Ultrafast signals in protein folding and the polypeptide contracted state

To test the significance of ultrafast protein folding signals (≪1 msec), we studied cytochrome c (Cyt c) and two Cyt c fragments with major C-terminal segments deleted. The fragments remain unfolded under all conditions and so could be used to define the unfolded baselines for protein fluorescence and circular dichroism (CD) as a function of denaturant concentration. When diluted from high to low denaturant in kinetic folding experiments, the fragments readjust to their new baseline values in a “burst phase” within the mixing dead time. The fragment burst phase reflects a contraction of the polypeptide from a more extended unfolded condition at high denaturant to a more contracted unfolded condition in the poorer, low denaturant solvent. Holo Cyt c exhibits fluorescence and CD burst phase signals that are essentially identical to the fragment signals over the whole range of final denaturant concentrations, evidently reflecting the same solvent-dependent, relatively nonspecific contraction and not the formation of a specific folding intermediate. The significance of fast folding signals in Cyt c and other proteins is discussed in relation to the hypothesis of an initial rate-limiting search-nucleation-collapse step in protein folding [Sosnick, T. R., Mayne, L. & Englander, S. W. (1996) Proteins Struct. Funct. Genet. 24, 413–426].

Multiple pathways for ultrafast transduction of light energy in the photosynthetic reaction center of Rhodobacter sphaeroides

A pathway of electron transfer is described that operates in the wild-type reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides. The pathway does not involve the excited state of the special pair dimer of bacteriochlorophylls (P*), but instead is driven by the excited state of the monomeric bacteriochlorophyll (BA*) present in the active branch of pigments along which electron transfer occurs. Pump-probe experiments were performed at 77 K on membrane-bound RCs by using different excitation wavelengths, to investigate the formation of the charge separated state P+HA−. In experiments in which P or BA was selectively excited at 880 nm or 796 nm, respectively, the formation of P+HA− was associated with similar time constants of 1.5 ps and 1.7 ps. However, the spectral changes associated with the two time constants are very different. Global analysis of the transient spectra shows that a mixture of P+BA− and P* is formed in parallel from BA* on a subpicosecond time scale. In contrast, excitation of the inactive branch monomeric bacteriochlorophyll (BB) and the high exciton component of P (P+) resulted in electron transfer only after relaxation to P*. The multiple pathways for primary electron transfer in the bacterial RC are discussed with regard to the mechanism of charge separation in the RC of photosystem II from higher plants.

An unusual pathway of excitation energy deactivation in carotenoids: Singlet-to-triplet conversion on an ultrafast timescale in a photosynthetic antenna

Carotenoids are important biomolecules that are ubiquitous in nature and find widespread application in medicine. In photosynthesis, they have a large role in light harvesting (LH) and photoprotection. They exert their LH function by donating their excited singlet state to nearby (bacterio)chlorophyll molecules. In photosynthetic bacteria, the efficiency of this energy transfer process can be as low as 30%. Here, we present evidence that an unusual pathway of excited state relaxation in carotenoids underlies this poor LH function, by which carotenoid triplet states are generated directly from carotenoid singlet states. This pathway, operative on a femtosecond and picosecond timescale, involves an intermediate state, which we identify as a new, hitherto uncharacterized carotenoid singlet excited state. In LH complex-bound carotenoids, this state is the precursor on the reaction pathway to the triplet state, whereas in extracted carotenoids in solution, this state returns to the singlet ground state without forming any triplets. We discuss the possible identity of this excited state and argue that fission of the singlet state into a pair of triplet states on individual carotenoid molecules constitutes the mechanism by which the triplets are generated. This is, to our knowledge, the first ever direct observation of a singlet-to-triplet conversion process on an ultrafast timescale in a photosynthetic antenna.

Computational adaptive optics for live three-dimensional biological imaging

Light microscopy of thick biological samples, such as tissues, is often limited by aberrations caused by refractive index variations within the sample itself. This problem is particularly severe for live imaging, a field of great current excitement due to the development of inherently fluorescent proteins. We describe a method of removing such aberrations computationally by mapping the refractive index of the sample using differential interference contrast microscopy, modeling the aberrations by ray tracing through this index map, and using space-variant deconvolution to remove aberrations. This approach will open possibilities to study weakly labeled molecules in difficult-to-image live specimens.

