The helix is a common secondary structural motif found in proteins, and the mechanism of helix-coil interconversion is key to understanding the protein-folding problem. We report the observation of the fast kinetics (nanosecond to millisecond) of helix melting in a small 21-residue alanine-based peptide. The unfolding reaction is initiated using a laser-induced temperature jump and probed using time-resolved infrared spectroscopy. The model peptide exhibits fast unfolding kinetics with a time constant of 160 +/- 60 ns at 28 degrees C in response to a laser-induced temperature jump of 18 degrees C which is completed within 20 ns. Using the unfolding time and the measured helix-coil equilibrium constant of the model peptide, a folding rate constant of approximately 6 x 10(7) s-1 (t1/2 = 16 ns) can be inferred for the helix formation reaction at 28 degrees C. These results demonstrate that secondary structure formation is fast enough to be a key event at early times in the protein-folding process and that helices are capable of forming before long range tertiary contacts are made.

The individual resonance Raman spectra of the PM568 and M412 forms of light-adapted purple membrane from Halobacterium halobium have been measured using the newly developed flow technique. For comparison purposes, the Raman spectra of the model chromophores, all-trans- and 13-cis retinal n-butylamine, both as protonated and unprotonated Schiff bases, have also been obtained. In agreement with previous work, the Raman data indicate that the retinal chromophore is linked to the purple membrane protein via a protonated. Schiff base in the case of the PM568 and an unprotonated Schiff base for the M412 form. The basic mechanism for color regulation in both forms appears to be electron delocalization. The spectral features of the two forms are different from each other and different from the model compound spectra.

Low-temperature resonance Raman spectroscopy has been used to study the conformation and interactions of retinal within its opsin binding site in disk membrane vesicles formed from bovine retinal rod outer segments. At 80°K, laser irradiation within the visible absorp-7 From the

We report the fast relaxation dynamics of ''native'' apomyoglobin (pH 5.3) following a 10-ns, laserinduced temperature jump. The structural dynamics are probed using time-resolved infrared spectroscopy. The infrared kinetics monitored within the amide I absorbance of the polypeptide backbone exhibit two distinct relaxation phases which have different spectral signatures and occur on very different time scales ( ‫؍‬ 1633 cm ؊1 , ‫؍‬ 48 ns; ‫؍‬ 1650 cm ؊1 , ‫؍‬ 132 s). We assign these two spectral components to discrete substructures in the protein: helical structure that is solvated (1633 cm ؊1 ) and native helix that is protected from solvation by interhelix tertiary interactions (1650 cm ؊1 ). Folding rate coefficients inferred from the observed relaxations at 60؇C are k f(solvated) ‫؍‬ (7 to 20) ؋ 10 6 s ؊1 and k f(native) ‫؍‬ 3.6 ؋ 10 3 s ؊1 , respectively. The faster rate is interpreted as the intrinsic rate of solvated helix formation, whereas the slower rate is interpreted as the rate of formation of tertiary contacts that determine a native helix. Thus, at 60؇C helix formation precedes the formation of tertiary structure by over three orders of magnitude in this protein. Furthermore, the distinct thermodynamics and kinetics observed for the apomyoglobin substructures suggest that they fold independently, or quasi-independently. The observation of inhomogeneous folding for apomyoglobin is remarkable, given the relatively small size and structural simplicity of this protein.The mechanisms by which a protein searches vast conformational space to attain its native fold in reasonable times and by which the three-dimensional structure is encoded in the primary sequence have not been resolved experimentally. In particular, the critical early-time structural dynamics which carry a protein along the pathway(s) from extended, disordered conformations to a compact fold are poorly characterized. A major impediment has been the conventional solutionmixing approach to initiation of a folding reaction, which imposes a short-time observation limit of greater than 1 ms.

