John M. Louis

Characterizing the unfolded states of proteins using single-molecule FRET spectroscopy and molecular simulations

To obtain quantitative information on the size and dynamics of unfolded proteins we combined single-molecule lifetime and intensity FRET measurements with molecular simulations. We compared the unfolded states of the 64-residue, α/β protein L and the 66-residue, all-β cold-shock protein CspTm. The average radius of gyration (Rg) calculated from FRET data on freely diffusing molecules was identical for the two unfolded proteins at guanidinium chloride concentrations >3 M, and the FRET-derived Rg of protein L agreed well with the Rg previously measured by equilibrium small-angle x-ray scattering. As the denaturant concentration was lowered, the mean FRET efficiency of the unfolded subpopulation increased, signaling collapse of the polypeptide chain, with protein L being slightly more compact than CspTm. A decrease in Rg with decreasing denaturant was also observed in all-atom molecular dynamics calculations in explicit water/urea solvent, and Langevin simulations of a simplified representation of the polypeptide suggest that collapse can result from either increased interresidue attraction or decreased excluded volume. In contrast to both the FRET and simulation results, previous time-resolved small-angle x-ray scattering experiments showed no collapse for protein L. Analysis of the donor fluorescence decay of the unfolded subpopulation of both proteins gives information about the end-to-end chain distribution and suggests that chain dynamics is slow compared with the donor life-time of ≈2 ns, whereas the bin-size independence of the small excess width above the shot noise for the FRET efficiency distributions may result from incomplete conformational averaging on even the 1-ms time scale.

Single-Molecule Fluorescence Experiments Determine Protein Folding Transition Path Times

A Fraction of Folding An energy barrier has to be crossed as a protein transforms between folded and unfolded states. Molecular dynamic simulations have observed sharp transitions, with barrier crossing times of less than a microsecond, a fraction of the total folding time; however, this time range has been inaccessible to single-molecule experiments. Chung et al. (p. 981) described single-molecule fluorescence experiments that allowed measurement of the transition-path time for a fast-folding protein and to reduce the upper bound for a slow-folding protein. Although the folding rates differed by a factor of 10,000, the transition-path times differ by less than a factor of 5, pointing to energy landscape theory for the explanation. Quickly and slowly folding proteins take the same time to cross the barrier from the unfolded to the folded state. The transition path is the tiny fraction of an equilibrium molecular trajectory when a transition occurs as the free-energy barrier between two states is crossed. It is a single-molecule property that contains all the mechanistic information on how a process occurs. As a step toward observing transition paths in protein folding, we determined the average transition-path time for a fast- and a slow-folding protein from a photon-by-photon analysis of fluorescence trajectories in single-molecule Förster resonance energy transfer experiments. Whereas the folding rate coefficients differ by a factor of 10,000, the transition-path times differ by a factor of less than 5, which shows that a fast- and a slow-folding protein take almost the same time to fold when folding actually happens. A very simple model based on energy landscape theory can explain this result.

Flap opening and dimer-interface flexibility in the free and inhibitor-bound HIV protease, and their implications for function

BACKGROUND (1)H and (15)N transverse relaxation measurements on perdeuterated proteins are ideally suited for detecting backbone conformational fluctuations on the millisecond-microsecond timescale. The identification of conformational exchange on this timescale by measuring the relaxation of both (1)H and (15)N holds great promise for the elucidation of functionally relevant conformational changes in proteins. RESULTS We measured the transverse (1)H and (15)N relaxation rates of backbone amides of HIV-1 protease in its free and inhibitor-bound forms. An analysis of these rates, obtained as a function of the effective rotating frame field, provided information about the timescale of structural fluctuations in several regions of the protein. The flaps that cover the active site of the inhibitor-bound protein undergo significant changes of backbone (φ,psi) angles, on the 100 micros timescale, in the free protein. In addition, the intermonomer beta-sheet interface of the bound form, which from protease structure studies appears to be rigid, was found to fluctuate on the millisecond timescale. CONCLUSIONS We present a working model of the flap-opening mechanism in free HIV-1 protease which involves a transition from a semi-open to an open conformation that is facilitated by interaction of the Phe53 ring with the substrate. We also identify a surprising fluctuation of the beta-sheet intermonomer interface that suggests a structural requirement for maturation of the protease. Thus, slow conformational fluctuations identified by (1)H and (15)N transverse relaxation measurements can be related to the biological functions of proteins.

