Proteins show diverse responses when placed under mechanical stress. The molecular origins of their differing mechanical resistance are still unclear, although the orientation of secondary structural elements relative to the applied force vector is thought to have an important function. Here, by using a method of protein immobilization that allows force to be applied to the same all-beta protein, E2lip3, in two different directions, we show that the energy landscape for mechanical unfolding is markedly anisotropic. These results, in combination with molecular dynamics (MD) simulations, reveal that the unfolding pathway depends on the pulling geometry and is associated with unfolding forces that differ by an order of magnitude. Thus, the mechanical resistance of a protein is not dictated solely by amino acid sequence, topology or unfolding rate constant, but depends critically on the direction of the applied extension.

beta-sheet proteins are generally more able to resist mechanical deformation than alpha-helical proteins. Experiments measuring the mechanical resistance of beta-sheet proteins extended by their termini led to the hypothesis that parallel, directly hydrogen-bonded terminal beta-strands provide the greatest mechanical strength. Here we test this hypothesis by measuring the mechanical properties of protein L, a domain with a topology predicted to be mechanically strong, but with no known mechanical function. A pentamer of this small, topologically simple protein is resistant to mechanical deformation over a wide range of extension rates. Molecular dynamics simulations show the energy landscape for protein L is highly restricted for mechanical unfolding and that this protein unfolds by the shearing apart of two structural units in a mechanism similar to that proposed for ubiquitin, which belongs to the same structural class as protein L, but unfolds at a significantly higher force. These data suggest that the mechanism of mechanical unfolding is conserved in proteins within the same fold family and demonstrate that although the topology and presence of a hydrogen-bonded clamp are of central importance in determining mechanical strength, hydrophobic interactions also play an important role in modulating the mechanical resistance of these similar proteins.

It is still unclear whether mechanical unfolding probes the same pathways as chemical denaturation. To address this point, we have constructed a concatamer of five mutant I27 domains (denoted (I27)(5)*) and used it for mechanical unfolding studies. This protein consists of four copies of the mutant C47S, C63S I27 and a single copy of C63S I27. These mutations severely destabilize I27 (DeltaDeltaG(UN) = 8.7 and 17.9 kJ mol(-1) for C63S I27 and C47S, C63S I27, respectively). Both mutations maintain the hydrogen bond network between the A' and G strands postulated to be the major region of mechanical resistance for I27. Measuring the speed dependence of the force required to unfold (I27)(5)* in triplicate using the atomic force microscope allowed a reliable assessment of the intrinsic unfolding rate constant of the protein to be obtained (2.0 x 10(-3) s(-1)). The rate constant of unfolding measured by chemical denaturation is over fivefold faster (1.1 x 10(-2) s(-1)), suggesting that these techniques probe different unfolding pathways. Also, by comparing the parameters obtained from the mechanical unfolding of a wild-type I27 concatamer with that of (I27)(5)*, we show that although the observed forces are considerably lower, core destabilization has little effect on determining the mechanical sensitivity of this domain.

1constant for internal return in 70% dioxane is estimated to be 40 times larger than k4. The fact that k4 is an apparent dissociation rate constant for an oriented complex held together by secondary valence forces makes it impossible to accurately compare k4 to the limiting values for internal return (k-,). The observation of a large isotope effect of 15% does not require that k 4 be much less than k-, since KirbyZo has observed similar effects of 5-16% (20) Craze, G.-A,; Kirby, A. J.; Osborne, R. J. Chem. Soc., Perkin Trans. 2 1978, 357.for SN2 displacement reactions involving formaldehyde methyl phenyl acetals. The large negative p of -4 that is observed in 70% dioxane suggests that if the reaction is SN2-like, the incoming and leaving groups are widely separated in space. This large amount of effective charge on the central carbon would be expected to have a significant effect on the zero-point energies for the two states under consideration, giving rise to the large isotope effect. If displacement reactions involving acetals commonly exhibit late, loose transition states such as this, large isotope effects such as those that are observed with glycosides would be anticipated. Abstract:The fluorescence decay kinetics of aqueous tryptophan and 3-methylindole have been determined as a function of pH and temperature by using a picosecond dye laser-single photon counting system with a time resolution of 50 ps. At pH 11, tryptophan exhibits a single exponential decay, with a lifetime of 9.1 ns at 18 O C . However, at pH 7 the decay is faster and definitely nonexponential; the values obtained from a biexponential fit to the data at pH 7 are T ] = 0.43 ns, 7 = 3.32 ns, a n d f = 0.19 at 18 O C . The behavior of a 3-methylindole closely resembles that of tryptophan at pH 11. A model for the photophysics of aqueous tryptophan is presented in which the excited-state decay constant at pH 11 (where the amino acid side chain is not protonated) is given by the superposition of three independent processes: fluorescence, intersystem crossing, and photoionization; of these processes only photoionization is temperature sensitive (E' = 51 kJ mol-'). In the region pH 4-8, where tryptophan exists in the zwitteridn form, a new nonradiative process is introduced, which involves intramolecular proton transfer from the -NH3+ group to the excited indole ring. The apparent activation energy for intramolecular quenching (E' = 16 kJ mol-') suggests that it is a predominantly diffusion-controlled process. It is proposed that the nonexponential decay observed for aqueous tryptophan at pH 7 arises from transient terms in the rate constant for intramolecular quenching. Quantum yields calculated from this model compare well with experimental values. IntroductionThe fluorescence of proteins is usually dominated by that of the tryptophan residues.' Both the fluorescence lifetime and quantum yield of a tryptophan residue are strongly influenced by the nature of its local environment, and this sensitivity is widely exploited through the use of tryp...

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