Electrostatic Interaction of Globins with Phospholipid Membranes

Biophysics - For the structures of sperm whale myoglobin (swMb), horse heart myoglobin (hhMb), hemoglobin I (HbI) from botfly Gasterophilus intestinalis (giHbI), and monomeric and dimeric...     

Transient spectroscopy of the reaction of cyanide with ferrous myoglobin. Effect of distal side residues

The reaction of cyanide metmyoglobin with dithionite conforms to a two-step sequential mechanism with formation of an unstable intermediate, identified as cyanide bound ferrous myoglobin. This reaction was investigated by stopped-flow time resolved spectroscopy using different myoglobins, i.e. those from horse heart, Aplysia limacina buccal muscle, and three recombinant derivatives of sperm whale skeletal muscle myoglobin (Mb) (the wild type and two mutants). The myoglobins from horse and sperm whale (wild type) have in the distal position (E7) a histidyl residue, which is missing in A. limacina Mb as well as the two sperm whale mutants (E7 His----Gly and E7 His----Val). All these proteins in the reduced form display an extremely low affinity for cyanide at pH less than 10. The differences in spectroscopy and kinetics of the ferrous cyanide complex of these myoglobins indicate a role of the distal pocket on the properties of the complex. The two mutants of sperm whale Mb are characterized by a rate constant for the decay of the unstable intermediate much faster than that of the wild type, at all pH values explored. Therefore, we envisage a specific role of the distal His (E7) in controlling the rate of cyanide dissociation and also find that this effect depends on the protonation of a single ionizable group, with pK = 7.2, attributed to the E7 imidazole ring. The results on A. limacina Mb, which displays the slowest rate of cyanide dissociation, suggests that a considerable stabilizing effect can be exerted by Arg E10 which, according to Bolognesi et al. (Bolognesi, M., Coda, A., Frigerio, F., Gatti, C., Ascenzi, P., and Brunori, M. (1990) J. Mol. Biol. 213, 621-625), interacts inside the pocket with fluoride bound to the ferric heme iron. A mechanism of control for the rate of dissociation of cyanide from ferrous myoglobin, involving protonation of the bound anion, is discussed.     

Identification of the myoglobin tyrosyl radical by immuno-spin trapping and its dimerization

5,5-Dimethyl-1-pyrroline N-oxide (DMPO) spin trapping in conjunction with antibodies specific for the DMPO nitrone epitope was used on hydrogen peroxide-treated sperm whale and horse heart myoglobins to determine the site of protein nitrone adduct formation. The present study demonstrates that the sperm whale myoglobin tyrosyl radical, formed by hydrogen peroxide-dependent self-peroxidation, can either react with another tyrosyl radical, resulting in a dityrosine cross-linkage, or react with the spin trap DMPO to form a diamagnetic nitrone adduct. The reaction of sperm whale myoglobin with equimolar hydrogen peroxide resulted in the formation of a myoglobin dimer detectable by electrophoresis/protein staining. Addition of DMPO resulted in the trapping of the globin radical, which was detected by Western blot. The location of this adduct was demonstrated to be at tyrosine-103 by MS/MS and site-specific mutagenicity. Interestingly, formation of the myoglobin dimer, which is known to be formed primarily by cross-linkage of tyrosine-151, was inhibited by the addition of DMPO.     

On the difference in stability between horse and sperm whale myoglobins

The work in the literature on apomyoglobin is almost equally divided between horse and sperm whale myoglobins. The two proteins share high homology, show similar folding behavior, and it is often assumed that all folding phenomena found with one protein will also be found with the other. We report data at equilibrium showing that horse myoglobin was 2.1 kcal/mol less stable than sperm whale myoglobin at pH 5.0, and aggregated at high concentrations as measured by gel filtration and analytical ultracentrifugation experiments. The higher stability of sperm whale myoglobin was identified for both apo and holo forms, and was independent of pH from 5 to 8 and of the presence of sodium chloride. We also show that the substitution of sperm whale myoglobin residues Ala15 and Ala74 to Gly, the residues found at positions 15 and 74 in horse myoglobin, decreased the stability by 1.0 kcal/mol, indicating that helix propensity is an important component of the explanation for the difference in stability between the two proteins.     