A simple apparatus for generating stretched polyacrylamide gels, yielding uniform alignment of proteins and detergent micelles.

Compressed and stretched polyacrylamide hydrogels previously have been shown to offer a robust method for aligning proteins. A simple, funnel-like apparatus is described for generating uniformly stretched hydrogels. For prolate-shaped proteins, gels stretched in the direction of the magnetic field yield two-fold larger alignment than gels compressed to the same aspect ratio in this direction. Empirically, protein alignment is found to be proportional to (c−2.3)2 [(do/dN)3-1], where do and dN are the diameters of the cylindrical gels before and after stretching, respectively, and c is the polyacrylamide weight fraction in percent. Low gel densities, in the 4–7% range, are found to have minimal effects on macromolecular rotational correlation times, τc, and no effect of the compression ratio on τc could be discerned over the range studied (do/dNle1.4). Application is demonstrated for a sample containing the first Ig-binding domain of protein G, and for a detergent-solubilized peptide.

Experimental determination of upper bound for transition path times in protein folding from single-molecule photon-by-photon trajectories

Transition paths are a uniquely single-molecule property not yet observed for any molecular process in solution. The duration of transition paths is the tiny fraction of the time in an equilibrium single-molecule trajectory when the process actually happens. Here, we report the determination of an upper bound for the transition path time for protein folding from photon-by-photon trajectories. FRET trajectories were measured on single molecules of the dye-labeled, 56-residue 2-state protein GB1, immobilized on a glass surface via a biotin-streptavidin-biotin linkage. Characterization of individual emitted photons by their wavelength, polarization, and absolute and relative time of arrival after picosecond excitation allowed the determination of distributions of FRET efficiencies, donor and acceptor lifetimes, steady state polarizations, and waiting times in the folded and unfolded states. Comparison with the results for freely diffusing molecules showed that immobilization has no detectable effect on the structure or dynamics of the unfolded protein and only a small effect on the folding/unfolding kinetics. Analysis of the photon-by-photon trajectories yields a transition path time <200 μs, >10,000 times shorter than the mean waiting time in the unfolded state (the inverse of the folding rate coefficient). Szabos theory for diffusive transition paths shows that this upper bound for the transition path time is consistent with previous estimates of the Kramers preexponential factor for the rate coefficient, and predicts that the transition path time is remarkably insensitive to the folding rate, with only a 2-fold difference for rate coefficients that differ by 105-fold.

Rapid structural fluctuations of the free HIV protease flaps in solution: relationship to crystal structures and comparison with predictions of dynamics calculations.

Crystal structures have shown that the HIV‐1 protease flaps, domains that control access to the active site, are closed when the active site is occupied by a ligand. Although flap structures ranging from closed to semi‐open are observed in the free protease, crystal structures reveal that even the semi‐open flaps block access to the active site, indicating that the flaps are mobile in solution. The goals of this paper are to characterize the secondary structure and fast (sub‐ns) dynamics of the flaps of the free protease in solution, to relate these results to X‐ray structures and to compare them with predictions of dynamics calculations. To this end we have obtained nearly complete backbone and many sidechain signal assignments of a fully active free‐protease construct that is stabilized against autoproteolysis by three point mutations. The secondary structure of this protein was characterized using the chemical shift index, measurements of 3hJNC′ couplings across hydrogen bonds, and NOESY connectivities. Analysis of these measurements indicates that the protease secondary structure becomes irregular near the flap tips, residues 49–53. Model‐free analysis of 15N relaxation parameters, T1, T2 (T1ρ) and 15N‐{1H} NOE, shows that residues in the flap tips are flexible on the sub‐ns time scale, in contrast with previous observations on the inhibitor‐bound protease. These results are compared with theoretical predictions of flap dynamics and the possible biological significance of the sub‐ns time scale dynamics of the flap tips is discussed.

