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How Fast Is Protein Hydrophobic Collapse?

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How Fast Is Protein Hydrophobic Collapse? How fast is protein hydrophobic collapse? Mourad Sadqi*, Lisa J. Lapidus†‡, and Victor Mun˜ oz*§ *Department of Chemistry and Biochemistry and Center for Biomolecular Structure and Organization, University of Maryland, College Park, MD 20742; and †Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 Edited by Michael Levitt, Stanford University School of Medicine, Stanford, CA, and approved August 18, 2003 (received for review June 21, 2003) One of the most recurring questions in protein folding refers to the Equilibrium FRET Experiments. Equilibrium FRET experiments interplay between formation of secondary structure and hydro- were performed at 25 ␮M concentration in citrate buffer at pH phobic collapse. In contrast with secondary structure, it is hard to 3.0 in an Applied Photophysics (Surrey, U.K.) PiStar instrument. isolate hydrophobic collapse from other folding events. We have Efficiency of energy transfer between naphtyl-alanine (donor) directly measured the dynamics of protein hydrophobic collapse in and dansyl-lysine (acceptor) was determined from the fluores- the absence of competing processes. Collapse was triggered with cence quantum yield of the donor by using the equation: laser-induced temperature jumps in the acid-denatured form of a Qda simple protein and monitored by fluorescence resonance energy E ϭ 1 Ϫ . [1] transfer between probes placed at the protein ends. The relaxation Qd time for hydrophobic collapse is only Ϸ60 ns at 305 K, even faster than secondary structure formation. At higher temperatures, as the The intrinsic quantum yield of the donor (Qd) was measured by protein becomes increasingly compact by a stronger hydrophobic exciting at 288 nm in the protein labeled at the N terminus with naphtyl-alanine and nonlabeled at the C terminus and using force, we observe a slowdown of the dynamics of collapse. This ϭ dynamic hydrophobic effect is a high-temperature analogue of the N-acetyl-trytophanamide (Q 0.13 at pH 7.0 and 298 K) as dynamic glass transition predicted by theory. Our results indicate reference. The quantum yield in the presence of acceptor (Qda) that in physiological conditions many proteins will initiate folding was measured in doubly labeled variant of the protein following the same procedure. Protein concentration was determined by by collapsing to an unstructured globule. Local motions will pre- absorbance. To minimize possible errors in concentration caused sumably drive the following search for native structure in the by the large pH sensitivity of dansyl’s absorbance, we determined collapsed globule. the concentration of both proteins at the maximum for naph- thylalanine (280 nm) and at the isosbestic point for the changes uring the folding reaction a protein must form its native in absorbance of dansyl as a function of pH (266 nm). The molar Dsecondary structure and collapse into a globular state. But, extinction coefficients of the two fluorophores at pH 3.0 at the ␧nafthyl ϭ Ϫ1⅐ Ϫ1 ␧nafthyl ϭ how do these two dynamic processes interlace with each other two wavelengths ( 266 3,595 M cm , 280 5,526 ␣ ␤ Ϫ1⅐ Ϫ1 ␧dansyl ϭ Ϫ1⅐ Ϫ1 ␧dansyl ϭ Ϫ1⅐ Ϫ1 (1)? The intrinsic time scales for -helix (2) and -hairpin (3) M cm , 266 4,528 M cm , 280 6,517 M cm ) formation have been measured in short peptides that form these were determined in our laboratory from the absorbance values secondary structure elements without the protein context. The of standard solutions of the free fluorophores. We used the dynamics of homopolymer collapse has been the subject of average of the values obtained with the two procedures, which intensive theoretical (4–6) and computational analysis (7). In agreed within 5%. The discrepancy between the FRET effi- proteins, hydrophobic collapse has been studied by using a ciency for BBL at pH 3.0 obtained here and that reported variety of computer simulations in different simplified protein previously (23) is caused by a previous slight overestimation of models (8–13). However, the experimental investigation of the molar extinction coefficient of dansyl at pH 3.0. This does not hydrophobic collapse has proven difficult. Most of what we know affect the previous data at pH 7.0. FRET efficiency as a function experimentally about protein reconfiguration dynamics arises of temperature was fitted to a Gaussian chain model by adjusting from detection of early phases in folding or from measurement the effective bond length at each temperature to reproduce the of rates of contact formation in unfolded polypeptides (14). How observed FRET. In these calculations we used an experimentally these observations relate to the dynamics of hydrophobic col- determined R0 of 1.7 nm for the naphthyl–dansyl pair at pH 3.0. lapse is unclear, because such experiments are either performed The low R0 at this pH is caused by