The solution structure and conformational dynamics of tumor necrosis factor-alpha and a (Cys69----Asp; Cys101----Arg) analog as examined by IR spectroscopy and hydrogen exchange.

An analog of human tumor necrosis factor-alpha (TNF-alpha) was created with Cys69 and Cys101 replaced with Asp and Arg respectively. We have undertaken a comparative study of the solution conformation and dynamics of the native and analog molecules using a combination of Fourier transform IR spectroscopy and hydrogen-deuterium (H-D) exchange kinetics. IR spectroscopic results indicate that the analog molecule adopts a gross structure similar to that of the native molecule but significant differences in the conformation of the beta-sheets are observed. Increased bandwidths observed for several of the amide I components also suggest a less rigid structure for the analog molecule. Further, by monitoring the frequency shifts of the individual amide I component bands as a function of hydrogen exchange, we have enhanced our ability to assign these components to individual protein secondary structures, particularly the high frequency beta-strand mode. Hydrogen exchange kinetic studies indicate that the Asp-Arg analog adopts a looser, more flexible solution structure relative to the natural sequence molecule.     

Hydrophobic core variant ubiquitin forms a molten globule conformation at acidic pH

The conformational properties of hydrophobic core variant ubiquitin (Val26 to Ala mutation) in an acidic solution were studied. The intrinsic tryptophan fluorescence emission spectrum, far-UV and near-UV circular dichroic spectra, the fluorescence emission spectrum of 8-anilinonaphthalene-1-sulfonic acid in the presence of V26A ubiquitin, and urea-induced unfolding measurements indicate this variant ubiquitin to be in the partially folded molten globule conformation in solution at pH 2. The folding kinetics from molten globule to the native state was nearly identical to those from the unfolded state to the native state. This observation suggests that the equilibrium molten globule state of hydrophobic core variant ubiquitin is an on-pathway folding intermediate.     

Molecular basis for the polymerization of octopus lens S-crystallin

S-Crystallin from octopus lens has a tertiary structure similar to sigma-class glutathione transferase (GST). However, after isolation from the lenses, S-crystallin was found to aggregate more easily than sigma-GST. In vitro experiments showed that the lens S-crystallin can be polymerized and finally denatured at increasing concentration of urea or guanidinium chloride (GdmCl). In the intermediate concentrations of urea or GdmCl, the polymerized form of S-crystallin is aggregated, as manifested by the increase in light scattering and precipitation of the protein. There is a delay time for the initiation of polymerization. Both the delay time and rate of polymerization depend on the protein concentration. The native protein showed a maximum fluorescence emission spectrum at 341 nm. The GdmCl-denatured protein exhibited two fluorescence maxima at 310 nm and 358 nm, respectively, whereas the urea-denatured protein showed a fluorescence peak at 358 nm with a small peak at 310 nm. The fluorescence intensity was quenched. Monomers, dimers, trimers, and polymers of the native protein were observed by negative-stain electron microscopic analysis. The aggregated form, however, showed irregular structure. The aggregate was solubilized in high concentrations of urea or GdmCl. The redissolved denatured protein showed an identical fluorescence spectrum to the protein solution that was directly denatured with high concentrations of urea or GdmCl. The denatured protein was readily refolded to its native state by diluting with buffer solution. The fluorescence spectrum of the renatured protein solution was similar to that of the native form. The phase diagrams for the S-crystallin in urea and GdmCl were constructed. Both salt concentration and pH value of the solution affect the polymerization rate, suggesting the participation of ionic interactions in the polymerization. Comparison of the molecular models of the S-crystallin and sigma-GST suggests that an extra ion-pair between Asp-101 and Arg-14 in S-crystallin contributes to stabilizing the protomer. Furthermore, the molecular surface of S-crystallin has a protruding Lys-208 on one side and a complementary patch of aspartate residues (Asp-90, Asp-94, Asp-101, Asp-102, Asp-179, and Asp-180) on the other side. We propose a molecular model for the S-crystallin polymer in vivo, which involves side-by-side associations of Lys-208 from one protomer and the aspartate patch from another protomer that allows the formation of a polymeric structure spontaneously into a liquid crystal structure in the lens.     

