Everett A. Lipman

Probing the free-energy surface for protein folding with single-molecule fluorescence spectroscopy

Protein folding is inherently a heterogeneous process because of the very large number of microscopic pathways that connect the myriad unfolded conformations to the unique conformation of the native structure. In a first step towards the long-range goal of describing the distribution of pathways experimentally, Förster resonance energy transfer (FRET) has been measured on single, freely diffusing molecules. Here we use this method to determine properties of the free-energy surface for folding that have not been obtained from ensemble experiments. We show that single-molecule FRET measurements of a small cold-shock protein expose equilibrium collapse of the unfolded polypeptide and allow us to calculate limits on the polypeptide reconfiguration time. From these results, limits on the height of the free-energy barrier to folding are obtained that are consistent with a simple statistical mechanical model, but not with the barriers derived from simulations using molecular dynamics. Unlike the activation energy, the free-energy barrier includes the activation entropy and thus has been elusive to experimental determination for any kinetic process in solution.

Quantifying internal friction in unfolded and intrinsically disordered proteins with single-molecule spectroscopy

Internal friction, which reflects the “roughness” of the energy landscape, plays an important role for proteins by modulating the dynamics of their folding and other conformational changes. However, the experimental quantification of internal friction and its contribution to folding dynamics has remained challenging. Here we use the combination of single-molecule Förster resonance energy transfer, nanosecond fluorescence correlation spectroscopy, and microfluidic mixing to determine the reconfiguration times of unfolded proteins and investigate the mechanisms of internal friction contributing to their dynamics. Using concepts from polymer dynamics, we determine internal friction with three complementary, largely independent, and consistent approaches as an additive contribution to the reconfiguration time of the unfolded state. We find that the magnitude of internal friction correlates with the compactness of the unfolded protein: its contribution dominates the reconfiguration time of approximately 100 ns of the compact unfolded state of a small cold shock protein under native conditions, but decreases for more expanded chains, and approaches zero both at high denaturant concentrations and in intrinsically disordered proteins that are expanded due to intramolecular charge repulsion. Our results suggest that internal friction in the unfolded state will be particularly relevant for the kinetics of proteins that fold in the microsecond range or faster. The low internal friction in expanded intrinsically disordered proteins may have implications for the dynamics of their interactions with cellular binding partners.

Localizing internal friction along the reaction coordinate of protein folding by combining ensemble and single-molecule fluorescence spectroscopy

Theory, simulations and experimental results have suggested an important role of internal friction in the kinetics of protein folding. Recent experiments on spectrin domains provided the first evidence for a pronounced contribution of internal friction in proteins that fold on the millisecond timescale. However, it has remained unclear how this contribution is distributed along the reaction and what influence it has on the folding dynamics. Here we use a combination of single-molecule Förster resonance energy transfer, nanosecond fluorescence correlation spectroscopy, microfluidic mixing and denaturant- and viscosity-dependent protein-folding kinetics to probe internal friction in the unfolded state and at the early and late transition states of slow- and fast-folding spectrin domains. We find that the internal friction affecting the folding rates of spectrin domains is highly localized to the early transition state, suggesting an important role of rather specific interactions in the rate-limiting conformational changes.

Single-molecule spectroscopy of protein folding in a chaperonin cage

Molecular chaperones are known to be essential for avoiding protein aggregation in vivo, but it is still unclear how they affect protein folding mechanisms. We use single-molecule Förster resonance energy transfer to follow the folding of a protein inside the GroEL/GroES chaperonin cavity over a time range from milliseconds to hours. Our results show that confinement in the chaperonin decelerates the folding of the C-terminal domain in the substrate protein rhodanese, but leaves the folding rate of the N-terminal domain unaffected. Microfluidic mixing experiments indicate that strong interactions of the substrate with the cavity walls impede the folding process, but the folding hierarchy is preserved. Our results imply that no universal chaperonin mechanism exists. Rather, a competition between intra- and intermolecular interactions determines the folding rates and mechanisms of a substrate inside the GroEL/GroES cage.

THE BERKELEY INFRARED SPATIAL INTERFEROMETER: A HETERODYNE STELLAR INTERFEROMETER FOR THE MID-INFRARED

A detailed description is given of the Infrared Spatial Interferometer (ISI), developed at the Space Sciences Laboratory of the University of California at Berkeley, which is a high spatial resolution interferometer for mid-infrared wavelengths. The instrumentation, its capabilities and performance, data analysis, science program, and future plans are all discussed. The systems use of heterodyne detection, analogous to that of a modern radio interferometer, is also compared with the homodyne or direct methods more commonly encountered in the visible and infrared. The ISI has been operating productively on Mount Wilson for the past 10 years measuring materials immediately surrounding stars and their changes as well as some stellar diameters. The new spectral capabilities described here, a recent increase in baseline length, and the upcoming expansion to a closure-phase imaging array provide important additional types of measurements.

