Dynamic Nonradiative Excitation Energy Transfer Research Articles

Fast Collapse but Slow Formation of Secondary Structure Elements in the Refolding Transition of E. coli Adenylate Kinase

The various models proposed for protein folding transition differ in their order of appearance of the basic steps during this process. In this study, steady state and time-resolved dynamic non-radiative excitation energy transfer (FRET and trFRET) combined with site specific labeling experiments were applied in order to characterize the initial transient ensemble of Escherichia coli adenylate kinase (AK) molecules upon shifting conditions from those favoring denaturation to refolding and from folding to denaturing. Three sets of labeled AK mutants were prepared, which were designed to probe the equilibrium and transient distributions of intramolecular segmental end-to-end distances. A 176 residue section (residues 28-203), which spans most of the 214 residue molecule, and two short secondary structure chain segments including an alpha-helix (residues 169-188) and a predominantly beta-strand region (residues 188-203), were labeled. Upon fast change of conditions from denaturing to folding, the end-to-end distance of the 176 residue chain section showed an immediate collapse to a mean value of 26 A. Under the same conditions, the two short secondary structure elements did not respond to this shift within the first ten milliseconds, and retained the characteristics of a fully unfolded state. Within the first 10 ms after changes of the solvent from folding to denaturing, only minor changes were observed at the local environments of residues 203 and 169. The response of these same local environments to the shift of conditions from denaturing to folding occurred within the dead time of the mixing device. Thus, the response of the CORE domain of AK to fast transfer from folding to unfolding conditions is slow at all three conformational levels that were probed, and for at least a few milliseconds the ensemble of folded molecules is maintained under unfolding conditions. A different order of the changes was observed upon initiation of refolding. The AK molecules undergo fast collapse to an ensemble of compact structures where the local environment of surface probes seems to be native-like but the two labeled secondary structure elements remain unfolded.

Applications of time-resolved resonance energy transfer measurements in studies of the molecular crowding effect.

The native structures of many globular proteins are only weakly stabilized and form in solution ensembles of multiple conformers. The energy differences between the conformers are assumed to be small. This is the case of flexible multidomain proteins where domain motions were observed. High concentrations of inert macrosolute, which create a crowded or confined environment, can cause shifts of the distribution of the conformers of such proteins towards the more compact structures. This effect may also promote compact structures in partially folded proteins. Time-resolved dynamic non-radiative excitation energy transfer (tr-RET) is suitable for detection of either subtle or major changes in distributions of intramolecular distances in protein molecules in solutions. Two experiments were performed which demonstrated the applicability of tr-RET for detection of the effect of macrosolutes on the conformational ensembles of flexible states of protein molecules. The distribution of distances between residues 203 and 169 in the CORE domain of E. coli adenylate kinase (AK) in the denatured state was determined in the presence of high concentrations of dextran 40. A significant shift of the mean of the distribution was observed without reduction of its width. This was interpreted as a shift to compact structure without change of the degree of disorder of the chain. In a second experiment the distribution of the distance between residues 55 and 169 in AK, which spans the cleft between the CORE and the AMPbind domains, was monitored. No clear effect of high concentrations of dextran 40 was found. These experiments show the strength of the application of tr-RET in investigation of changes in the sub-states of flexible conformations of globular protein. Networks of pairs of labeled sites can be prepared and tr-RET experiments can be performed in order to search for the segments of the protein molecules, which respond to the presence of inert macromolecules in their environment.

Distributions of intramolecular distances in the reduced and denatured states of bovine pancreatic ribonuclease A. Folding initiation structures in the C-terminal portions of the reduced protein.

The purpose of this investigation is to characterize the reduced state of RNase A (r-RNase A) in terms of (i) intramolecular distances, (ii) the sequence of formation of stable loops in the initial stages of folding, and (iii) the unfolding transitions induced by GdnHCl. This is accomplished by identifying specific subdomain structures and local and long-range interactions that direct the folding process of this protein and lead to the native fold and formation of the disulfide bonds. Eleven pairs of dispersed sites in the RNase A molecule were labeled with fluorescent donor and acceptor probes, and the distributions of intramolecular distances (IDDs) were determined by means of time-resolved dynamic nonradiative excitation energy transfer (TR-FRET) measurements. The mutants were designed to search for (a) a possible nonrandom fold of the backbone in the collapsed state and (b) possible loops stabilized by long-range interactions. It was found that, under folding conditions, (i) the labeled mutants of r-RNase A in refolding buffer (the R(N) state) exhibit features of specific (nonrandom) compact but very dispersed subdomain structures (indicated by short mean distances, broad IDDs, and a weak dependence of the mean distances on segment length), (ii) the backbone fold in the C-terminal beta-like portion of the molecule appears to adopt a native-like overall fold, (iii) the N-terminal alpha-like portion of the chain is separated from the C-terminal core by very large intramolecular distances, larger than those in the crystal structure, and (iv) perturbations by addition of GdnHCl reveal several conformational transitions in different sections of the chain. Addition of GdnHCl to the native disulfide-intact protein provided a reference state for the extent of expansion of intramolecular distances under denaturing conditions. In conclusion, r-RNase A under folding conditions (the R(N) state) is poised for the final folding step(s) with a native-like trace of the chain fold but a large separation between the two subdomains which is then decreased upon introduction of three of the four native disulfide cross-links.