Illuminating the Off-Pathway Nature of the Molten Globule Folding Intermediate of an α-β Parallel Protein

Partially folded protein species transiently form during folding of most proteins. Often, these species are molten globules, which may be on- or off-pathway to the native state. Molten globules are ensembles of interconverting protein conformers that have a substantial amount of secondary structure, but lack virtually all tertiary side-chain packing characteristics of natively folded proteins. Due to solvent-exposed hydrophobic groups, molten globules are prone to aggregation, which can have detrimental effects on organisms. The molten globule observed during folding of the 179-residue apoflavodoxin from Azotobacter vinelandii is off-pathway, as it has to unfold before native protein can form. Here, we study folding of apoflavodoxin and characterize its molten globule using fluorescence spectroscopy and Förster Resonance Energy Transfer (FRET). Apoflavodoxin is site-specifically labeled with fluorescent donor and acceptor dyes, utilizing dye-inaccessibility of Cys69 in cofactor-bound protein. Donor (i.e., Alexa Fluor 488) is covalently attached to Cys69 in all apoflavodoxin variants used. Acceptor (i.e., Alexa Fluor 568) is coupled to Cys1, Cys131 and Cys178, respectively. Our FRET data show that apoflavodoxin’s molten globule forms in a non-cooperative manner and that its N-terminal 69 residues fold last. In addition, striking conformational differences between molten globule and native protein are revealed, because the inter-label distances sampled in the 111-residue C-terminal segment of the molten globule are shorter than observed for native apoflavodoxin. Thus, FRET sheds light on the off-pathway nature of the molten globule during folding of an α-β parallel protein.     

Two-State Folding of Lysozyme Versus Multiple-State Folding of α-Lactalbumin Illustrated by the Technique of Disulfide Scrambling

The folding of lysozyme and of alpha-lactalbumin exhibits vastly different kinetics and pathways. Existing evidence indicates that folding intermediates of alphaLA form a well-populated equilibrium molten globule state that is absent in the case of hen lysozyme. We demonstrate here such divergent folding mechanisms of lysozyme and alphaLA using the technique of disulfide scrambling. Two extensively unfolded homologous isomers (beads-form) of lysozyme (Cys6-Cys30, Cys64-Cys76, Cys80-Cys94, Cys115-Cys127) and alphaLA (Cys6-Cys28, Cys61-Cys73, Cys77-Cys91, Cys111-Cys120) were allowed to refold in parallel to form the native protein. Folding kinetics was measured by the recovery of the native structure. Folding intermediates, which illustrate the folding pathway, were trapped by quenching disulfide shuffling and were analyzed by reversed-phase high-pressure liquid chromatography. The results revealed that under identical folding conditions, the folding rate of lysozyme is about 30-fold faster than that of alphaLA. Folding intermediates of lysozyme are far less heterogeneous and sparsely populated than those of alphaLA. Numerous predominant on-pathway and off-pathway intermediates observed along the folding pathway of alphaLA are conspicuously absent in the case of lysozyme. The difference is most striking under fast folding conditions performed in the presence of protein disulfide isomerase. Under these conditions, folding of lysozyme undergoes a near two-state mechanism without accumulation of stable folding intermediates.     

Electron Paramagnetic Resonance and Fluorescence Studies of the Conformation of Aspartate Aminotransferase Bound to GroEL