A comparative study on axial coordination and ligand binding in ferric mini myoglobin and horse heart myoglobin

The absorption and resonance Raman spectra and the azide binding kinetics of ferric horse heart myoglobin (Mb) and mini myoglobin (a chemically truncated form of horse heart Mb containing residues 32-139) have been compared. The steady-state spectra show that an additional six-coordinated low-spin form (not present in entire horse heart Mb, which is purely six-coordinated high spin) predominates in mini Mb. The distal histidine is possibly the sixth ligand in this species. The presence of two species corresponds to a kinetic biphasicity for mini Mb that is not observed for horse heart Mb. Azide binds to horse heart Mb much more slowly than to sperm whale Mb. This difference may result from a sterically hindered distal pocket in horse heart Mb. In both cases, the rate constants level off at high azide concentrations, implying the existence of a rate-limiting step (likely referable to the dissociation of the axial sixth ligand). The faster rate constant of mini Mb is similar to that of sperm whale Mb, whereas the slower one is similar to that of entire horse heart Mb.     

Decay Studies of Dmpo-Spin Adducts of Free Radicals Produced by Reactions of Metmyoglobin and Methemoglobin with Hydrogen Peroxide

The 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) spin adduct of myoglobin (Mb) or hemoglobin (Hb) was formed when metmyoglobin (MetMb) or methemoglobin (MetHb) reacted with H₂O₂ in the presence of DMPO, and both decayed with half-life of a few minutes. The DMPO spin adduct of Mb decayed with biphasic kinetics with k₁ = 0.645 min^-1 and k₂ = 0.012 min^-1, indicating that the spin adduct consisted of two kinetically heterogeneous species, stable and unstable ones. The DPMO spin adduct of Hb, however, was homogeneous. Decay of both spin adducts was accelerated in the presence of tyrosine, tryptophan or cysteine, but not phenylalanine, methionine or histidine. The decay obeyed the first order kinetics at varying concentrations of the spin adducts. The decay was accelerated by denaturation and proteolysis of protein moiety. The decay rate was not affected by the extra addition of MetMb or MetHb to each spin adduct. The decay rate of the spin adduct of Mb was increased by hematin in the presence of H₂O₂ and decreased by catalase. Decay of stable spin adduct of Mb, however, was not significantly changed under any experimental conditions used. These results led us to conclude that instability of the DMPO-spin adducts of Mb and Hb is due to intramolecular redox reactions between the spin adducts and amino acid residues and/or products of the reaction between heme and H₂O₂.     

Multiple conformational states in myoglobin revealed by frequency domain fluorometry

The tryptophanyl fluorescence decays of two myoglobins, i.e., sperm whale and tuna myoglobin, have been examined in the frequency domain with an apparatus which utilizes the harmonic content of a mode-locked laser. Data analysis was performed in terms of continuous distribution of lifetime having a Lorentzian shape. Data relative to sperm whale myoglobin, which possesses two tryptophanyl residues, i.e., Trp-A-5 and -A-12, provided a broad lifetime distribution including decay rates from a few picoseconds to about 10 ns. By contrast, the tryptophanyl lifetime distribution of tuna myoglobin, which contains only Trp-A-12, showed two well-separated and narrow Lorentzian components having centers at about 50 ps and 3.37 ns, respectively. In both cases, the chi 2 obtained from distribution analysis was lower than that provided by a fit using the sum of exponential components. The long-lived components present in the fluorescence decay of the two myoglobins do not correspond to any of those observed for the apoproteins at neutral pH. The tryptophanyl lifetime distribution of sperm whale apomyoglobin consists of two separated Lorentzian components centered at 2.25 and 5.4 ns, whereas that of tuna apomyoglobin consists of a single Lorentzian component, whose center is at 2.19 ns. Acidification of apomyoglobin to pH 3.5 produced a shift of the distribution centers toward longer lifetimes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative study of tyrosine radicals in hemoglobin and myoglobins treated with hydrogen peroxide