Determination of intramolecular distance distribution during protein folding on the millisecond timescale

A method for determination of transient (on the millisecond timescale) intramolecular distance distributions (IDDs) by time-resolved dynamic non-radiative excitation energy transfer measurements was developed. The time-course of the development of the IDD between residues 73 and 203 in the CORE domain of Escherichia coli adenylate kinase throughout refolding from the GuHCl-induced denatured state was determined. The mean of the apparent IDD reduced to a value close to its magnitude in the native protein, within 2 ms (the dead-time of the instrument). At that time the width of that distribution was rather large (16 ± 2 Å). The large width implies that the intramolecular diffusion coefficient of the labeled segment does not exceed 10 −7 cm 2/second. In a second slower phase of the refolding transition, the width was reduced to its native value (6 ± 4 Å).

The problem of protein folding and dynamics: time-resolved dynamic nonradiative excitation energy transfer measurements

The proteins are molecular machines that are self-assembled infinitely diverse, give rise to complex structures of any size, and have well-controlled vectorial modes of conformational changes, driven by thermal motions. Numerous experiments show that the pathway of folding of globular proteins, from the un-ordered state to the native conformations, and their modes of intramolecular motions, are determined by the sequence of the amino acyl residues (the monomers) in the polypeptide chain (the genetic information) of each protein. Attempts to decipher the mechanism by which the multiple transitions, on a wide range of time and distance scales are directed, depend on development of new methods for determination of distributions of intermolecular distances in flexible molecules and their time dependence. Methods based on time resolved measurements of intramolecular dynamic nonradiative excitation energy transfer, combined with protein engineering and site directed labeling were developed. Global analysis of data sets obtained by these measurements yield distributions of probabilities of segments end-to-end distances (EEDP) (in the range of 8 to 80 W chain). Methods for AMP determination of rates of conformational fluctuations and of the folding transitions, down to picosecond time scale, by means of this approach were also developed. Application of this experimental approach in the study of the denatured state of single-domain globular proteins, show that even in the denatured states, these proteins have compact structures. The bovine pancreatic trypsin inhibitor (BPTI) showed in the denatured state at least two conformational subpopulations, one was native-like and the other was characterized by EEDP distributions corresponding to an unfolded polypeptide chain.

Domain closure in adenylate kinase.

The method of time-resolved dynamic nonradiative excitation energy transfer (ET) was used to analyze the proposed domain closure in adenylate kinase (AK). A highly active mutant of Escherichia coli AK, (C77S, V169W, A55C)-AK, was prepared, in which the solvent- accessible residues valine 169 and alanine 55 were replaced by tryptophan (the donor of excitation energy) and cysteine, respectively. The latter was subsequently labeled with either 5- or 4-acetamidosalicylic acid (the acceptor). From the comparative analysis of AK crystal structures [Schulz, G.E., Müller, C.W., & Diederichs, K. (1990) J. Mol. Biol. 213, 627-630] (apo-AK,AK.AMP complex and AK.AP5A [P1,P5-di(adenosine-5') pentaphosphate] complex), "sequential formation" of the pseudoternary AK.AP5A complex is followed by two- step domain closure. The domain closure reduces interdomain distances in a two-step manner. Specifically, the distance between C alpha-atoms of the residues 169 and 55 (numbers correspond to those of E. coli AK) is decreased from 23.6 A in the apo-enzyme to 16.2 A upon the formation of the AK.AMP complex and to 12.3 A upon the further formation of the pseudoternary AK.AP5A complex. Time-resolved dynamic nonradiative excitation energy transfer was measured for the following ligand forms of the labeled derivative of the mutant enzyme: the apo-enzyme, the enzyme-MgATP complex, the enzyme.AMP complex, and the enzyme.AP5A "ternary" complex. The transfer efficiencies, which were determined in these experiments, were approximately 7.5%, 22%, 33%, and 65%, respectively. Global analyses of the time resolved ET experiments with the same ligand forms yielded intermolecular distance distributions with corresponding means of 31, 23, 19, and 12 A and full widths at half- maximum of 29, 24, 14, and 11 A. The data confirmed the proposed stepwise manner of the domain closure of the enzyme and revealed the presence of multiple conformations of E. coli AK in solution.

Nonlocal interactions stabilize long range loops in the initial folding intermediates of reduced bovine pancreatic trypsin inhibitor.