The interaction of the precursor to mitochondrial aspartate aminotransferase (pmAAT) with GroEL has been studied by electron paramagnetic resonance (EPR) and fluorescence spectroscopy. In the native protein, the spin probe was immobilized when attached to Cys166 at the domain interface, but was fully mobile when introduced at Cys(−19) in the N-terminal presequence peptide. Unfolding of the protein resulted in a highly mobile EPR spectrum for probes introduced at either site. However, the nitroxide group in GroEL-bound pmAAT showed either intermediate or high mobility depending on the spin probe used. Power saturation experiments indicated that the accessibility of the nitroxide side chain to Ni(EDDA) in the GroEL–pmAAT complex was higher than in the native state when in position 166 but lower when at position −19. Similar results were obtained in fluorescence quenching experiments. These data suggest that GroEL binds partly folded states of pmAAT with the presequence peptide probably in direct contact with GroEL. GroES and ATP, but not AMP–PNP or ADP, support refolding of pmAAT. During refolding, the rate of recovery of the native spectroscopic properties of labeled Cys166 is nearly identical to the rate-limiting reactivation step. Thus, correct docking of the large and small domains of pmAAT may be a key structural event in the regain of catalytic activity.     

Effect of an alternative disulfide bond on the structure, stability, and folding of human lysozyme

Human lysozyme has four disulfide bonds, one of which, Cys65-Cys81, is included in a long loop of the beta-domain. A cysteine-scanning mutagenesis in which the position of Cys65 was shifted within a continuous segment from positions 61 to 67, with fixed Cys81, has previously shown that only the mutant W64CC65A, which has a nonnative Cys64-Cys81 disulfide, can be correctly folded and secreted by yeast. Here, using the W64CC65A mutant, we investigated the effects of an alternative disulfide bond on the structure, stability, and folding of human lysozyme using circular dichroism (CD) and fluorescence spectroscopy combined with a stopped-flow technique. Although the mutant is expected to have a different main-chain structure from that of the wild-type protein around the loop region, far- and near-UV CD spectra show that the native state of the mutant has tightly packed side chains and secondary structure similar to that of the wild-type. Guanidine hydrochloride-induced equilibrium unfolding transition of the mutant is reversible, showing high stability and cooperativity of folding. In the kinetic folding reaction, both proteins accumulate a similar burst-phase intermediate having pronounced secondary structure within the dead time of the measurement and fold into the native structure by means of a similar folding mechanism. Both the kinetic refolding and unfolding reactions of the mutant protein are faster than those of the wild-type, but the increase in the unfolding rate is larger than that of the refolding rate. The Gibbs' free-energy diagrams obtained from the kinetic analysis suggest that the structure around the loop region in the beta-domain of human lysozyme is formed after the transition state of folding, and thus, the effect of the alternative disulfide bond on the structure, stability, and folding of human lysozyme appears mainly in the native state.     

Two-dimensional 1H nuclear magnetic resonance study of the (5-55) single-disulphide folding intermediate of bovine pancreatic trypsin inhibitor

An analogue of the bovine pancreatic trypsin inhibitor (BPTI) folding intermediate that contains only the disulphide bond between Cys5 and Cys55 has been prepared in Escherichia coli by protein engineering methods, with the other four Cys residues replaced by Ser. Two-dimensional 1H nuclear magnetic resonance studies of the analogue have resulted in essentially complete resonance assignments of the folded form of the protein. The folded protein has a compact conformation that is structurally very similar to that of native BPTI, although there are subtle differences and the folded conformation is not very stable. Approximately half of the protein molecules are unfolded at 3 degrees C, and this proportion increases at higher temperatures. The folded and unfolded conformations are in slow exchange. The conformational properties of the analogue can explain many aspects of the kinetic role that the normal (5-55) intermediate plays in the folding of BPTI.     

Compact molten globule-like state of hUBF HMG Box1 at extremely low pH

Using far and near-UV CD, ANS fluorescence and 2D NMR spectroscopy, an acid-induced partly folded state (A state) at extremely low pH for hUBF HMG Box1 was identified and characterized. As compared to the native state (N), the A state has similar secondary structure, less compact pack with larger amounts of exposed hydrophobic surface, and narrower chemical shift dispersion in (1)H-(15)N HSQC spectrum, which implies that it is a molten globule (MG)-like species. On the other hand, substantial tertiary contacts and cooperative thermal denaturing transition indicate that the A state is closer-relative to the classic MG-to the native folded state. In addition, when the solution pH is adjusted to neutrality, the protein in the A state refolds to the native state easily. All these data suggest that the A state of hUBF HMG Box1 could represent a potential folding intermediate on protein folding pathway.     

