Jan Kubelka

Single-molecule spectroscopy of the temperature-induced collapse of unfolded proteins.

We used single-molecule FRET in combination with other biophysical methods and molecular simulations to investigate the effect of temperature on the dimensions of unfolded proteins. With single-molecule FRET, this question can be addressed even under near-native conditions, where most molecules are folded, allowing us to probe a wide range of denaturant concentrations and temperatures. We find a compaction of the unfolded state of a small cold shock protein with increasing temperature in both the presence and the absence of denaturant, with good agreement between the results from single-molecule FRET and dynamic light scattering. Although dissociation of denaturant from the polypeptide chain with increasing temperature accounts for part of the compaction, the results indicate an important role for additional temperature-dependent interactions within the unfolded chain. The observation of a collapse of a similar extent in the extremely hydrophilic, intrinsically disordered protein prothymosin α suggests that the hydrophobic effect is not the sole source of the underlying interactions. Circular dichroism spectroscopy and replica exchange molecular dynamics simulations in explicit water show changes in secondary structure content with increasing temperature and suggest a contribution of intramolecular hydrogen bonding to unfolded state collapse.

Chemical, physical, and theoretical kinetics of an ultrafast folding protein

An extensive set of equilibrium and kinetic data is presented and analyzed for an ultrafast folding protein—the villin subdomain. The equilibrium data consist of the excess heat capacity, tryptophan fluorescence quantum yield, and natural circular-dichroism spectrum as a function of temperature, and the kinetic data consist of time courses of the quantum yield from nanosecond-laser temperature-jump experiments. The data are well fit with three kinds of models—a three-state chemical-kinetics model, a physical-kinetics model, and an Ising-like theoretical model that considers 105 possible conformations (microstates). In both the physical-kinetics and theoretical models, folding is described as diffusion on a one-dimensional free-energy surface. In the physical-kinetics model the reaction coordinate is unspecified, whereas in the theoretical model, order parameters, either the fraction of native contacts or the number of native residues, are used as reaction coordinates. The validity of these two reaction coordinates is demonstrated from calculation of the splitting probability from the rate matrix of the master equation for all 105 microstates. The analysis of the data on site-directed mutants using the chemical-kinetics model provides information on the structure of the transition-state ensemble; the physical-kinetics model allows an estimate of the height of the free-energy barrier separating the folded and unfolded states; and the theoretical model provides a detailed picture of the free-energy surface and a residue-by-residue description of the evolution of the folded structure, yet contains many fewer adjustable parameters than either the chemical- or physical-kinetics models.

Effect of Asphaltene Structure on Association and Aggregation Using Molecular Dynamics

The aggregation of asphaltenes has been established for decades by numerous experimental techniques; however, very few studies have been performed on the association free energy and asphaltene aggregation in solvents. The lack of reliable and coherent data on the free energy of association and aggregation size of asphaltene has imposed severe limitations on the thermodynamic modeling of asphaltene phase behavior. Current thermodynamic models either consider asphaltenes as non-associating components or use fitting parameters to characterize the association. In this work, the relations between Gibbs free energy of asphaltene association and asphaltene molecular structure are studied using molecular dynamics (MD). The free energy of association is computed from the potential of mean force profile along the separation distance between the centers of mass of two asphaltene molecules using the umbrella sampling technique in the GROMACS simulation package. The average aggregation number for asphaltene nanoaggregates and clusters is also calculated through MD simulations of 36 asphaltene molecules in toluene and heptane in order to estimate the effects of association free energy and steric repulsion on the aggregation behavior of asphaltenes. Our simulation results confirm that the interactions between aromatic cores of asphaltene molecules are the major driving force for association as the energy of association increases substantially with the number of aromatic rings. Moreover, heteroatoms attached to the aromatic cores have much more influence on the association free energy than to ones attached to the aliphatic chains. The length and number of aliphatic chains do not seem to have a noticeable effect on asphaltene dimerization; however, they have a profound effect on asphaltene aggregation size since steric repulsion can prevent asphaltenes from forming T-shape configurations and therefore decrease the aggregation size of asphaltenes significantly. Our MD simulation results show for the first time asphaltene precipitation in heptane as an explicit solvent, and predict three distinct stages of aggregation (nanoaggregation, clustering, and flocculation) as proposed by the modified Yen model. Finally, the association free energy for asphaltenes in heptane is higher than that in toluene, which is consistent with asphaltene aggregate sizes obtained from MD simulations.

