Recent theories of protein folding suggest that individual proteins within a large ensemble may follow different routes in conformation space from the unfolded state toward the native state and vice versa. Herein, we introduce a new type of kinetics experiment that shows how different unfolding pathways can be selected by varying the initial reaction conditions. The relaxation kinetics of the major cold shock protein of Escherichia coli (CspA) in response to a laser-induced temperature jump are exponential for small temperature jumps, indicative of folding through a two-state mechanism. However, for larger jumps, the kinetics become strongly nonexponential, implying the existence of multiple unfolding pathways. We provide evidence that both unfolding across an energy barrier and diffusive downhill unfolding can occur simultaneously in the same ensemble and provide the experimental requirements for these to be observed.

Energy landscapesA simple system, such as the hydrogen atom, possesses a unique structure with a unique energy. A complex system, such as a cluster of many atoms or a biomolecule, behaves differently: The atoms can often arrange themselves into different conformations, which often have very similar energies. In an ensemble of such clusters or biomolecules, many different conformations can therefore occur simultaneously. The different conformations have the same chemical formula, but different structures and energies. Each particular conformation can at any instant of time be described by the coordinates of all atoms. The energy landscape is defined as the potential energy of the system as a function of all coordinates. Different conformations, called conformational substates, are usually separated by an energy barrier. The energy landscape is important not only because it characterizes the different possible structures, but also because it determines the dynamics of the system. This means that the structural degrees of freedom of the system are determined by the structure of its energy landscape (for instance, the number of barriers and their heights) just like the difficulty of a hike depends on the structure of the landscape in three dimensions. Often it is useful to make analogies between energy landscapes and ordinary landscapes. Compare skiing in Illinois or The Netherlands to skiing in Colorado or Switzerland. Similar comparisons can be made between the energy landscapes of different systems, for instance a cluster and a protein.The complexity of the energy landscape rapidly increases with the number of atoms and the interactions between them. Consider two extreme situations, the ammonia molecule and proteins. In the ammonia molecule, NH 3 , the four atoms form a pyramid and the nitrogen atom can be on one side or the other of the plane formed by the three hydrogen atoms. The molecule consequently has two substates that are structurally different, but are related by mirror symmetry and have the same energy. The existence of the two substates forms the basis for the ammonia maser, a device for generating coherent microwave radiation 2 . The two ammonia substates can be represented by the simple energy landscape shown in Fig. 1a. Native proteins, in contrast, can assume a very large number of slightly different conformations and their energy landscape is very complicated 3,4 . A highly simplified protein energy landscape is shown in Fig. 1b. Each valley, or conformational substate, represents a particular arrangement of the atoms of the protein.The different conformational substates are separated by barriers and a typical protein energy landscape is characterized by a wide distribution of barrier heights. The extreme complexity of the energy landscape of a folded and working protein is revealed by both experiments and computer simulations. The results of a simulation of the structural dynamics within the folded state of a small protein, crambin, consisting of 46 amino acids, is shown in Fig. 2 in the form of ...

In this paper we demonstrate how the potential energy surface of a protein, which determines its conformational degrees of freedom, can be constructed from a series of advanced nonlinear optical experiments. The energy landscape of myoglobin was probed by studying its low-temperature structural dynamics, using several spectral hole burning and photon echo techniques. The spectral diffusion of the heme group of the protein was studied on a time scale ranging from nanoseconds to several days while covering a temperature range from 100 mK to 23 K. The spectral line broadening, as measured in three-pulse stimulated photon echo experiments, occurs in a stepwise fashion, while the exact time dependence of the line width is critically dependent on temperature. From these results we obtained the energy barriers between the conformational states of the protein. Aging time dependent hole-burning experiments show that, at 100 mK, it takes several days for the protein to reach thermal equilibrium. When, after this period a spectral hole is burned, the line broadening induced by welldefined temperature cycles is partly reversed over a period of several hours. From this we conclude that a rough structure is superimposed on the overall shape of the potential energy surface of the protein. By combining the hole burning and photon echo results, we construct a detailed image of this energy landscape, supporting the general concept of a structural hierarchy. More specifically, we show that the number of conformational substates in the lower hierarchical tiers is much lower than was previously anticipated and, in fact, is comparable to the number of taxonomic substates.

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