Tobin R. Sosnick

Mechanisms and uses of hydrogen exchange.

Recent work has largely completed our understanding of the hydrogen-exchange chemistry of unstructured proteins and nucleic acids. Some of the high-energy structural fluctuations that determine the hydrogen-exchange behavior of native macromolecules have been explained; others remain elusive. A growing number of applications are exploiting hydrogen-exchange behavior to study difficult molecular systems and elicit otherwise inaccessible information on protein structure, dynamics and energetics.

Molecular collapse: The rate-limiting step in two-state cytochrome c folding

Experiments with cytochrome c (cyt c) show that an initial folding event, molecular collapse, is not an energetically downhill continuum as commonly presumed but represents a large‐scale, time‐consuming, cooperative barrier‐crossing process. In the absence of later misfold‐reorganization barriers, the early collapse barrier limits cyt c folding to a time scale of milliseconds. The collapse process itself appears to be limited by an uphill search for some coarsely determined transition state structure that can nucleate subsequent energetically downhill folding events. An earlier “burst phase” event at strongly native conditions appears to be a non‐specific response of the unfolded chain to reduced denaturant concentration. The molecular collapse process may or may not require the co‐formation of the amino‐ and carboxyl‐terminal helices, which are present in an initial metastable intermediate directly following the rate‐limiting collapse. After the collapse‐nucleation event, folding can proceed rapidly in an apparent two‐state manner, probably by way of a predetermined sequence of metastable intermediates that leads to the native protein structure (Bai et al., Science 269:192–197, 1995).

Light-activated DNA binding in a designed allosteric protein

An understanding of how allostery, the conformational coupling of distant functional sites, arises in highly evolvable systems is of considerable interest in areas ranging from cell biology to protein design and signaling networks. We reasoned that the rigidity and defined geometry of an α-helical domain linker would make it effective as a conduit for allosteric signals. To test this idea, we rationally designed 12 fusions between the naturally photoactive LOV2 domain from Avena sativa phototropin 1 and the Escherichia coli trp repressor. When illuminated, one of the fusions selectively binds operator DNA and protects it from nuclease digestion. The ready success of our rational design strategy suggests that the helical “allosteric lever arm” is a general scheme for coupling the function of two proteins.

TULIPs: Tunable, light-controlled interacting protein tags for cell biology

Naturally photoswitchable proteins offer a means of directly manipulating the formation of protein complexes that drive a diversity of cellular processes. We developed tunable light-inducible dimerization tags (TULIPs) based on a synthetic interaction between the LOV2 domain of Avena sativa phototropin 1 (AsLOV2) and an engineered PDZ domain (ePDZ). TULIPs can recruit proteins to diverse structures in living yeast and mammalian cells, either globally or with precise spatial control using a steerable laser. The equilibrium binding and kinetic parameters of the interaction are tunable by mutation, making TULIPs readily adaptable to signaling pathways with varying sensitivities and response times. We demonstrate the utility of TULIPs by conferring light sensitivity to functionally distinct components of the yeast mating pathway and by directing the site of cell polarization.

Protein folding : Defining a "standard" set of experimental conditions and a preliminary kinetic data set of two-state proteins

Recent years have seen the publication of both empirical and theoretical relationships predicting the rates with which proteins fold. Our ability to test and refine these relationships has been limited, however, by a variety of difficulties associated with the comparison of folding and unfolding rates, thermodynamics, and structure across diverse sets of proteins. These difficulties include the wide, potentially confounding range of experimental conditions and methods employed to date and the difficulty of obtaining correct and complete sequence and structural details for the characterized constructs. The lack of a single approach to data analysis and error estimation, or even of a common set of units and reporting standards, further hinders comparative studies of folding. In an effort to overcome these problems, we define here a “consensus” set of experimental conditions (25°C at pH 7.0, 50 mM buffer), data analysis methods, and data reporting standards that we hope will provide a benchmark for experimental studies. We take the first step in this initiative by describing the folding kinetics of 30 apparently two‐state proteins or protein domains under the consensus conditions. The goal of our efforts is to set uniform standards for the experimental community and to initiate an accumulating, self‐consistent data set that will aid ongoing efforts to understand the folding process.

Intermediates and kinetic traps in the folding of a large ribozyme revealed by circular dichroism and UV absorbance spectroscopies and catalytic activity

The folding thermodynamics and kinetics for the ribozyme from Bacillus subtilis RNase P are analyzed using circular dichroism and UV absorbance spectroscopies and catalytic activity. At 37 °C, the addition of Mg2+ (Kd ∼ 50 μM) to the unfolded state produces an intermediate state within 1 ms which contains a comparable amount of secondary structure as the native ribozyme. The subsequent transition to the native state (Kd[Mg] ∼ 0.8 mM, Hill coefficient ∼ 3.5) has a half-life of hundreds of seconds as measured by circular dichroism at 278 nm and by a ribozyme activity assay. Surprisingly, the formation of the native structure is accelerated strongly by the addition of a denaturant; ∼30-fold at 4.5 M urea. Thus, the rate-limiting step entails the disruption of a considerable number of interactions. The folding of this, and presumably other large RNAs, is slow due to the structural rearrangement of kinetically trapped species. Taken together with previous submillisecond relaxation kinetics of tRNA tertiary structure, we suggest that error-free RNA folding can be on the order of milliseconds.

Fast and slow intermediate accumulation and the initial barrier mechanism in protein folding.

Do stable intermediates form very early in the protein folding process? New results and a quantity of literature that bear on this issue are examined here. Results available provide little support for early intermediate accumulation before an initial search-dependent nucleation barrier.

Rationally improving LOV domain-based photoswitches

Genetically encoded protein photosensors are promising tools for engineering optical control of cellular behavior; we are only beginning to understand how to couple these light detectors to effectors of choice. Here we report a method that increases the dynamic range of an artificial photoswitch based on the LOV2 domain of Avena sativa phototropin 1 (AsLOV2). This approach can potentially be used to improve many AsLOV2-based photoswitches.

RNA folding: models and perspectives

Intrinsic events during RNA folding include conformational search and metal ion binding. Several experimentally testable models have been proposed to explain how large ribozymes accomplish folding. Future challenges include the validation of these models, and the correlation of experimental results and theoretical simulations.

The burst phase in ribonuclease A folding and solvent dependence of the unfolded state

Submillisecond burst phase signals measured in kinetic protein folding experiments have been widely interpreted in terms of the fast formation of productive folding intermediates. Experimental comparisons with non-folding polypeptide chains show that, for ribonuclease A and cytochrome c, these signals in fact reflect a shift from one biased ensemble of the unfolded state to another as a function of change in denaturant concentration.