The reactions of hydrogen peroxide with human methemoglobin, sperm whale metmyoglobin, and horse heart metmyoglobin were studied by electron paramagnetic resonance (EPR) spectroscopy at 10 K and room temperature. The singlet EPR signal, one of the three signals seen in these systems at 10 K, is characterized by a poorly resolved, but still detectable, hyperfine structure that can be used to assign it to a tyrosyl radical. The singlet is detectable as a quintet at room temperature in methemoglobin with identical spectral features to those of the well characterized tyrosyl radical in photosystem II. Hyperfine splitting constants found for Tyr radicals were used to find the rotation angle of the phenoxyl group. Analysis of these angles in the crystal structures suggests that the radical resides on Tyr151 in sperm whale myoglobin, Tyr133 in soybean leghemoglobin, and either alphaTyr42, betaTyr35, or betaTyr130 in hemoglobin. In the sperm whale metmyoglobin Tyr103Phe mutant, there is no detectable tyrosyl radical present. Yet the rotation angle of Tyr103 (134 degrees) is too large to account for the observed EPR spectrum in the wild type. Tyr103 is the closest to the heme. We suggest that Tyr103 is the initial site of the radical, which then rapidly migrates to Tyr151.     

Unusually weak oxygen binding, physical properties, partial sequence, autoxidation rate and a potential phosphorylation site of beluga whale (Delphinapterus leucas) myoglobin

We purified myoglobin from beluga whale (Delphinapterus leucas) muscle (longissimus dorsi) with size exclusion and cation exchange chromatographies. The molecular mass was determined by mass spectrometry (17,081 Da) and the isoelectric pH (9.4) by capillary isoelectric focusing. The near-complete amino acid sequence was determined and a phylogeny indicated that beluga was in the same clad as Dall's and harbor porpoises. There were consensus motifs for a phosphorylation site on the protein surface with the most likely site at serine-117. This motif was common to all cetacean myoglobins examined. Two oxygen-binding studies at 37 degrees C indicated dissociation constants (20.5 and 23.6 microM) 5.7-6.6 times larger than horse myoglobin (3.6 microM). The autoxidation rate of beluga myoglobin at 37 degrees C, pH 7.2 was 0.218+/-0.028 h(-1), 1/3 larger than reported for myoglobin of terrestrial mammals. There was no clear sequence change to explain the difference in oxygen binding or autoxidation although substitutions (N66 and T67) in an invariant rich sequence (HGNTV) distal to the heme may play a role. Structural models based on the protein sequence and constructed on topologies of known templates (horse and sperm whale crystal structures) were not adequate to assess perturbation of the heme pocket.     

Titration behavior of individual tyrosine residues of myoglobins from sperm whale, horse, and red kangaroo.

The titration behavior of individual tyrosine residues of myoglobins has been studied by observing the pH dependence of the chemical shifts of Czeta and Cgamma of these residues in natural abundance of 13C Fourier transform NMR spectra (at 15.18 MHz, in 20-mm sample tubes, at 37 degrees) of cyanoferrimyoglobins from sperm whale, horse, and red kangaroo. A comparison of the pH dependence of the spectra of the three proteins yielded specific assignments for the resonance of Tyr-151 (sperm whale) and Tyr-103 (sperm whale and horse). Selective proton decoupling yielded specific assignments for Czeta of Tyr-146 of the cyanoferrimyoglobins from horse and kangaroo, but not the corresponding assignment for sperm whale. The pH dependence of the chemical shifts indicated that only Tyr-151 and Tyr-103 are titratable tyrosine residues. Even at pH 12, Tyr-146 did not begin to titrate. The titration behavior of C zeta and Cgamma of Tyr-151 of sperm whale cyanoferrimyoglobin yielded a single pK value of 10.6. The pH dependence of the chemical shift of each of the resonances of Tyr-103 of the cyanoferrimyoglobins from horse and sperm whale could not be fitted with the use of a single pK value, but was consistent with two pK values (about 9.8 and 11.6). Furthermore, the resonances of Czeta and Cgamma of Tyr-103 broadened at high pH. The titration behavior of the tyrosines of sperm whale carbon monoxide myoglobin and horse ferrimyoglobin was also examined. A comparison of all the experimental results indicated that Tyr-151 is exposed to solvent, Tyr-146 is not exposed, and Tyr-103 exhibits intermediate behavior. These results for myoglobins in solution are consistent with expectations based on the crystal structure.     