Diffractive optics-based heterodyne-detected four-wave mixing signals of protein motion: From “protein quakes” to ligand escape for myoglobin

Ligand transport through myoglobin (Mb) has been observed by using optically heterodyne-detected transient grating spectroscopy. Experimental implementation using diffractive optics has provided unprecedented sensitivity for the study of protein motions by enabling the passive phase locking of the four beams that constitute the experiment, and an unambiguous separation of the Real and Imaginary parts of the signal. Ligand photodissociation of carboxymyoglobin (MbCO) induces a sequence of events involving the relaxation of the protein structure to accommodate ligand escape. These motions show up in the Real part of the signal. The ligand (CO) transport process involves an initial, small amplitude, change in volume, reflecting the transit time of the ligand through the protein, followed by a significantly larger volume change with ligand escape to the surrounding water. The latter process is well described by a single exponential process of 725 ± 15 ns at room temperature. The overall dynamics provide a distinctive signature that can be understood in the context of segmental protein fluctuations that aid ligand escape via a few specific cavities, and they suggest the existence of discrete escape pathways.

Ultrafast detection and control of molecular dynamics

Many elementary chemical and physical processes such as the breaking of a chemical bond or the vibrational motion of atoms within a molecule take place on a femtosecond (fs = 10−15 s) or picosecond (ps = 10−12 s) time scale. It is now possible to monitor these events as a function of time with temporal resolution well below 100 fs. This capability is based on the pump-probe technique where one optical pulse triggers a reaction and a second delayed optical pulse probes the changes that ensue. To illustrate this capability, the dynamics of ligand motion within a protein are presented. Moving beyond casual observation of a reaction to active control of its outcome requires additional experimental and theoretical effort. To illustrate the concept of control, the effect of optical pulse duration on the vibrational dynamics of a tri-atomic molecule are discussed. The experimental and theoretical resources currently available are poised to make the dream of reaction control a reality for certain molecular systems.

Ultrafast diffraction and structural dynamics: The nature of complex molecules far from equilibrium

Studies of molecular structures at or near their equilibrium configurations have long provided information on their geometry in terms of bond distances and angles. Far-from-equilibrium structures are relatively unknown—especially for complex systems—and generally, neither their dynamics nor their average geometries can be extrapolated from equilibrium values. For such nonequilibrium structures, vibrational amplitudes and bond distances play a central role in phenomena such as energy redistribution and chemical reactivity. Ultrafast electron diffraction, which was developed to study transient molecular structures, provides a direct method for probing the nature of complex molecules far from equilibrium. Here we present our ultrafast electron diffraction observations of transient structures for two cyclic hydrocarbons. At high internal energies of ≈4 eV, these molecules display markedly different behavior. For 1,3,5-cycloheptatriene, excitation results in the formation of hot ground-state structures with bond distances similar to those of the initial structure, but with nearly three times the average vibrational amplitude. Energy is redistributed within 5 ps, but with a negative temperature characterizing the nonequilibrium population. In contrast, the ring-opening reaction of 1,3-cyclohexadiene is shown to result in hot structures with a C—C bond distance of over 1.7 Å, which is 0.2 Å away from any expected equilibrium value. Even up to 400 ps, energy remains trapped in large-amplitude motions comprised of torsion and asymmetric stretching. These studies promise a new direction for studying structural dynamics in nonequilibrium complex systems.

Ultrafast thermally induced unfolding of RNase A.

A temperature jump (T-jump) method capable of initiating thermally induced processes on the picosecond time scale in aqueous solutions is introduced. Protein solutions are heated by energy from a laser pulse that is absorbed by homogeneously dispersed molecules of the dye crystal violet. These act as transducers by releasing the energy as heat to cause a T-jump of up to 10 K with a time resolution of 70 ps. The method was applied to the unfolding of RNase A. At pH 5.7 and 59 degrees C, a T-jump of 3-6 K induced unfolding which was detected by picosecond transient infrared spectroscopy of the amide I region between 1600 and 1700 cm-1. The difference spectral profile at 3.5 ns closely resembled that found for the equilibrium (native-unfolded) states. The signal at 1633 cm-1, corresponding to the beta-sheet structure, achieved 15 +/- 2% of the decrease found at equilibrium, within 5.5 ns. However, no decrease in absorbance was detected until 1 ns after the T-ump. The disruption of beta-sheet therefore appears to be subject to a delay of approximately 1 ns. Prior to 1 ns after the T-jump, water might be accessing the intact hydrophobic regions.