Intrinsic tryptophans of CRABPI as probes of structure and folding.

The native state fluorescence and CD spectra of the predominantly beta-sheet cellular retinoic acid-binding protein I (CRABPI) include contributions from its three tryptophan residues and are influenced by the positions of these residues in the three-dimensional structure. Using a combination of spectroscopic approaches and single Trp-mutants of CRABPI, we have deconvoluted these spectra and uncovered several features that have aided in our analysis of the development of structure in the folding pathway of CRABPI. The emission spectrum of native CRABPI is dominated by Trp 7. Trp 109 is fluorescence-silent due to its interaction with the guanidino group of Arg 111. Although the far-UV CD spectrum of CRABPI is largely determined by the protein's secondary structure, aromatic clustering around Trp 87 and the aromatic-charge interaction between Arg 111 and Trp 109 give rise to a characteristic feature in the CD spectrum at 228 nm. The near-UV CD bands of CRABPI arise largely from additive contributions of the three tryptophan residues. Trp 7 and Trp 87 give a negative CD band at 275 nm. The near-UV CD band from Trp 109 is positive and shifted to longer wavelengths (to 302 nm) due to the charge-aromatic interaction between Arg 111 and Trp 109. Our deconvolution of the equilibrium spectra have been used to interpret kinetic folding experiments monitored by stopped-flow fluorescence. These dynamic experiments suggest the early evolution of a well-populated, hydrophobically collapsed intermediate, which undergoes global rearrangement to form the fully folded structure. The results presented here suggest several additional strategies for dissecting the folding pathway of CRABPI.     

Conformational restrictions on the pathway of folding and unfolding of the pancreatic trypsin inhibitor

The kinetic roles of the partially folded, intermediate protein species with two disulphide bonds in folding and unfolding of the pancreatic trypsin inhibitor have been investigated further. Formation of a second disulphide bond between Cys5 and Cys55 during refolding of the reduced inhibitor, which would yield the species with the 30–51 and 5–55 disulphide bonds and, possibly, the native-like conformation of the protein, is not significant. Instead, three other second disulphide bonds (5–14, 5–38 and 14–38) are formed approximately 10⁵ times more readily, but each of these two-disulphide species then rearranges intramolecularly to the native-like, two-disulphide intermediate. Therefore, the reduced protein does not simply form sequentially the three disulphide bonds of the native state. Unfolding of the native state takes place by the reverse of this process.The kinetic importance for folding and unfolding of this transition between two-disulphide intermediates under the conditions used here was illustrated experimentally by a modified form of the inhibitor in which the thiols of Cys14 and Cys38 were blocked irreversibly. In the folded conformation, this modified protein is more stable to unfolding than normal, but after unfolding cannot readily regain the native-like conformation, because Cys14 or Cys38 are required to be involved in disulphide bonds during the interconversion of the two-disulphide intermediates.Some conception of the conformational transitions that take place at each stage of the folding transition may be inferred from the relative propensities of the six cysteine residues to make or rearrange disulphide bonds. It is concluded that the inhibitor probably does not refold by sequential adoption of the native conformation by the unfolded polypeptide chain. Instead, it appears that essentially all elements of the native conformation are attained simultaneously in the final stage of folding, within an unstable and flexible, yet relatively compact, form of the entire polypeptide chain produced by weak interactions between groups distant in the primary structure.     

