Role of Electrostatics in Protein-RNA Binding: The Global vs the Local Energy Landscape.

U1A protein-stem loop 2 RNA association is a basic step in the assembly of the spliceosomal U1 small nuclear ribonucleoprotein. Long-range electrostatic interactions due to the positive charge of U1A are thought to provide high binding affinity for the negatively charged RNA. Short range interactions, such as hydrogen bonds and contacts between RNA bases and protein side chains, favor a specific binding site. Here, we propose that electrostatic interactions are as important as local contacts in biasing the protein-RNA energy landscape toward a specific binding site. We show by using molecular dynamics simulations that deletion of two long-range electrostatic interactions (K22Q and K50Q) leads to mutant-specific alternative RNA bound states. One of these states preserves short-range interactions with aromatic residues in the original binding site, while the other one does not. We test the computational prediction with experimental temperature-jump kinetics using a tryptophan probe in the U1A-RNA binding site. The two mutants show the distinct predicted kinetic behaviors. Thus, the stem loop 2 RNA has multiple binding sites on a rough RNA-protein binding landscape. We speculate that the rough protein-RNA binding landscape, when biased to different local minima by electrostatics, could be one way that protein-RNA interactions evolve toward new binding sites and novel function.

Estimation of Relative Protein-RNA Binding Strengths from Fluctuations in the Bound State.

Protein-RNA complexes are increasingly important in our understanding of cell signaling, metabolism, and transcription. Electrostatic interactions play dominant role in stabilizing such complexes. Using conventional computational approaches, very long simulations of both bound and unbound states are required to obtain accurate estimates of complex dissociation constants (Kd). Here, we derive a simple formula that offers an alternative approach based on the theory of fluctuations. Our method extracts a strong correlate to experimental Kd values using short molecular dynamics simulations of the bound complex only. To test our method, we compared the computed relative Kd values to our experimentally measured values for the U1A-Stem Loop 2 (SL2) RNA complex, which is one of the most-studied protein-RNA complexes. Additionally we also included several experimental values from the literature, to enlarge the data set. We obtain a correlation of r = 0.93 between theoretical and measured estimates of Kd values of the mutated U1A protein-RNA complexes relative to the wild type dissociation constant. The proposed method increases the efficiency of relative Kd values estimation for multiple protein mutants, allowing its applicability to protein engineering projects.

Subcellular modulation of protein VlsE stability and folding kinetics.

The interior of a cell interacts differently with proteins than a dilute buffer because of a wide variety of macromolecules, chaperones, and osmolytes that crowd and interact with polypeptide chains. We compare folding of fluorescent constructs of protein VlsE among three environments inside cells. The nucleus increases the stability of VlsE relative to the cytoplasm, but slows down folding kinetics. VlsE is also more stable in the endoplasmic reticulum, but unlike PGK, tends to aggregate there. Although fluorescent-tagged VlsE and PGK show opposite stability trends from in vitro to the cytoplasm, their trends from cytoplasm to nucleus are similar.

Native conformational dynamics of the spliceosomal U1A protein.

The complex of spliceosomal U1A protein and its cognate SL2 RNA is a prototype system for protein-RNA binding studies. A major question is whether U1A protein alone is capable of undergoing conformational dynamics similar to structural rearrangements upon RNA binding. Using a fast temperature jump and tryptophan fluorescence detection, we uncover a ∼20 µs conformational transition for the Lys22Gln/Phe56Trp-only mutant of U1A, yet a Phe56Trp-only control mutant does not show the transition. To explain this observation, we performed extensive molecular dynamics (MD) simulations. The simulations explain why only the Lys22Gln/Phe56Trp-only mutant shows a fluorescence signal: in the other mutant, the tryptophan probe is not quenched upon structural rearrangement. The simulations support helix C movement as the underlying structural rearrangement, although the simulated time scale is faster than experimentally detected. On the basis of our MD results, we propose a reversible two-pathway three-state transition for the helix C movement and assign T-jump kinetics to a closed to semi-closed transition of the helix. Our result provides a specific example of how alternative protein conformations on the native side of the folding barrier can be functionally important, for example in conformational selection by a binding partner.

