Eefei Chen

Early Events, Kinetic Intermediates and the Mechanism of Protein Folding in Cytochrome c

Kinetic studies of the early events in cytochrome c folding are reviewed with a focus on the evidence for folding intermediates on the submillisecond timescale. Evidence from time-resolved absorption, circular dichroism, magnetic circular dichroism, fluorescence energy and electron transfer, small-angle X-ray scattering and amide hydrogen exchange studies on the t ≤ 1 ms timescale reveals a picture of cytochrome c folding that starts with the ~ 1-μs conformational diffusion dynamics of the unfolded chains. A fractional population of the unfolded chains collapses on the 1 – 100 μs timescale to a compact intermediate IC containing some native-like secondary structure. Although the existence and nature of IC as a discrete folding intermediate remains controversial, there is extensive high time-resolution kinetic evidence for the rapid formation of IC as a true intermediate, i.e., a metastable state separated from the unfolded state by a discrete free energy barrier. Final folding to the native state takes place on millisecond and longer timescales, depending on the presence of kinetic traps such as heme misligation and proline mis-isomerization. The high folding rates observed in equilibrium molten globule models suggest that IC may be a productive folding intermediate. Whether it is an obligatory step on the pathway to the high free energy barrier associated with millisecond timescale folding to the native state, however, remains to be determined.

Nanosecond time‐resolved circular dichroism measurements using an upconverted Ti:sapphire laser

Several years ago a time‐resolved circular dichroism technique for the far ultraviolet spectral region with submicrosecond (10−7 s) time resolution was developed using a xenon flash lamp probe source for measurements of circular dichroism (CD) signals. Recent improvements in Ti:sapphire lasers, providing the ability to frequency‐convert the fundamental outputs to produce second, third, and fourth harmonic pulses, allow single wavelength measurements of CD with nanosecond (10−9 s) time resolution over a broad spectral region (205–910 nm). This provides a powerful technique to study fast biophysical phenomena such as protein folding processes. In this article, the methodology and preliminary application of this new technique are presented.

The Folding Kinetics of the SDS-Induced Molten Globule Form of Reduced Cytochrome c

The folding of reduced cytochrome c (redcyt c) is increasingly being recognized as undergoing a mechanism that deviates from a two-state process. In previous far-UV TRORD studies of redcyt c folding, a rapidly forming intermediate was attributed to the appearance of a molten-globule-like (MG) state [Chen, E., Goldbeck, R. A., and Kliger, D. S. (2003) J. Phys. Chem. A 107, 8149-8155]. A slow folding phase (>1 ms) was identified with the formation of native (N) secondary structure from that MG form. Here, using 0.65 mM SDS to induce the MG state in oxidized cytochrome c, folding of redcyt c was triggered with fast photoreduction and probed from early microseconds to milliseconds using far-UV TRORD spectroscopy. The kinetics of the reaction are described with a time constant of 50 +/- 16 ms, which corresponds to 1 +/- 0.6 ms upon extrapolation of the data to zero SDS concentration. The latter folding time is about 5 times faster than the calculated GuHCl-free time constant of 5.5 +/- 1.4 ms for slow-phase folding obtained in our previous study. This ratio of rates would be consistent with a scenario in which 20-30% MG that is suggested to form in the fast phase of redcyt c folding in GuHCl is an obligatory intermediate. The native state forms from this obligatory intermediate with an observed rate, k(f) = fk(G-->N) where f is the fractional population of MG and k(G-->N) is the microscopic rate for MG --> N. Calculation and comparison of the m(#)/m values show agreement within the uncertainties between the SDS ( approximately 0.5) and GuHCl ( approximately 0.3) based redcyt c folding experiments, suggesting that the two experiments report on comparable intermediates. The m values were obtained from far-UV CD SDS titration experiments, from which calculated thermodynamic parameters allowed estimation of the reduction potential for the MG state to be approximately 155 mV (-15 kJ/mol) vs NHE which, like the reduction potential for the native state, is more favorable than that for the unfolded protein.

