The mechanisms by which a protein searches vast conformational space to reach its native, functional folded state have not yet been resolved experimentally.  In particular, the critical early stages leading the disordered structure of the unfolded polypeptide to the establishment of a compact fold are still poorly characterized.
One of the major impedments has been the use of conventional rapid-mixing techniques, which impose a limit to the shortest observable times to about 1 ms.  Mixing-initiated refolding generally leads to a fast, sub-millisecond burst phase during which much of the secondary structure is formed.  The kinetic details of this nano- to micro-second step are essentially inaccessible to traditional relaxation techniques.
Recently, the development of modern laser T-jump instrumentation, coupled with various optical detection, has allowed to study the response to a fast increase in the temperature of several model systems (Walters, Paula et al. 1995; Muņoz, Thompson et al. 1997; Thompson, Eaton et al. 1997) as well as proteins (Phillips, Mizutani et al. 1995; Ballew, Sabelko et al. 1996; Gilmanshin, Williams et al. 1997) on the nano- to microsecond time scale.  Using the fluorescence emission of the N-terminal probe 4-(methylamino)benzoic acid Thompson et al. (Thompson, Eaton et al. 1997) characterized the kinetics of the helix-coil transition of a 21-residue Alanine peptide.  The interpretation of the results with a "kinetic zipper" model allows the identification of two exponential relaxations with comparable amplitudes and a temperature dependence exhibiting a maximum of about 20 ns near the midpont of the melting transition.  The faster relaxation corresponds to the unzipping (and zipping) of the helix ends in response to the temperature jump, whereas the slower phase is associated with the equilibration of helix-containing and non-helix-containing structures by passage over the nucleation free energy barrier.  The decay of the average helix content is dominated by the slower process.  This last rate is in agreement with the data reported by Williams et al. (Williams, Causgrove et al. 1996) who monitored the helix melting by infrared transient absorption in the amide I region and found for this process an apparent lifetime of about 160 ns.
Temperature induced unfolding of a hairpin was investigated by Munoz et al. (Muņoz, Thompson et al. 1997) by means of the fluorescence emission of the single tryptophan in the C-terminal fragment (41-56) of protein G B1.  The rate constants for folding and unfolding were determined at the midpoint of the melting transition and found 1/6 ms-1 , substantially slower than the case of the helix-coil transition.
Besides model compounds studies, only a few applications to proteins have been so far reported.  Phillips et al. investigated the response of RNase A to a laser T-jump on the picosecond time scale by monitoring the infrared transient absorption in the amide I band.  The authors found that the disruption of the ?-sheet structure doesn't occur before 1 ns after the T-jump and that about 15 % of the change in structure observed at equilibrium is achieved 5.5 ns after the perturbation.
The thermally induced folding of apomyoglobin has ben been studied by Ballew et al. (Ballew, Sabelko et al. 1996; Ballew, Sabelko et al. 1996) by monitoring the fluorescence emission of tryptophan and by Gilmanshin et al. (Gilmanshin, Williams et al. 1997) by measuring the amide I transient absorption.  Infrared absorption shows that temperature induced refolding of apomyoglobin is characterized by a complex behaviour with a fast step (48 ns) followed by a much slower phase (132 ms).  On the other hand, tryptophan fluorescence emission also shows a biphasic behaviour, but the rate constants appear completely different ( 250 ns and 3.5 ms).

Ultrafast perturbations of the protein stability can be obtained also by means of laser-induced pH jump, a methodology in which an aqueous solution containing a suitable photolabile caged compound (either proton or hydroxide) is flashed with a nanosecond UV laser (Gutman and Nachliel 1990 714).  In a series of works, Gutman and coworkers have investigated a number of proton transfer reactions, also involving biological macromolecules and supramolecular assemblies, using aromatic alcohols or eterosubstituted compounds and pulsed UV lasers to induce either a pH or a pOH jump.  Although the technique was developed already during the 80’s, no applications to the problem of the protein folding have been reported so far.

In this work we have used a nanosecond UV laser and photolabile caged protons to perturb the native state of apoMb monitoring the structural response of the protein by means of time resolved photoacoustics (Braslavsky and Heibel 1992).  We have recently applied this experimental methodology to follow proton transfer reactions in aqueous solutions, characterizing the solvation of photoinduced charges (Small and Kurian 1995; Bonetti, Vecli et al. 1997; Abbruzzetti, Viappiani et al. 1998; Losi and Viappiani 1998), the formation of water molecules from proton and hydroxide (Bonetti, Vecli et al. 1997; Abbruzzetti, Viappiani et al. 1998) and the reaction with poly-L-lyisine (Abbruzzetti, Viappiani et al. 1998).

Apomyoglobin (apoMb), which is prepared by removing the heme group from myoglobin, adopts an intermediate (I) conformation at pH 4 that has been the subject of numerous structural, thermodynamic, kinetic and theoretical studies (Barrick, Hughson et al. 1994; Privalov 1996).
The holoprotein contains eight strands of mostly a-helical segments, labeled A-H.  According to the available NMR, CD and calorimetric evidence, near neutral pH apoMb adopts a structure that is similar to the one of the native holoprotein (Hughson, Wright et al. 1990; Barrick and Baldwin 1993; Cocco and Lecomte 1994; Johnson and Walsh 1994; Privalov 1996).  This structure is characterized by a compact, hydrophobic core, consisting of at least the very stable A, G and H helices, with roughly the same secondary structure content and tertiary fold as myoglobin.  Although A, G and H helices form a distinct compact subdomain in the holoprotein, when isolated as fragments these helices are unstable (Barrick and Baldwin 1993).
ApoMb I shows decreased helix content and lacks the tight side-chain packing characteristic of globular protein cores.
Extensive studies of the kinetics of apoMb folding using conventional stopped flow initiation methods, SAXS and hydrogen-deuterium pulsed labeling (Jennings and Wright 1993; Eliezer, Jennings et al. 1995) have revealed the rapid, submillisecond development of a compact acid intermediate on the kinetic folding pathway.  Pulsed hydrogen exchange experiments (Jennings and Wright 1993) identified an early folding intermediate with a very similar pattern of amide NH protection to that seen for the equilibrium intermediate (Hughson, Wright et al. 1990), suggesting that the equilibrium intermediate is also a kinetic folding intermediate.
Besides being induced by acidification of the solution, the intermediate I of apoMb is observed also at high concentrations of denaturing agents as urea (Jennings and Wright 1993; Barrick, Hughson et al. 1994) and GuHCl.
The diffusion of a proton in the heme binding site of apoMb was investigated by Gutman and coworkers using the caged compound pyranine bound to this hydrophobic pocket and inducing the proton transfer reaction with a pulsed UV laser (Shimoni, Nachliel et al. 1993).  The reactions were monitored by means of transient absorption and time resolved fluorescence.
However, to our knowledge, this is the first application of the laser pH jump technique with time resolved photoacoustics detection to the apoMb unfolding problem and, more generally, to the protein folding problem.

References