Ian S. Millett

Exploring the folding landscape of a structured RNA

Structured RNAs achieve their active states by traversing complex, multidimensional energetic landscapes. Here we probe the folding landscape of the Tetrahymena ribozyme by using a powerful approach: the folding of single ribozyme molecules is followed beginning from distinct regions of the folding landscape. The experiments, combined with small-angle x-ray scattering results, show that the landscape contains discrete folding pathways. These pathways are separated by large free-energy barriers that prevent interconversion between them, indicating that the pathways lie in deep channels in the folding landscape. Chemical protection and mutagenesis experiments are then used to elucidate the structural features that determine which folding pathway is followed. Strikingly, a specific long-range tertiary contact can either help folding or hinder folding, depending on when it is formed during the process. Together these results provide an unprecedented view of the topology of an RNA folding landscape and the RNA structural features that underlie this multidimensional landscape.

Rapid compaction during RNA folding

We have used small angle x-ray scattering and computer simulations with a coarse-grained model to provide a time-resolved picture of the global folding process of the Tetrahymena group I RNA over a time window of more than five orders of magnitude. A substantial phase of compaction is observed on the low millisecond timescale, and the overall compaction and global shape changes are largely complete within one second, earlier than any known tertiary contacts are formed. This finding indicates that the RNA forms a nonspecifically collapsed intermediate and then searches for its tertiary contacts within a highly restricted subset of conformational space. The collapsed intermediate early in folding of this RNA is grossly akin to molten globule intermediates in protein folding.

Toward a taxonomy of the denatured state: small angle scattering studies of unfolded proteins.

Publisher Summary This chapter reviews recent small-angle X-ray and neutron scattering (SAXS and SANS) studies of putatively “fully” unfolded states formed at equilibrium. It discusses the taxonomy of unfolded states that is chemically denatured state, thermally denatured state, pressure-denatured state, cold-unfolded states, and intrinsically unfolded proteins. The majority of SAXS and SANS studies of the unfolded state focus on the ensembles of states induced by chemical denaturants such as urea, GuHCl, extremes of pH, and organic cosolvents. As urea and GuHCl dominate spectroscopic studies of protein folding thermodynamics and kinetics, these denaturants have similarly been employed in the vast majority of small-angle scattering studies as well. The extraordinary solubility of unfolded proteins at high levels of urea or GuHCl provides an added technical benefit. Limited SAXS studies suggest that urea and GuHCl produce indistinguishable denatured states. Studies of thermally denatured proteins remain technically challenging owing to the propensity of thermally unfolded proteins to aggregate. Despite this potential difficulty, small-angle scattering techniques have been employed in the characterization of a number of thermally unfolded states. Because the molar volume of an unfolded protein is less than that of the native state, increasing pressure leads to denaturation. High-pressure SAXS was employed to monitor the pressure-induced unfolding of Snase.

Chain collapse can occur concomitantly with the rate-limiting step in protein folding

We have directly characterized the extent of chain collapse early in the folding of protein L using time-resolved small angle X-ray scattering. We find that, immediately after the initiation of refolding, the protein exhibits dimensions indistinguishable from those observed under highly denaturing, equilibrium conditions and that this expanded initial state collapses with the same rate as that of the overall folding reaction. The observation that chain compaction need not significantly precede the rate-limiting step of folding demonstrates that rapid chain collapse is not an obligatory feature of protein folding reactions.

Small angle X-ray scattering reveals a compact intermediate in RNA folding

We have used small angle X-ray scattering (SAXS) to monitor changes in the overall size and shape of the Tetrahymena ribozyme as it folds. The native ribozyme, formed in the presence of Mg2+, is much more compact and globular than the ensemble of unfolded conformations. Time-resolved measurements show that most of the compaction occurs at least 20-fold faster than the overall folding to the native state, suggesting that a compact intermediate or family of intermediates is formed early and then rearranges in the slow steps that limit the overall folding rate. These results lead to a kinetic folding model in which an initial ‘electrostatic collapse’ of the RNA is followed by slower rearrangements of elements that are initially mispositioned.