Nanosecond laser temperature-jump optical rotatory dispersion: Application to early events in protein folding/unfolding

Nanosecond time-resolved optical rotatory dispersion (TRORD) techniques are coupled with laser temperature-jump (T-jump) triggering in an instrument that measures ultrafast protein folding-unfolding dynamics with high specificity to secondary structure. Far-ultraviolet (UV) ORD can be measured with this instrument over a wide wavelength range at times as early as 35 ns after a 3 ns laser T-jump pulse. The fundamental of a Nd:YAG laser is passed through a D2-filled Raman shifter to generate a pulse of 1.5μm infrared (IR) light that is efficiently absorbed by water. The resulting T-jump is stable for at least 1 ms before decaying back to the starting temperature with a time constant of ∼30ms. The ability to measure entire TRORD band shapes during this temporal window makes it possible to distinguish between changes in the signal due to a genuine unfolding or refolding process and changes due to artifacts. The technique, applicable to a wide variety of proteins, is demonstrated here in submillisecond unfoldi...

Probing early events in ferrous cytochrome c folding with time-resolved natural and magnetic circular dichroism spectroscopies.

In a 1998 collaboration with Tony Fink, we coupled nanosecond circular dichroism methods (TRCD) with a CO-photolysis system for quickly triggering folding in cytochrome c (cyt c) in order to make the first time-resolved far-UV CD measurement of early secondary structure formation in a protein. The small signal observed in that initial study, approximately 10% of native helicity, became the seed for increasingly robust results from subsequent studies bringing additional natural and magnetic circular polarization dichroism and optical rotatory dispersion detection methods (e.g., TRORD, TRMCD, and TRMORD), coupled to fast photolysis and photoreduction triggers, to the study of early folding events. Nanosecond polarization methods are reviewed here in the context of the range of initiation methods and structure-sensitive probes currently available for fast folding studies. We also review the impact of experimental results from fast polarization studies on questions in folding dynamics such as the possibility of multiple folding pathways implied by energy landscape models, the sequence dependence of ultrafast helix formation, and the simultaneity of chain collapse and secondary structure formation implicit in molten globule models for kinetic folding intermediates.

Short-lived alpha-helical intermediates in the folding of beta-sheet proteins.

Several lines of evidence point strongly toward the importance of highly alpha-helical intermediates in the folding of all globular proteins, regardless of their native structure. However, experimental refolding studies demonstrate no observable alpha-helical intermediate during refolding of some beta-sheet proteins and have dampened enthusiasm for this model of protein folding. In this study, beta-sheet proteins were hypothesized to have potential to form amphiphilic helices at a period of <3.6 residues/turn that matches or exceeds the potential at 3.6 residues/turn. Hypothetically, such potential is the basis for an effective and unidirectional mechanism by which highly alpha-helical intermediates might be rapidly disassembled during folding and potentially accounts for the difficulty in detecting highly alpha-helical intermediates during the folding of some proteins. The presence of this potential was confirmed, indicating that a model entailing ubiquitous formation of alpha-helical intermediates during the folding of globular proteins predicts previously unrecognized features of primary structure. Further, the folding of fatty acid binding protein, a predominantly beta-sheet protein that exhibits no apparent highly alpha-helical intermediate during folding, was dramatically accelerated by 2,2,2-trifluoroethanol, a solvent that stabilizes alpha-helical structure. This observation suggests that formation of an alpha-helix can be a rate-limiting step during folding of a predominantly beta-sheet protein and further supports the role of highly alpha-helical intermediates in the folding of all globular proteins.

Time-resolved near UV circular dichroism and absorption studies of carbonmonoxymyoglobin photolysis intermediates

Abstract Time-resolved circular dichroism (TRCD) spectra of ligand photolysis intermediates of horse skeletal carbonmonoxymyoglobin (MbCO) in the near UV (250–300 run) region are obtained to probe the environment of protein aromatic side chains as a function of time. Since X-ray data reveal compact structures for MbCO and deoxyMb with no apparent pathway for ligand migration through the protein matrix, dynamic protein fluctuations of side chains must be important in facilitating the ligand binding process. Near UV TRCD spectral data are fit to two exponential components with lifetimes of 110±35 μs and 1.5 ± 0.3 ms. Time-resolved absorption spectra in this wavelength region were fit to three lifetimes of 340 ± 100 ns, 830± 240μs and 2.1 ± 0.2 ms. The 830 μs and 2.1 ms processes are associated with bimolecular ligand rebinding and are consistent with lifetimes obtained from spectra in the Soret region of 200 ± 120 ns, 680 ± 80 μs and 1.8 ± 0.05 ms. The absence of the ca. 700μs component in the TRCD data indicates that bimolecular rebinding induces changes in the heme interaction with surrounding polar residues and not with aromatic residues. The 110μs TRCD transient is discussed in terms of motions of tyrosines and/or nearby aromatic groups that are involved in the early stages of ligand rebinding.