The myoglobin protein radical. Coupling of Tyr-103 to Tyr-151 in the H2O2-mediated cross-linking of sperm whale myoglobin

Sperm whale metmyoglobin, which has tyrosine residues at positions 103, 146, and 151, dimerizes in the presence of H2O2. Equine metmyoglobin, which lacks Tyr-151, and red kangaroo metmyoglobin, which lacks Tyr-103 and Tyr-151, do not dimerize in the presence of H2O2. The dityrosine content of the sperm whale myoglobin dimer shows that it is primarily held together by dityrosine cross-links, although more tyrosine residues are lost than are accounted for by dityrosine formation. Digestion of the myoglobin dimer with chymotrypsin yields a peptide with the fluorescence spectrum of dityrosine. The amino acid composition, amino acid sequence, and mass spectrum of the peptide show that cross-linking involves covalent bond formation between Tyr-103 of one myoglobin chain and Tyr-151 of the other. Replacement of the prosthetic group of sperm whale myoglobin with zinc protoporphyrin IX prevents H2O2-induced dimerization even when intact horse metmyoglobin is present in the incubation. This suggests that the tyrosine radicals required for the dimerization reaction are generated by intra- rather than intermolecular electron transfer to the ferryl heme. Rapid electron transfer from Tyr-103 to the ferryl heme followed by slower electron transfer from Tyr-151 to Tyr-103 is most consistent with the present results.     

Fluorescence decay kinetics of the tryptophyl residues of myoglobin: effect of heme ligation and evidence for discrete lifetime components

The fluorescence decay kinetics of the tryptophyl residues of sperm whale and yellowfin tuna myoglobin have been determined by using time-correlated single photon counting, with picosecond resolution. Purification by HPLC techniques resulted in the isolation of samples that exclusively displayed picosecond decay kinetics. Lifetimes of 24.4 ps for Trp14 and 122.0 ps for Trp7 were found for oxy sperm whale myoglobin (pH 7), which agree with theoretical predictions [Hochstrasser, R. M., & Negus, D. K. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4399-4403]. The effects of ligand binding and pH on the decay kinetics were investigated, and the results were shown to be consistent with the known crystal structures. Data for the met form of sperm whale myoglobin were analyzed both in terms of a sum of discrete exponential components and as a continuous gamma distribution of exponential decays. The results were not found to support the existence of multiple, structurally distinct conformation states in myoglobin.     

Binding of alkylisocyanides with soybean leghemoglobin. Comparisons with sperm whale myoglobin

The binding of various linear and branched chain alkylisocyanides to soybean leghemoglobin has been studied with respect to association and dissociation kinetics and the results compared with those obtained in parallel on sperm whale and horse heart myoglobins; the linear ligands used (methyl to n-heptyl) cover a greater distribution of chain lengths than hitherto used. The association rate constants are much higher for leghemoglobin than for myoglobin, while the dissociation rates are slower. For a given protein, the dissociation rate constants are not much different when different isocyanides are used (except for methyl), whereas the association rates show complex behavior in relation with the alkyl chain length; singular differences are observed between leghemoglobin and sperm whale myoglobin in this regard. For myoglobin, the binding rate constants decrease from methyl to n-propyl, but remain approximately the same when the ligand carries a still longer alkyl chain. In contrast, for leghemoglobin, although the rate constants decrease from methyl to n-propyl, they show a progressive and important rise with longer alkyl substituents: n-butyl and n-pentyl.     