NONSPHERICAL STRUCTURES AND TEMPORAL VARIATIONS IN THE DUST SHELL OF o CETI OBSERVED WITH A LONG BASELINE INTERFEROMETER AT 11 MICRONS

Visibility observations at 11 km of o Ceti have been made with the University of California (Berkeley) Infrared Spatial Interferometer during the time period 1988E1995. The observed visibilities change dra- matically from one epoch to another and are not consistent with simple heating or cooling of the dust with change in luminosity as a function of stellar phase. Instead, large temporal variations in the density of dust within a few stellar radii of the photosphere of o Ceti have occurred. Spherically symmetric models of the dust distribution with two dust shells, one within three stellar radii of the photosphere of the star, the other approximately 10 stellar radii from the star, can account reasonably well for the observed changes. Four types of axially symmetric radiative transfer models were also compared with the dataEa spherical shell with an ellipsoidal inner cavity, a disk, a spherical shell with one or two inhomogeneities or clumps, and a set of thin partial shells with a -xed distance between them. Of the four models, only the one with the ellipsoidal inner cavity is excluded. The data were best--tted with the last two models, which emphasize inhomogeneities or clumps. To -t the observed temporal changes in the visibility data, all models must include a change in the densityEincreasing and decreasingEof dust close to the photosphere of the star. The axially symmetric models had clumps placed at distances from the star in agreement with distances of the spherical models. Good -ts to the observed broadband spec- trum of the star were also obtained with these models. Subject headings: circumstellar matter E infrared: stars E stars: individual (o Ceti) E stars: mass loss E stars: variables: long-period variables E techniques: interferometric

A microfluidic mixing system for single-molecule measurements

This article describes the design and fabrication of a microfluidic mixing system optimized for ultrasensitive optical measurements. Channels are replica-molded in polydimethylsiloxane elastomer and sealed with fused-silica coverglass. The resulting devices have broad chemical compatibility and extremely low fluorescence background, enabling measurements of individual molecules under well-characterized nonequilibrium conditions. Fluid delivery and pressure connections are made using an interface that allows for rapid assembly, rapid sample exchange, and modular device replacement while providing access for high numerical aperture optics.

Nonuniform Dust Outflow Observed around Infrared Object NML Cygni

Measurements by the University of California Berkeley Infrared Spatial Interferometer at 11.15 μm have yielded strong evidence for multiple dust shells and/or significant asymmetric dust emission around NML Cyg. New observations reported also include multiple 8-13 μm spectra taken from 1994-1995 and N-band (10.2 μm) photometry from 1980-1992. These and past measurements are analyzed and fitted to a model of the dust distribution around NML Cyg. No spherically symmetric single dust shell model is found consistent with both near- and mid-infrared observations. However, a circularly symmetric maximum entropy reconstruction of the 11 μm brightness distribution suggests a double-shell model for the dust distribution. Such a model, consisting of a geometrically thin shell of intermediate optical depth (τ11 μm ~ 1.9) plus an outer shell (τ11 μm ~ 0.33), is consistent not only with the 11 μm visibility data but also with near-infrared speckle measurements, the broadband spectrum, and the 9.7 μm silicate feature. The outer shell, or large-scale structure, is revealed only by long-baseline interferometry at 11 μm, being too cold (~400 K) to contribute in the near-infrared and having no unambiguous spectral signature in the mid-infrared. The optical constants of Ossenkopf, Henning, & Mathis proved superior to the Draine & Lee (1984) constants in fitting the detailed shape of the silicate feature and broadband spectrum for this object. Recent observations of H2O maser emission around NML Cyg by Richards, Yates, & Cohen (1996) are consistent with the location of the two dust shells and provide further evidence for the two-shell model.

MULTIPLE DUST SHELLS AND MOTIONS AROUND IK TAURI AS SEEN BY INFRARED INTERFEROMETRY

A visibility curve of IK Tau has been measured with the ISI, an 11 μm stellar interferometer, over a period of several years. Time variations in 11 μm flux were also measured. The results indicate an approximately periodic distribution of dust shells around the star, with shells separated by 200-250 mas and a diameter of about 200 mas for the innermost shell. Some shell motion has been detected, and if velocities are the same as those measured for CO gas and OH masers, the motion implies that the distance to IK Tau is about 265 pc and that shells have been emitted at times separated by about 12 yr, which is considerably longer than the stars luminosity period of 470 days.

Mid-Infrared Interferometry on Spectral Lines. II. Continuum (Dust) Emission Around IRC +10216 and VY Canis Majoris

The University of California Berkeley Infrared Spatial Interferometer has measured the mid-infrared visibilities of the carbon star IRC +10216 and the red supergiant VY CMa. The dust shells around these sources have been previously shown to be time variable, and these new data are used to probe the evolution of the dust shells on a decade timescale, complementing contemporaneous studies at other wavelengths. Self-consistent, spherically symmetric models at maximum and minimum light both show the inner radius of the IRC +10216 dust shell to be much larger (150 mas) than expected from the dust-condensation temperature, implying that dust production has slowed or stopped in recent years. Apparently, dust does not form every pulsational cycle (638 days), and these mid-infrared results are consistent with recent near-infrared imaging, which indicates little or no new dust production in the last 3 yr. Spherically symmetric models failed to fit recent VY CMa data, implying that emission from the inner dust shell is highly asymmetric and/or time variable.