Ligand Induced Circular Dichroism and Circularly Polarized Luminescence in CdSe Quantum Dots

Chiral thiol capping ligands L- and D-cysteines induced modular chiroptical properties in achiral cadmium selenide quantum dots (CdSe QDs). Cys-CdSe prepared from achiral oleic acid capped CdSe by postsynthetic ligand exchange displayed size-dependent electronic circular dichroism (CD) and circularly polarized luminescence (CPL). Opposite CPL signals were measured for the CdSe QDs capped with D- and L-cysteine. The CD profile and CD anisotropy varied with size of CdSe nanocrystals with largest anisotropy observed for CdSe nanoparticles of 4.4 nm. Magic angle spinning solid state NMR (MAS ssNMR) experiments suggested bidentate interaction between cysteine and the surface of CdSe. Time Dependent Density Functional Theory (TDDFT) calculations verified that attachment of L- and D-cysteine to the surface of model (CdSe)13 nanoclusters induces measurable opposite CD signals for the exitonic band of the nanocluster. The origin of the induced chirality is consistent with the hybridization of highest occupied CdSe molecular orbitals with those of the chiral ligand.

Infrared, vibrational circular dichroism, and Raman spectral simulations for β-sheet structures with various isotopic labels, interstrand, and stacking arrangements using density functional theory.

Infrared (IR), Raman, and vibrational circular dichroism (VCD) spectral variations for different β-sheet structures were studied using simulations based on density functional theory (DFT) force field and intensity computations. The DFT vibrational parameters were obtained for β-sheet fragments containing nine-amides and constrained to a variety of conformations and strand arrangements. These were subsequently transferred onto corresponding larger β-sheet models, normally consisting of five strands with ten amides each, for spectral simulations. Further extension to fibril models composed of multiple stacked β-sheets was achieved by combining the transfer of DFT parameters for each sheet with dipole coupling methods for interactions between sheets. IR spectra of the amide I show different splitting patterns for parallel and antiparallel β-sheets, and their VCD, in the absence of intersheet stacking, have distinct sign variations. Isotopic labeling by (13)C of selected residues yields spectral shifts and intensity changes uniquely sensitive to relative alignment of strands (registry) for antiparallel sheets. Stacking of multiple planar sheets maintains the qualitative spectral character of the single sheet but evidences some reduction in the exciton splitting of the amide I mode. Rotating sheets with respect to each other leads to a significant VCD enhancement, whose sign pattern and intensity is dependent on the handedness and degree of rotation. For twisted β-sheets, a significant VCD enhancement is computed even for sheets stacked with either the same or opposite alignments and the inter-sheet rotation, depending on the sense, can either further increase or weaken the enhanced VCD intensity. In twisted, stacked structures (without rotation), similar VCD amide I patterns (positive couplets) are predicted for both parallel and antiparallel sheets, but different IR intensity distributions still enable their differentiation. Our simulation results prove useful for interpreting experimental vibrational spectra in terms of β-sheet and fibril structure, as illustrated in the accompanying paper.

Simulations of oligopeptide vibrational CD: effects of isotopic labeling.

Simulated ir absorption and vibrational CD (VCD) spectra of four alanine‐based octapeptides, each having its main chain constrained to a different secondary structure conformation, were analyzed and compared with experimental results for several different peptides. The octapeptide simulations were based on transfer of property tensors from a series of ab initio calculations for a short L‐alanine based segment containing 3 peptide bonds with relative ϕ, ψ angles fixed to those appropriate for α‐helix, 310‐helix, ProII‐like helix, and β‐sheet‐like strand. The tripeptide force field (FF) and atomic polar tensors were obtained with density functional theory techniques at the BPW91/6‐31G** level and the atomic axial tensor at the mixed BPW91/6‐31G**/HF/6‐31G level. Allowing for frequency correction due to the FF limitations, the octapeptide results obtained are qualitatively consistent with experimental observations for ir and VCD spectra of polypeptides and oligopeptides in established conformations. In all cases, the correct VCD sign patterns for the amide I and II bands were predicted, but the intensities did have some variation from the experimental patterns. Predicted VCD changes upon deuteration of either the peptide or side‐chains as well as for 13C isotopic labeling of the amide CO at specific sites in the peptide chain were computed for analysis of experimental observations. A combination of theoretical modeling with experimental data for labeled compounds leads both to enhanced resolution of component transitions and added conformational applicability of the VCD spectra.