Acid Bohr effects in myoglobin characterized by proton NMR hyperfine shifts and oxygen binding studies.

Proton NMR studies of sperm whale and horse deoxymyoglobin have revealed that both proteins exhibit a single, well defined, pH-induced structural change. The changes in hyperfine shifts are clearly observed not only at the heme peripheral substituents, but also at the proximal histidyl imidazole, which suggest that heme-apoprotein contacts are looser in the acidic than alkaline conformations. The hyperfine shift changes are modulated by a single titratable group with a pK of approx. 5.7 in both proteins. Oxygen binding studies of sperm whale myoglobin over a range of temperature and pH showed that, while the oxygen affinity was independent of pH at 25 degrees C, it increased below pH 7 at 0 degrees C and decreased below pH 7 at 37 degrees C. Hence, sperm whale myoglobin exhibits a small acid Bohr effect which most likely arises from the characterized structural changes in the deoxy proteins. While horse myoglobin failed to exhibit a resolvable acid Bohr effect between 0 and 37 degrees C, it did show a weak alkaline Bohr effect at 25 degrees C which disappeared at lower temperatures. Since the oxygen affinity changed smoothly over several pH units, this alkaline Bohr effect can not be associated with any well defined conformational change detected by NMR.     

Assignment of 1H NMR resonances of histidine and other aromatic residues in met-, cyano-, oxy-, and (carbon monoxy)myoglobins

The resolved 1H NMR resonances of the aromatic region in the 270-MHz NMR spectrum of sperm whale, horse, and pig metmyoglobin (metMb) have been assigned, including the observable H-2 and H-4 histidine resonances, the tryptophan H-2 resonances, and upfield-shifted resonances from one tyrosine residue. The use of different Mb species, carboxymethylation, and matching of pK values allows the assignment of the H-4 resonances, which agree in only three cases out of seven with scalar-correlated two-dimensional NMR spectroscopy assignments by others. The conversion to hydroxymyoglobin at high pH involves rearrangements throughout the molecule and is observed by many assigned residues. In sperm whale ferric cyanomyoglobin, nine H-2 and eight H-4 histidine resonances have been assigned, including the His-97 H-2 resonance and tyrosine resonances from residues 103 and 146. The hyperfine-shifted resonances from heme and near-heme protons observe a shift with a pK = 5.3 +/- 0.3 (probably due to deprotonation of His-97, pK = 5.6) and another shift at pK = 10.8 +/- 0.3. The spectrum of high-spin ferrous sperm whale deoxymyoglobin is very similar to that of metMb, which allows the assignment of seven surface histidine H-2 and H-4 resonances and also resonances from the two tryptophan residues and one tyrosine. In diamagnetic sperm whale (carbon monoxy)myoglobin (COMb), 10 His H-2 and 11 His H-4 resonances are observed, and 8 H-2 and 9 H-4 resonances are assigned, including His-64 H-4, the distal histidine. This important resonance is not observed in sperm whale oxymyoglobin, which in general shows very similar titration curves to COMb. Histidine-36 shows unusual titration behavior in the paramagnetic derivatives but normal behavior in the diamagnetic derivatives, which is discussed in the accompanying paper [Bradbury, J. H., & Carver, J. A. (1984) Biochemistry (following paper in this issue)].     

Studies of individual carbon sites of proteins in solution by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Strategies for assignments.