NMR study of the tetrameric KcsA potassium channel in detergent micelles

Nuclear magnetic resonance (NMR) studies of large membrane‐associated proteins are limited by the difficulties in preparation of stable protein–detergent mixed micelles and by line broadening, which is typical of these macroassemblies. We have used the 68‐kDa homotetrameric KcsA, a thermostable N‐terminal deletion mutant of a bacterial potassium channel from Streptomyces lividans, as a model system for applying NMR methods to membrane proteins. Optimization of measurement conditions enabled us to perform the backbone assignment of KcsA in SDS micelles and establish its secondary structure, which was found to closely agree with the KcsA crystal structure. The C‐terminal cytoplasmic domain, absent in the original structure, contains a 14‐residue helix that could participate in tetramerization by forming an intersubunit four‐helix bundle. A quantitative estimate of cross‐ relaxation between detergent and KcsA backbone amide protons, together with relaxation and light scattering data, suggests SDS–KcsA mixed micelles form an oblate spheroid with ∼180 SDS molecules per channel. K+ ions bind to the micelle‐solubilized channel with a KD of 3 ± 0.5 mM, resulting in chemical shift changes in the selectivity filter. Related pH‐induced changes in chemical shift along the “outer” transmembrane helix and the cytoplasmic membrane interface hint at a possible structural explanation for the observed pH‐gating of the potassium channel.

Autoprocessing of HIV-1 protease is tightly coupled to protein folding

In the Gag-Pol polyprotein of HIV-1, the 99-amino acid protease is flanked at its N-terminus by a transframe region (TFR) composed of the transframe octapeptide (TFP) and 48 amino acids of the p6pol, separated by a protease cleavage site. The intact precursor (TFP-p6pol-PR) has very low dimer stability relative to that of the mature enzyme and exhibits negligible levels of stable tertiary structure. Thus, the TFR functions by destabilizing the native structure, unlike proregions found in zymogen forms of monomeric aspartic proteases. Cleavage at the p6pol-PR site to release a free N-terminus of protease is concomitant with the appearance of enzymatic activity and formation of a stable tertiary structure that is characteristic of the mature protease as demonstrated by nuclear magnetic resonance. The release of the mature protease from the precursor can either occur in two steps at pH values of 4 to 6 or in a single step above pH 6. The mature protease forms a dimer through a four-stranded β-sheet at the interface. Residues 1–4 of the mature protease from each subunit constitute the outer strands of the β-sheet, and are essential for maintaining the stability of the free protease but are not a prerequisite for the formation of tertiary structure and catalytic activity. Our experimental results provide the basis for the model proposed here for the regulation of the HIV-1 protease in the viral replication cycle.

Structure and dynamics of KH domains from FBP bound to single-stranded DNA

Gene regulation can be tightly controlled by recognition of DNA deformations that are induced by stress generated during transcription. The KH domains of the FUSE-binding protein (FBP), a regulator of c-myc expression, bind in vivo and in vitro to the single-stranded far-upstream element (FUSE), 1,500 base pairs upstream from the c-myc promoter. FBP bound to FUSE acts through TFIIH at the promoter. Here we report the solution structure of a complex between the KH3 and KH4 domains of FBP and a 29-base single-stranded DNA from FUSE. The KH domains recognize two sites, 9–10 bases in length, separated by 5 bases, with KH4 bound to the 5′ site and KH3 to the 3′ site. The central portion of each site comprises a tetrad of sequence 5′d-ATTC for KH4 and 5′d-TTTT for KH3. Dynamics measurements show that the two KH domains bind as articulated modules to single-stranded DNA, providing a flexible framework with which to recognize transient, moving targets.

Dimerization of the class A G protein-coupled neurotensin receptor NTS1 alters G protein interaction

G protein-coupled receptors (GPCRs) have been found as monomers but also as dimers or higher-order oligomers in cells. The relevance of the monomeric or dimeric receptor state for G protein activation is currently under debate for class A rhodopsin-like GPCRs. Clarification of this issue requires the availability of well defined receptor preparations as monomers or dimers and an assessment of their ligand-binding and G protein-coupling properties. We show by pharmacological and hydrodynamic experiments that purified neurotensin receptor NTS1, a class A GPCR, dimerizes in detergent solution in a concentration-dependent manner, with an apparent affinity in the low nanomolar range. At low receptor concentrations, NTS1 binds the agonist neurotensin with a Hill slope of ≈1; at higher receptor concentrations, neurotensin binding displays positive cooperativity with a Hill slope of ≈2. NTS1 monomers activate Gαqβ1γ2, whereas receptor dimers catalyze nucleotide exchange with lower affinity. Our results demonstrate that NTS1 dimerization per se is not a prerequisite for G protein activation.