Reversible denaturation of Aequorea green-fluorescent protein: physical separation and characterization of the renatured protein

The green-fluorescent protein (GFP) that functions as a bioluminescence energy transfer acceptor in the jellyfish Aequorea has been renatured with up to 90% yield following acid, base, or guanidine denaturation. Renaturation, following pH neutralization or simple dilution of guanidine, proceeds with a half-recovery time of less than 5 min as measured by the return of visible fluorescence. Residual unrenatured protein has been quantitatively removed by chromatography on Sephadex G-75. The chromatographed, renatured GFP has corrected fluorescence excitation and emission spectra identical with those of the native protein at pH 7.0 (excitation lambda max = 398 nm; emission lambda max = 508 nm) and also at pH 12.2 (excitation lambda max = 476 nm; emission lambda max = 505 nm). With its peak position red-shifted 78 nm at pH 12.2, the Aequorea GFP excitation spectrum more closely resembles the excitation spectra of Renilla (sea pansy) and Phialidium (hydromedusan) GFPs at neutral pH. Visible absorption spectra of the native and renatured Aequorea green-fluorescent proteins at pH 7.0 are also identical, suggesting that the chromophore binding site has returned to its native state. Small differences in far-UV absorption and circular dichroism spectra, however, indicate that the renatured protein has not fully regained its native secondary structure.     

Conformational dynamics of the GdmHCl-induced molten globule state of creatine kinase monitored by hydrogen exchange and mass spectrometry

Our understanding of the mechanism of protein folding can be improved by the characterization of folding intermediate states. Intrinsic tryptophan fluorescence measurements of equilibrium GdmHCl-induced unfolding of MM-CK allow for the construction of a "phase diagram", which shows the presence of five different conformational states, including three partially folded intermediates. However, only three states are detected by using pulsed-labeled H-D exchange analyzed by electrospray ionization mass spectrometry. One of them is the native state, and the two other species are present in proportions strongly dependent on the GdmHCl concentration and denaturation time. The low-mass peak is due to a largely exchange-incompetent state, which has gained only approximately 10 deuteriums more than the native protein. This population of MM-CK molecules has undergone a small conformational change induced by low GdmHCl concentrations. However, this limited change is in itself not sufficient to inactivate the enzyme or is easily reversible. The high-mass peak corresponds to a population of MM-CK that is fully deuterated. The comparison of fluorescence, activity, and H-D exchange measurements shows that the maximally populated intermediate at 0.8 M GdmHCl has the characteristics of a molten globule. It has no activity; it has 55% of its native alpha-helices and a maximum fluorescence emission wavelength of approximately 341 nm, and it binds ANS strongly. However, no protection against exchange is detected under the conditions used in this work. This paradox, the presence of significant residual secondary and tertiary structures detected by optical probes and the total deuteration of its amide protons detected by H-D exchange and mass spectrometry, could be explained by a highly dynamic MM-CK molten globule.     

Dissecting a role of evolutionary-conserved but noncritical disulfide bridges in cysteine-rich peptides using ω-conotoxin GVIA and its selenocysteine analogs

Conotoxins comprise a large group of peptidic neurotoxins that use diverse disulfide-rich scaffolds. Each scaffold is determined by an evolutionarily conserved pattern of cysteine residues. Although many structure-activity relationship studies confirm the functional and structural importance of disulfide crosslinks, there is growing evidence that not all disulfide bridges are critical in maintaining activities of conotoxins. To answer the fundamental biological question of what the role of noncritical disulfide bridges is, we investigated function and folding of disulfide-depleted analogs of ω-conotoxin GVIA (GVIA) that belongs to an inhibitory cystine knot motif family and blocks N-type calcium channels. Removal of a noncritical Cys1-Cys16 disulfide bridge in GVIA or its selenopeptide analog had, as predicted, rather minimal effects on the inhibitory activity on calcium channels, as well as on in vivo activity following intracranial administration. However, the disulfide-depleted GVIA exhibited significantly lower folding yields for forming the remaining two native disulfide bridges. The disulfide-depleted selenoconotoxin GVIA analog also folded with significantly lower yields, suggesting that the functionally noncritical disulfide pair plays an important cooperative role in forming the native disulfide scaffold. Taken together, our results suggest that distinct disulfide bridges may be evolutionarily preserved by the oxidative folding or/and stabilization of the bioactive conformation of a disulfide-rich scaffold.     

