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confidence: 95%

Core-Directed Protein Design. I. An Experimental Method for Selecting Stable Proteins from Combinatorial Libraries

et al. 1999

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The design of proteins represents a significant challenge to modern-day structural biology. A major obstacle here is the specification of well-packed hydrophobic cores to drive the folding and stabilization of the target. Computational approaches have been used to alleviate this by testing alternate sequences prior to the production and characterization of a few proteins. Here we present the experimental counterpart of this approach. We selected stable variants from a library of ubiquitin hydrophobic-core mutants as follows. Hexahistidine-tagged proteins were displayed on the surface of phage. These proteinphage were immobilized onto Ni-coated surfaces. The bound fusion-phage were treated with protease to remove unstable or poorly folded proteins. Stable phage fusions were eluted and infected into Escherichia coli, which allowed amplification for further selection, sequencing, or protein expression. Two Ni-derivatized supports were tested: Ni-NTA chips for surface plasmon resonance (SPR) and Ni-NTA agarose beads. SPR had an advantage in that the selection process could be monitored directly. This allowed individual clones and experimental conditions to be tested rapidly prior to preparative panning of the library, which was carried out using Ni-NTA agarose beads. We demonstrate the method by selecting stable core mutants of ubiquitin, the characterization of which is described in the following paper [Finucane, M. D., and Woolfson, D. N. (1999) Biochemistry 38, XXXXX-XXXXX]. As our method selects only on the basis of structure and stability, it will be of use in improving the stabilities and structural specificities of proteins of de novo design, and in establishing rules that link sequence and structure.In de novo protein design attempts are made to construct amino acid sequences that adopt prescribed folds. There are two principal reasons for conducting such studies. First, by designing proteins we test hypotheses of how sequence relates to structure. As sequence data are accumulating much faster than structural information, it is becoming even more critical to determine rules that aid protein structure prediction and modeling. Second, successful protein designs would present routes to new proteins with ranges of structures and functions that extend beyond those found in nature.

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“…Although mutation of a handful of surface residues is conditionally lethal [6], the structure is remarkably resilient to core mutation, with the fold remaining intact when both overpacked and under-packed [10,11]. Experiments conducted on a F45W tryptophan mutant of ubiquitin, here referred to as the pseudo-wild type (WT*), revealed that hydrophobic-core mutations of WT* destabilise the protein, suggesting these residues might be conserved to maximise protein stability [12].…”

Section: Introductionmentioning

confidence: 99%

Destabilised mutants of ubiquitin gain equal stability in crowded solutions

Roberts¹,

Jackson²

2007

Biophysical Chemistry

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT AbstractThis paper investigates the thermodynamic and kinetic response of WT* ubiquitin (F45W) and three mutants to high concentrations of glucose, sucrose and dextran under physiological temperature and pH. WT* ubiquitin was stabilised by the same amount when comparing each cosolute on a weight to volume ratio, with cosolute effects largely independent of denaturant concentration. The energy difference between the mutants and WT* ubiquitin also remained the same in high concentrations of cosolute. An apparent decrease in transition-state surface burial in the presence of the cosolutes was attributed to increased compaction of the denatured state, and not to the Hammond effect. Together, these results suggest higher thermodynamic stabilities and folding rates for proteins in vivo compared to in vitro, in addition to more compact denatured states. Because the effects of mutation are the same in dilute solution and crowded conditions used to mimic the cellular environment, there is validity in using measurements of mutant stabilities made in dilute solutions to inform on how the mutations may affect stability in vivo.

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Core-Directed Protein Design. II. Rescue of a Multiply Mutated and Destabilized Variant of Ubiquitin

Cited by 47 publications

References 51 publications

Allosteric Switching by Mutually Exclusive Folding of Protein Domains

Allosteric Switching by Mutually Exclusive Folding of Protein Domains

Core-Directed Protein Design. I. An Experimental Method for Selecting Stable Proteins from Combinatorial Libraries

Destabilised mutants of ubiquitin gain equal stability in crowded solutions

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