the shift of the dansyl in competition with other folding events (15–21) or with no absorption band to the blue, which results in smaller spectral driving force for collapse because of the presence of chemical overlap with naphthyl’s fluorescence. The quantum yield of denaturants (22). The ‘‘ideal’’ experiment to measure directly naphthyl is almost temperature independent, so R0 can be BIOPHYSICS the dynamics of hydrophobic collapse could be summarized in assumed constant with temperature. To calculate the end-to-end four requirements: (i) an archetypal protein that can be fully distance from FRET efficiency we have used an orientation ␬2 ͞ denatured with no chemical denaturants, (ii) a spectroscopic factor ( )of23, assuming conditions of isotropic dynamic probe to monitor molecular dimensions, (iii) a simple strategy averaging. These conditions are typically met when at least one for tuning the degree of collapse, and (iv) a technique to trigger of the two fluorophores is freely rotating at rates faster than the fluorescence lifetime. This is probably the case for both fluoro- changes in collapse with sufficient time resolution. phores at pH 3.0, as indicated by their invariant quantum yield Here we study a small acid-denatured protein that collapses as upon incorporation to the protein. Furthermore, ␬2 ϭ 2͞3isa temperature increases. We trigger collapse in nanoseconds by reasonable assumption even for fixed fluorophores if the accep- using a laser-induced temperature jump (T jump) instrument. The subsequent relaxation dynamics are inferred from the time-dependent changes in the efficiency of fluorescence This paper was submitted directly (Track II) to the PNAS office. resonance energy transfer (FRET) between two terminal Abbreviations: FRET, fluorescence resonance energy transfer; T jump, temperature jump; fluorophores. SVD, singular value decomposition. ‡Present address: Physics Department, Stanford University, 382 Via Pueblo Mall, Stanford, Materials and Methods CA 94305-4060. NMR Experiments. NMR experiments were carried out in a Bruker §To whom correspondence should be addressed. E-mail: [email protected]. 500-MHz instrument as described (23). © 2003 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.2033863100 PNAS ͉ October 14, 2003 ͉ vol. 100 ͉ no. 21 ͉ 12117–12122 Downloaded by guest on September 27, 2021 tor has multiple absorption transition dipoles, and therefore mixed polarizations, such as the dansyl moiety. The spectrum and quantum yield of dansyl are very sensitive to the polariz- ability of its chemical environment (23). The fact that at pH 3.0 these properties remain unchanged at all temperatures for the dansyl group attached to the protein indicates that there are no interactions between the two fluorophores or between dansyl and other hydrophobic residues of the denatured protein. Laser-Induced T-Jump Experiments. The laser-induced T-jump instrument used here is based on the general principles as described (24). There are two major differences between our instrument and previous ones. The first one is the use of an optical parametric oscillator to shift the fundamental of the heating Q-switched Nd-YAG laser to a frequency that overlaps with the first vibrational overtone of water (i.e., 1,570 nm). The second difference is that our instrument uses another Q- switched Nd-YAG laser to excite the donor. The fundamental of this laser is frequency quadrupled to 266 nm, and then shifted to 288 nm by first Stokes stimulated Raman emission in a methane containing Raman cell. Excitation with light of 288 nm minimizes the photodamage of the fluorophores. Total fluorescence is collected at 90° and then imaged into a spectrograph and a charge-coupled device camera, providing complete fluorescence spectra with nanosecond resolution. The spectral resolution of this instrument is extremely useful to check for transient spectral changes in the fluorophores, which would indicate the presence of transient interactions Fig. 1. 1D proton NMR in BBL. (A) Detail of the aliphatic region of the NMR occurring during the time scale of the kinetic experiment. No spectra of BBL at pH 3.0 and three temperatures: 273 K (blue), 303 K (green), spectral changes were observed in any of the T-jump and 333 K (red). (B) Detail of the aliphatic region of BBL at pH 5.3 and the same experiments of acid-denatured BBL. A complete layout of three temperatures: 273 K (blue), 303 K (green), and 333 K (red). The 3D the instrument and a more detailed description of its features structure of BBL was originally determined at pH 5.3 and 293 K (26). The are available in Supporting Text and Figs. 5–7, which are absence of ring-current effects because of the lack of aromatic residues in the core of BBL results in a less spread out aliphatic region of the proton NMR published as supporting information on the PNAS web site, spectrum. Nevertheless, at pH 5.3 significant changes in the chemical shifts of www.pnas.org.
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