A Two-step Mechanism for the Folding of Actin by the Yeast Cytosolic Chaperonin

Actin requires the chaperonin containing TCP1 (CCT), a hexadecameric ATPase essential for cell viability in eukaryotes, to fold to its native state. Following binding of unfolded actin to CCT, the cavity of the chaperone closes and actin is folded and released in an ATP-dependent folding cycle. In yeast, CCT forms a ternary complex with the phosducin-like protein PLP2p to fold actin, and together they can return nascent or chemically denatured actin to its native state in a pure in vitro folding assay. The complexity of the CCT-actin system makes the study of the actin folding mechanism technically challenging. We have established a novel spectroscopic assay through selectively labeling the C terminus of yeast actin with acrylodan and observe significant changes in the acrylodan fluorescence emission spectrum as actin is chemically unfolded and then refolded by the chaperonin. The variation in the polarity of the environment surrounding the fluorescent probe during the unfolding/folding processes has allowed us to monitor actin as it folds on CCT. The rate of actin folding at a range of temperatures and ATP concentrations has been determined for both wild type CCT and a mutant CCT, CCT4anc2, defective in folding actin in vivo. Binding of the non-hydrolysable ATP analog adenosine 5′-(β,γ-imino)triphosphate to the ternary complex leads to 3-fold faster release of actin from CCT following addition of ATP, suggesting a two-step folding process with a conformational change occurring upon closure of the cavity and a subsequent final folding step involving packing of the C terminus to the native-like state.     

Fluorescence energy transfer indicates similar transient and equilibrium intermediates in staphylococcal nuclease folding

Fluorescence resonance energy transfer (FRET) is one of the few methods available to measure the rate at which a folding protein collapses. Using staphylococcal nuclease in which a cysteine residue was engineered in place of Lys64, permitted FRET measurements of the distance between the donor tryptophan 140 and 5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1-sulfonic acid-labeled Cys64. These measurements were undertaken on both equilibrium partially folded intermediates at low pH (A states), as well as transient intermediates during stopped-flow refolding. The results indicate that there is an initial collapse of the protein in the deadtime of the stopped-flow instrument, corresponding to a regain of approximately 60% of the native signal, followed by three slower transients. This is in contrast to circular dichroism measurements which show only 20-25% regain of the native secondary structure in the burst phase. Thus hydrophobic collapse precedes the formation of substantial secondary structure. The first two detected transient intermediate species have FRET properties essentially identical with those of the previously characterized equilibrium A state intermediates, suggesting similar structures between the equilibrium and transient intermediates.The effects of anions on the folding of acid-unfolded staphylococcal nuclease, and urea on the unfolding of the resulting A states, indicates that in folding the protein becomes compact prior to formation of major secondary structure, whereas in unfolding the protein expands prior to major loss of secondary structure. Comparison of the kinetics of refolding of staphylococcal nuclease, monitored by FRET, and for a proline-free variant, indicate that folding occurs via two partially folded intermediates leading to a native-like species with one (or more) proline residues in a non-native conformation. For the A states an excellent correlation between compactness measured by FRET, and compactness determined from small-angle X-ray scattering, was observed. Further, a linear relationship between compactness and free energy of unfolding was noted. Formation of soluble aggregates of the A states led to dramatic enhancement of the FRET, consistent with intermolecular fluorescence energy transfer.     