Biophysical and structural considerations for protein sequence evolution

BackgroundProtein sequence evolution is constrained by the biophysics of folding and function, causing interdependence between interacting sites in the sequence. However, current site-independent models of sequence evolutions do not take this into account. Recent attempts to integrate the influence of structure and biophysics into phylogenetic models via statistical/informational approaches have not resulted in expected improvements in model performance. This suggests that further innovations are needed for progress in this field.ResultsHere we develop a coarse-grained physics-based model of protein folding and binding function, and compare it to a popular informational model. We find that both models violate the assumption of the native sequence being close to a thermodynamic optimum, causing directional selection away from the native state. Sampling and simulation show that the physics-based model is more specific for fold-defining interactions that vary less among residue type. The informational model diffuses further in sequence space with fewer barriers and tends to provide less support for an invariant sites model, although amino acid substitutions are generally conservative. Both approaches produce sequences with natural features like dN/dS < 1 and gamma-distributed rates across sites.ConclusionsSimple coarse-grained models of protein folding can describe some natural features of evolving proteins but are currently not accurate enough to use in evolutionary inference. This is partly due to improper packing of the hydrophobic core. We suggest possible improvements on the representation of structure, folding energy, and binding function, as regards both native and non-native conformations, and describe a large number of possible applications for such a model.

Time-resolved methods in biophysics. 9. Laser temperature-jump methods for investigating biomolecular dynamics

Many important biochemical processes occur on the time-scales of nanoseconds and microseconds. The introduction of the laser temperature-jump (T-jump) to biophysics more than a decade ago opened these previously inaccessible time regimes up to direct experimental observation. Since then, laser T-jump methodology has evolved into one of the most versatile and generally applicable methods for studying fast biomolecular kinetics. This perspective is a review of the principles and applications of the laser T-jump technique in biophysics. A brief overview of the T-jump relaxation kinetics and the historical development of laser T-jump methodology is presented. The physical principles and practical experimental considerations that are important for the design of the laser T-jump experiments are summarized. These include the Raman conversion for generating heating pulses, considerations of size, duration and uniformity of the temperature jump, as well as potential adverse effects due to photo-acoustic waves, cavitation and thermal lensing, and their elimination. The laser T-jump apparatus developed at the NIH Laboratory of Chemical Physics is described in detail along with a brief survey of other laser T-jump designs in use today. Finally, applications of the laser T-jump in biophysics are reviewed, with an emphasis on the broad range of problems where the laser T-jump methodology has provided important new results and insights into the dynamics of the biomolecular processes.

Novel Use of a Static Modification of Two-Dimensional Correlation Analysis. Part I: Comparison of the Secondary Structure Sensitivity of Electronic Circular Dichroism, FT-IR, and Raman Spectra of Proteins

A modification of Nodas algorithm that allows for calculation of two-dimensional (2D) correlation maps is presented as a method for analysis of a series of (static) spectra of proteins. In this approach, fractional secondary structure was used as the perturbation to generate the 2D correlation. The functional dependence of the spectral intensities on secondary structure is approximated by an even-order polynomial fit to the protein spectra at each spectral frequency. These functions are used to calculate the 2D correlation and disrelation maps, and their regression coefficients are used to weight the results to minimize artifacts. Electronic circular dichroism (ECD), Fourier transform infrared (FT-IR) (amide I and II regions), and Raman spectra of up to 22 proteins are used in the study. Spectral regions identified by the α-helix- and β-sheet-based 2D correlation maps are in agreement with established interpretation of ECD and FT-IR spectra in terms of secondary structure and provide insight into secondary structure assignment for a broad range of Raman bands. Comparison of our functional fit method, specifically designed to identify synchronous correlations, with Nodas Fourier transform-based method, which generates asynchronous maps as well, is discussed.

Insight into the packing pattern of β2 fibrils: A model study of glutamic acid rich oligomers with 13C isotopic edited vibrational spectroscopy

Polyglutamic acid at low pH forms aggregates and self-assembles into a spiral, fibril-like superstructure formed as a β2-type sheet conformation that has a more compact intersheet packing than commonly found. This is stabilized by three-centered bifurcated hydrogen bonding of the amide carbonyl involving the protonated glutamic acid side chain. We report vibrational spectroscopic results and analyses for oligopeptides rich in glutamic acid enhanced with (13)C isotope labeling in a study modeling low pH poly-Glu self-assembly. Our results indicate bifurcated H-bonding and β2 aggregation can be attained in these model decamers, confirming they have the same conformations as poly-Glu. We also prepared conventional β1-sheet aggregates by rapid precipitation from the residual peptides in the higher pH supernatant. By comparing the isotope-enhanced IR and VCD spectra with theoretical predictions, we deduced that the oligo-Glu β2 structure is based on stacked, twisted, antiparallel β-sheets. The best fit to theoretical predictions was obtained for the strands being out of register, sequentially stepped by one residue, in a ladder-like fashion. The alternate β1 conformer for this oligopeptide was similarly shown to be antiparallel but was less ordered and apparently had a different registry in its aggregate structure.