Platymonas subcordiformis channelrhodopsin-2 function: I. The photochemical reaction cycle

Background: Channelrhodopsins (ChRs) are light-gated cation channels widely used in optogenetics. Results: Time-resolved absorption spectroscopy of the high efficiency PsChR2 throughout the visible spectrum from 100 ns to 3 s was used to resolve spectra and kinetics of all photointermediates. Conclusion: The photochemical reactions are described by the superposition of two parallel photocycles. Significance: The parallel-cycle model opens new perspectives in understanding the mechanism of channelrhodopsins. The photocycle kinetics of Platymonas subcordiformis channelrhodopsin-2 (PsChR2), among the most highly efficient light-gated cation channels and the most blue-shifted channelrhodopsin, was studied by time-resolved absorption spectroscopy in the 340–650-nm range and in the 100-ns to 3-s time window. Global exponential fitting of the time dependence of spectral changes revealed six lifetimes: 0.60 μs, 5.3 μs, 170 μs, 1.4 ms, 6.7 ms, and 1.4 s. The sequential intermediates derived for a single unidirectional cycle scheme based on these lifetimes were found to contain mixtures of K, L, M, O, and P molecular states, named in analogy to photointermediates in the bacteriorhodopsin photocycle. The photochemistry is described by the superposition of two independent parallel photocycles. The analysis revealed that 30% of the photoexcited receptor molecules followed Cycle 1 through the K, M, O, and P states, whereas 70% followed Cycle 2 through the K, L, M, and O states. The recovered state, R, is spectrally close, but not identical, to the dark state on the seconds time scale. The two-cycle model of this high efficiency channelrhodopsin-2 (ChR) opens new perspectives in understanding the mechanism of channelrhodopsin function.

Nanosecond time-resolved polarization spectroscopies: Tools for probing protein reaction mechanisms

Polarization methods, introduced in the 1800s, offered one of the earliest ways to examine protein structure. Since then, many other structure-sensitive probes have been developed, but circular dichroism (CD) remains a powerful technique because of its versatility and the specificity of protein structural information that can be explored. With improvements in time resolution, from millisecond to picosecond CD measurements, it has proven to be an important tool for studying the mechanism of folding and function in many biomolecules. For example, nanosecond time-resolved CD (TRCD) studies of the sub-microsecond events of reduced cytochrome c folding have provided direct experimental evidence of kinetic heterogeneity, which is an inherent property of the diffusional nature of early folding dynamics on the energy landscape. In addition, TRCD has been applied to the study of many biochemical processes, such as ligand rebinding in hemoglobin and myoglobin and signaling state formation in photoactive yellow protein and prototropin 1 LOV2. The basic approach to TRCD has also been extended to include a repertoire of nanosecond polarization spectroscopies: optical rotatory dispersion (ORD), magnetic CD and ORD, and linear dichroism. This article will discuss the details of the polarization methods used in this laboratory, as well as the coupling of time-resolved ORD with the temperature-jump trigger so that protein folding can be studied in a larger number of proteins.

Probing kinetic mechanisms of protein function and folding with time-resolved natural and magnetic chiroptical spectroscopies.

Recent and ongoing developments in time-resolved spectroscopy have made it possible to monitor circular dichroism, magnetic circular dichroism, optical rotatory dispersion, and magnetic optical rotatory dispersion with nanosecond time resolution. These techniques have been applied to determine structural changes associated with the function of several proteins as well as to determine the nature of early events in protein folding. These studies have required new approaches in triggering protein reactions as well as the development of time-resolved techniques for polarization spectroscopies with sufficient time resolution and sensitivity to probe protein structural changes.