Protein folding dynamics in the cell.

Protein folding is a remarkably fast unimolecular reaction, spanning microseconds to hours at room temperature. Thus, free energy differences and activation barriers on the free energy landscape of proteins are rather small. This opens up the possibility of living cells modulating their protein's landscapes, providing cells another way to control the function of their proteomes after transcriptional control, translational control, and post-translational modification. In this Feature Article, we discuss advances in physicochemical studies of protein stability and folding inside living cells. We focus in particular on our studies using fast relaxation imaging (FREI). Although the effect of the cell on protein free energy landscapes is only a few kT, the strong cooperativity of many folding and binding processes allows small modulation of the energy and entropy to produce a large population modulation. Lastly, we discuss some biomolecular processes that are particularly likely to be affected by in-cell modulation of the proteome, and thus of interest for quantitative physical chemistry studies.

The extracellular protein VlsE is destabilized inside cells.

We use U2OS cells as in vivo "test tubes" to study how the same cytoplasmic environment has opposite effects on the stability of two different proteins. Protein folding stability and kinetics were compared by fast relaxation imaging, which combines a temperature jump with fluorescence microscopy of FRET (Förster resonance energy transfer)-labeled proteins. While the stability of the cytoplasmic enzyme PGK (phosphoglycerate kinase) increases in cells, the stability of the cell surface antigen VlsE, which presumably did not evolve for stability inside cells, decreases. VlsE folding also slows down more than PGK folding in cells, relative to their respective aqueous buffer kinetics. Our FRET measurements provide evidence that VlsE is more compact inside cells than in aqueous buffer. Two kinetically distinct protein populations exist inside cells, making a connection with previous in vitro crowding studies. In addition, we confirm previous studies showing that VlsE is stabilized by 150mg/mL of the carbohydrate crowder Ficoll, even though it is destabilized in the cytoplasm relative to aqueous buffer. We propose two mechanisms for the observed destabilization of VlsE in U2OS cells: long-range interactions competing with crowding or shape-dependent crowding favoring more compact states inside the cell over the elongated aqueous buffer native state.

Chiral spiroaminoborate ester as a highly enantioselective and efficient catalyst for the borane reduction of furyl, thiophene, chroman and thiochroman containing ketones.

Prochiral heteroaryl ketones containing furan, thiophene, chroman and thiochroman moieties were successfully reduced in the presence of 1 - 10 mol % of spiroaminoborate ester 1 with different borane sources to afford non-racemic alcohols in up to 99% ee. In addition, modest enantioselectivity, around 80% ee, was achieved in the reduction of linear α,ß-unsaturated heteroaryl ketones.

Spiroborate esters in the borane-mediated asymmetric synthesis of pyridyl and related heterocyclic alcohols.

The effectiveness of several spiroborate ester catalysts was investigated in the asymmetric borane reduction of 2-, 3-, 4-acetylpyridines under different reaction conditions. Highly enantiomerically enriched 1-(2-, 3- and 4-pyridyl)ethanols and 1-(heterocyclic)ethanols were obtained using 1 to 10% catalytic loads of the spiroborate 5 derived from diphenylprolinol and ethylene glycol.

Enantioselective Reduction of Prochiral Ketones using Spiroborate Esters as Catalysts.

Novel spiroborate esters derived nonracemic 1,2-aminoalcohols and ethylene glycol are reported as highly effective catalysts for the asymmetric borane reduction of a variety of prochiral ketones with borane-dimethyl sulfide complex at room temperature. Optically active alcohols were obtained in excellent chemical yields using 0.1 to 10 mol % of catalysts with up to 99% ee.