Elucidation of the Molecular Mechanism During the Early Events in Immunoglobulin Light Chain Amyloid Fibrillation: Evidence for an Off-pathway Oligomer at Acidic pH

Light chain amyloidosis involves the systemic pathologic deposition of monoclonal light chain variable domains of immunoglobulins as insoluble fibrils. The variable domain LEN was obtained from a patient who had no overt amyloidosis; however, LEN forms fibrils in vitro, under mildly destabilizing conditions. The in vitro kinetics of fibrillation were investigated using a wide variety of probes. The rate of fibril formation was highly dependent on the initial protein concentration. In contrast to most amyloid systems, the kinetics became slower with increasing LEN concentrations. At high protein concentrations a significant lag in time was observed between the conformational changes and the formation of fibrils, consistent with the formation of soluble off-pathway oligomeric species and a branched pathway. The presence of off-pathway species was confirmed by small angle x-ray scattering. At low protein concentrations the structural rearrangements were concurrent with fibril formation, indicating the absence of formation of the off-pathway species. The data are consistent with a model for fibrillation in which a dimeric form of LEN (at high protein concentration) inhibits fibril formation by interaction with an intermediate on the fibrillation pathway and leads to formation of the off-pathway intermediate.

Natively unfolded C-terminal domain of caldesmon remains substantially unstructured after the effective binding to calmodulin

The structure of C‐terminal domain (CaD136, C‐terminal residues 636–771) of chicken gizzard caldesmon has been analyzed by a variety of physico‐chemical methods. We are showing here that CaD136 does not have globular structure, has low secondary structure content, is essentially noncompact, as it follows from high Rg and RS values, and is characterized by the absence of distinct heat absorption peaks, i.e. it belongs to the family of natively unfolded (or intrinsically unstructured) proteins. Surprisingly, effective binding of single calmodulin molecule (Kd = 1.4 ± 0.2 μM) leads only to a very moderate folding of this protein and CaD136 remains substantially unfolded within its tight complex with calmodulin. The biological significance of these observations is discussed. Proteins 2003.

Effect of association state and conformational stability on the kinetics of immunoglobulin light chain amyloid fibril formation at physiological pH

Light chain amyloidosis involves the systemic deposition of fibrils in patients overproducing monoclonal immunoglobulin light chains. The kinetics of fibril formation of LEN, a benign light chain variable domain, were investigated at physiological pH in the presence of urea. Despite the lack of in vivofibril formation, LEN readily forms fibrils in vitro under mildly destabilizing conditions. The effect of low to moderate concentrations of urea on the conformation, association state, stability, and kinetics of fibrillation of LEN were investigated. The conformation of LEN was only slightly affected by the addition of up to 4 m urea. The fibrillation kinetics were highly dependent on protein and urea concentrations, becoming faster with decreasing protein concentration and increasing urea concentration. Changes in spectral probes were concomitant to fibril formation throughout the protein and urea concentration ranges, indicating the absence of off-pathway oligomeric species or amorphous aggregates prior to fibril formation. Reducing the amount of dimers initially present in solution by either decreasing the protein concentration or adding urea resulted in faster fibril formation. Thus, increasing concentrations of urea, by triggering dissociation of dimeric LEN, lead to increased rates of fibrillation.

Expression, purification, and characterization of Thermotoga maritima membrane proteins for structure determination

Structural studies of integral membrane proteins typically rely upon detergent micelles as faithful mimics of the native lipid bilayer. Therefore, membrane protein structure determination would be greatly facilitated by biophysical techniques that are capable of evaluating and assessing the fold and oligomeric state of these proteins solubilized in detergent micelles. In this study, an approach to the characterization of detergent‐solubilized integral membrane proteins is presented. Eight Thermotoga maritima membrane proteins were screened for solubility in 11 detergents, and the resulting soluble protein–detergent complexes were characterized with small angle X‐ray scattering (SAXS), nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) spectroscopy, and chemical cross‐linking to evaluate the homogeneity, oligomeric state, radius of gyration, and overall fold. A new application of SAXS is presented, which does not require density matching, and NMR methods, typically used to evaluate soluble proteins, are successfully applied to detergent‐solubilized membrane proteins. Although detergents with longer alkyl chains solubilized the most proteins, further characterization indicates that some of these protein–detergent complexes are not well suited for NMR structure determination due to conformational exchange and protein oligomerization. These results emphasize the need to screen several different detergents and to characterize the protein–detergent complex in order to pursue structural studies. Finally, the physical characterization of the protein–detergent complexes indicates optimal solution conditions for further structural studies for three of the eight overexpressed membrane proteins.

Sample holder for small-angle x-ray scattering static and flow cell measurements

We present the design of a sample holder for small-angle x-ray scattering (SAXS) that can be used for both static and flow cell measurements, allowing to switch between these two types of measurement without having to realign the detector and camera geometry. The device makes possible high signal-to-noise experiments with sample volumes as small as 16μl and can be thermocontrolled using a standard circulating water bath. The setup has been used successfully for a range of biological SAXS measurements, including peptides, detergent micelles, membrane proteins, and nucleic acids. As a performance test, we present scattering data for horse heart cytochrome c, collected at the BESSRC CAT beam line 12-ID of the Advanced Photon Source. The design drawings are provided in the supplementary material.