A search for the topology of the chain folding of reduced bovine pancreatic trypsin inhibitor (BPTI), in unfolded and partially folded states, was done by means of time resolved dynamic nonradiative excitation energy transfer (ET) measurements. Four double labeled BPTI derivatives were used in which the donor was attached to the N-terminal arginine residue and the acceptor was specifically attached to one of the lysine residues. The four derivatives form a series of labeled backbone segments of increasing length spanning the full lengths of the BPTI chain: 15, 26, 41, and 46 residues. The intramolecular segmental end-to-end distance (EED) distributions were determined for all the derivatives by global analysis of the decay curves of both the donor and the acceptor in the reduced state, in low (0.5 M) guanidinium chloride (GuHCl) concentrations at pH 3.6 and 2.1 (R and A states, respectively). The results show that, in the partial folding conditions of low GuHCl concentration, reduced BPTI is in a compact state, but in this state the polypeptide chain is not in a condensed statistical coil conformation. Two distinct subpopulations were found for the four intramolecular EED distributions. One subpopulation was compact, with native-like EED distribution, while the second was unfolded. The pairs of sites, residues 1 and 26 and residues 1 and 46, showed close proximity in the dominant subpopulation. These contacts form two loops (probably collapsed): one consists of the first 26 residues, and the second comprises the full length of the chain from the N- to the C-terminal segments, which is in fact made up to two shorter loops (1-26 and 27-46). The N-terminal 15 residue segment was relaxed into statistical coil-like non-native conformation, in contrast to its extended conformation in the native state. The effect of temperature in the range of 2-60 degrees C was small; the folded subpopulations were stable over this range. These results show that in BPTI the compact conformations found under unfolding and partially folding conditions have native-like chain topology. Under the conditions of transition to partially folding conditions the compact conformation is stabilized, not only by the hydrophobic collapse and the local interaction but also by nonlocal interactions (NLIs). Few specific, very stable NLIs between the three segments which form the main structural elements of the native conformation direct the formation of native-like topology of the chain in the transition.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparation and Characterization of Three Fluorescent Labels for Proteins, Suitable for Structural Studies

Three 7-aminocoumarin derivatives, 7-carboxymethyl-amino-4-trifluoromethyl-2 H-1-benzopyran-2-one [2], 7-carboxymethylamino-4-methyl-2 H-1-benzopyran-k2-one [4], and 7-dimethylamino-4-formyl-2 H-1-benzopyran-2-one [6], were prepared. The reagents can be attached to primary amino groups of proteins. The labeled derivatives are stable and are characterized by a strong absorption band in the near uv (300-400 nm) and an emission band in the blue region (440-510 nm) with a large Stokes′ shift. The spectral characteristics of these probes make them useful in applications of dynamic nonradiative excitation energy transfer measurements for conformational studies and analytical procedures based on simultaneous detection of labeled components in mixtures.

Detection of local structures in reduced unfolded bovine pancreatic trypsin inhibitor.

The structure of BPTI and reduced BPTI in concentrated guanidinium HCl (GUHCl) in the presence of glycerol has been probed by measurements of dynamic nonradiative excitation energy transfer between probes attached to its amino groups. Interprobe distance distributions were obtained from analysis of donor fluorescence decay curves and used to characterize local structures in unordered states of the protein. Site specifically fluorescently labeled BPTI derivatives (1-n)BPTI (n = 15, 20, 41, 46) were used, each carrying a 2-methoxy-naphthyl-1-methylenyl group (MNA) at the N-terminal amino group of arg1 and 7-(dimethylamino)-coumarin-4-yl-acetyl residue (DA-coum) at one of its epsilon-NH2 groups of the lysine side chains. Analysis of donor fluorescence decay kinetics gave the interprobe distance distributions in the native and denatured states. The N-terminal-segment, residues 1-15, is in an extended conformation (with an average interprobe distance of 34 +/- 2 A) in the native state. Upon unfolding by reduction with DTT or beta-mercapto ethanol in 6 M GUHCl/glycerol mixture, the conformation of this segment relaxed to a state characterized by a reduced average interprobe distance and a larger width of the distances distribution. The average distance between residues 1 and 26, i.e., between the N-terminus and the turn of the twisted beta sheet element (residues 18-35), increased upon unfolding. At -30 degrees C in the above solvent, the distribution between these two sites was probably composed of two conformational subpopulations. About 45 +/- 20% of the molecules were characterized by a short interprobe distance (like the native state) representing a compact conformation, and 55 +/- 20% of the molecules showed large interprobe distances representing an expanded (unfolded) conformation. Thus local structures seem to exist in reduced denatured BPTI even under denaturing conditions in 6 M GUHCl/glycerol mixtures. Some of those structures are unstable in guanidinium isothiocyanate (GUSCN). The method introduced here is suitable for probing local structures and very long range interactions in unfolded proteins and for search for folding initiation sites (FISs) and early folding intermediates.