Visual pigments are a class of proteins found in the membranes of photoreceptor cells (for reviews, see refs. 1 and 2). Their chromophoric unit is 11-cis-retinal covalently bound in the form of a Schiff base to the c-amino group of a lysine. The absorption of a photon by a visual pigment initiates a sequence of biochemical events that eventually lead to the generation of a neural signal by a photoreceptor cell. The identity of the primary photochemical event has been a subject of considerable interest and controversy. It was originally suggested that the primary event was an isomerization of the chromophore from its 1 1-cis to an all-trans conformation (3, 4). The strongest evidence favoring this mechanism was the observation (based on spectral data at low temperature) that an artificial pigment containing a 9-cis chromophore had the same photoproduct as rhodopsin itself. It was quite reasonably concluded that the most plausible common photoproduct formed from the two cis isomers is a trans isomer. A number of picosecond absorption studies of the primary event have raised widespread doubts as to the validity of the original model. It was found (5) that the primary event is complete in less than a few picoseconds at room temperature, and it was argued that this is too short a time for isomerization to occur [although other picosecond studies have reached the opposite conclusion (6)]. More recent evidence has come from the observation that at low temperature the rate of the process is significantly inhibited by deuterium replacement of the exchangeable protons on the pigment (7). Since only one proton on the chromophore is exchangeable, it is unlikely that this would have a measurable effect on the rate of isomerization. The picosecond measurements have generated numerous models (7-11) whose major feature is a photochemical proton transfer followed by a thermal cis-trans isomerization that occurs at a later stage. However, the original evidence upon which the suggestion of a cis-trans isomerization was originally based has never been discredited and, thus, it appears necessary to find a molecular model that is consistent with the entire body of available evidence. The purpose of this paper is to present such a model.There are, in fact, a fairly large number of observations that can be used in the construction and evaluation of alternative models. For example, we have shown, by using simple thermodynamic arguments, that a significant fraction of the photon's energy is "stored" in the primary photoproduct (12). Clearly a mechanism for energy storage must be an important component of any model that is proposed. Another energetic constraint may be derived from psychophysical and electrophysiological measurements of the level of thermal noise in photoreceptor cells. We show below that these observations may be interpreted directly in molecular terms and that they require an unusually high thermal activation energy for the primary event that appears to be inconsistent with many of the models that have been proposed.The ...

R. Brian Dyer received the Ph.D. degree in inorganic chemistry from Duke University in 1985. After spending 2 years in the Inorganic and Structural Chemistry Group (INC-4) at Los Alamos National Laboratory as a postdoc, he moved to the chemical and laser sciences division as a staff member. He is currently on staff in the biosciences and biotechnology group (CST-4). He was recently awarded the Los Alamos Fellows Prize for his work on molecular dynamics and protein folding.

Most experimental studies on the dynamics of protein folding have been confined to timescales of 1 ms and longer. Yet it is obvious that many phenomena that are obligatory elements of the folding process occur on much faster timescales. For example, it is also now clear that the formation of secondary and tertiary structures can occur on nanosecond and microsecond times, respectively. Although fast events are essential to, and sometimes dominate, the overall folding process, with a few exceptions their experimental study has become possible only recently with the development of appropriate techniques. This review discusses new approaches that are capable of initiating and monitoring the fast events in protein folding with temporal resolution down to picoseconds. The first important results from those techniques, which have been obtained for the folding of some globular proteins and polypeptide models, are also discussed.

The resonance Raman spectra of bovine metarhodopsin I and metarhodopsin II have been measured. The spectra are compared with model chromophore resonance Raman data. It was found that metarhodopsin I is linked to opsin via a protonated Schiff base linkage, whereas metarhodopsin II is linked by an unprotonated Schiff base. A recent suggestion that the chromophore of metarhodopsin II is retinal is explicitly disproved. The chromophores of both metarhodopsins are found to have an essentially all-trans conformation. The basic mechanism for color regulation in both forms appears to be electron delocalization. The data tend to support the model of cis-trans isomerization as the primary mechanism for vision. Also, the conclusions and inferences of this work on energy uses and storage by rhodopsin in neural generation are discussed.