The acid-induced folded state of Sac7d is the native state

Sac7d unfolds at low pH in the absence of salt, with the greatest extent of unfolding obtained at pH 2. We have previously shown that the acid unfolded protein is induced to refold by decreasing the pH to 0 or by addition of salt (McCrary BS, Bedell J. Edmondson SP, Shriver JW, 1998, J Mol Biol 276:203-224). Both near-ultraviolet circular dichroism spectra and ANS fluorescence enhancements indicate that the acid- and salt-induced folded states have a native fold and are not molten globular. 1H,15N heteronuclear single quantum coherence NMR spectra confirm that the native, acid-, and salt-induced folded states are essentially identical. The most significant differences in amide 1H and 15N chemical shifts are attributed to hydrogen bonding to titrating carboxyl side chains and through-bond inductive effects. The 1H NMR chemical shifts of protons affected by ring currents in the hydrophobic core of the acid- and salt-induced folded states are identical to those observed in the native. The radius of gyration of the acid-induced folded state at pH 0 is shown to be identical to that of the native state at pH 7 by small angle X-ray scattering. We conclude that acid-induced collapse of Sac7d does not lead to a molten globule but proceeds directly to the native state. The folding of Sac7d as a function of pH and anion concentration is summarized with a phase diagram that is similar to those observed for other proteins that undergo acid-induced folding except that the A-state is encompassed by the native state. These results demonstrate that formation of a molten globule is not a general property of proteins that are refolded by acid.     

Energetic coupling between native-state prolyl isomerization and conformational protein folding

In folded proteins, prolyl peptide bonds are usually thought to be either trans or cis because only one of the isomers can be accommodated in the native folded protein. For the N-terminal domain of the gene-3 protein of the filamentous phage fd (N2 domain), Pro161 resides at the tip of a beta hairpin and was found to be cis in the crystal structure of this protein. Here we show that Pro161 exists in both the cis and the trans conformations in the folded form of the N2 domain. We investigated how conformational folding and prolyl isomerization are coupled in the unfolding and refolding of N2 domain. A combination of single-mixing and double-mixing unfolding and refolding experiments showed that, in unfolded N2 domain, 7% of the molecules contain a cis-Pro161 and 93% of the molecules contain a trans-Pro161. During refolding, the fraction of molecules with a cis-Pro161 increases to 85%. This implies that 10.3 kJ mol(-1) of the folding free energy was used to drive this 75-fold change in the Pro161 cis/trans equilibrium constant during folding. The stabilities of the forms with the cis and the trans isomers of Pro161 and their folding kinetics could be determined separately because their conformational folding is much faster than the prolyl isomerization reactions in the native and the unfolded proteins. The energetic coupling between conformational folding and Pro161 isomerization is already fully established in the transition state of folding, and the two isomeric forms are thus truly native forms. The folding kinetics are well described by a four-species box model, in which the N2 molecules with either isomer of Pro161 can fold to the native state and in which cis/trans isomerization occurs in both the unfolded and the folded proteins.     

Concatenation of cyan and yellow fluorescent proteins for efficient resonance energy transfer

Highly efficient fluorescence resonance energy transfer between cyan(CFP) and yellow fluorescent proteins (YFP), the cyan- and yellow-emitting variants of the Aequorea green fluorescent protein, respectively, was achieved by tightly concatenating the two proteins. After the C-terminus of CFP and the N-terminus of YFP were truncated by 11 and 5 amino acids, respectively, the proteins were fused through a leucine-glutamate dipeptide. The resulting chimeric protein, which we called Cy11.5, exhibited a simple emission spectrum that peaked at 527 nm when the protein was excited at 436 nm. The time-resolved emission of Cy11.5 was measured using a streak camera. After excitation of Cy11.5 with a 400 nm ultrashort pulse, a fast decay of the CFP emission and a concomitant rise of the YFP emission were observed with a lifetime of 66 ps. By contrast, the emission from CFP alone showed a decay component with a lifetime of 2.9 ns. We concluded that in fully folded Cy11.5 molecules, intramolecular FRET occurred with an efficiency of 98%. Importantly, most Cy11.5 molecules were properly folded, and the protein was highly resistant to all of the tested proteases. In living cells, therefore, Cy11.5 behaved as a single fluorescent protein with a broad excitation spectrum. Moreover, Cy11.5 was used as an optical highlighter after photobleaching of YFP. When HeLa cells expressing Cy11.5 were irradiated at 514.5 nm, a 10-fold increase in the 475 nm fluorescence intensity was observed. These features make Cy11.5 useful as an optical highlighter and a new-colored fluorescent protein for multicolor imaging.