Natural abundance 13C Fourier transform NMR spectra (at 15.18 MHz, in 20-mm sample tubes) of aqueous native proteins yield numerous narrow single carbon resonances of nonprotonated aromatic carbons. Techniques for the assignment of these resonances are presented. Each technique is applied to one or more of the following proteins: ferricytochrome c from horse heart and Candida krusei, ferrocytochrome c and cyanoferricytochrome c from horse heart, lysozyme from hen egg white, cyanoferrimyoglobins from horse and sperm whale skeletal muscle, and carbon monoxide myoglobin from horse. In all of the protein spectra we have examined, methine aromatic carbons give rise to broad bands. Studies of the narrow resonances of nonprotonated aromatic carbons of proteins are facilitated by removal of these broad bands by means of the convolution-difference method, preferably from spectra recorded under conditions of noise-modulated off-resonance proton decoupling. We present a summary of the chemical shift ranges for the various types of nonprotonated aromatic carbons of amino acid residues and hemes of diamagnetic proteins, based on our results for hen egg white lysozyme, horse heart ferrocytochrome c, horse carbon monoxide myoglobin, and carbon monoxide hemoglobins from various species...     

Solution 1H NMR investigation of the seating and rotational "hopping" of centrosymmetric etioheme-I in myoglobin: effect of globin origin and its oxidation/spin state on heme dynamics

Solution 1H NMR spectroscopy was used to investigate the heme active-site structure and dynamics of rotation about the Fe-His bond of centrosymmetric etioheme-I reconstituted into sperm whale and horse myoglobin (Mb). Comparison of the NOESY cross-peak pattern and paramagnetic relaxation properties of the cyanomet complexes confirm a heme pocket that is essentially the same as Mb with either native protoheme or etioheme-I. Dipolar contacts between etioheme and the conserved heme pocket residues establish a unique seating of etioheme that conserves the orientation of the N-Fe-N vector relative to the axial His plane, with ethyl groups occupying the vinyl positions of protoheme. Saturation transfer between methyls on adjacent pyrroles in etioheme-reconstituted horse Mb in all accessible oxidation/spin states reveals rotational hopping rates that decrease dramatically with either loss of ligands or reduction of the heme, and correlate qualitatively with expectations based on the Fe-His bond strength and the rate of heme dissociation from Mb. The rate of hopping for etioheme in metMbCN, in contrast to hemes with propionates, is the same in the sperm whale and horse proteins.     

1H NMR resonance assignments in a paramagnetic heme protein by two-dimensional spectroscopy: heme resonances in equine met-azido myoglobin

Specific heme protons for the majority of resonances in the downfield resolved region of equine met-azido myoglobin have been assigned using solely the two-dimensional 1H NMR experiments NOESY and COSY. Metazido myoglobin provides a useful test case for the applicability of these techniques to paramagnetic proteins for the following reasons. First met-azido myoglobin is a mixed spin-state protein, with significantly shorter relaxation times and broadened lines relative to pure low-spin systems (eg., met-cyano myoglobin). Second, met-azido hemoglobin and met-azido myoglobin are important as models for the physiological forms of hemoglobin. Third, a few sperm whale met-azido myoglobin resonances have been previously assigned, which permits a comparison of assignments for these similar proteins, and a check of the method presented here.     

Spectral evidence for sub-picosecond iron displacement after ligand detachment from hemoproteins by femtosecond light pulses.

We have measured spectral and kinetic differences in protoheme, sperm whale or horse heart myoglobin and human hemoglobin following photodissociation induced by optical pulses of 80 fs duration. Full ligation was performed with oxygen or carbon monoxide. Femtosecond kinetics and transient difference spectra revealed the appearance of a deoxy species with tau approximately equal to 250-300 fs. The transient deoxy species in myoglobin and hemoglobin evidenced a 3-4 nm red shift of their delta A spectra compared with the equilibrium delta A spectrum. This shift was not observed after photodissociation of the carbon monoxide liganded protoheme. We proposed that the 250 fs time constant corresponding to the appearance of the deoxy-like species is related to the displacement of the ferrous iron out of the heme plane. Consequently, the small red shift of the delta A spectra observed in photodissociated hemoproteins may be tentatively attributed to changes in the vibrational modes of either the proximal histidine-Fe2+ bond and/or of the N4 porph-Fe-N epsilon His (F8) bent.     

Structure-dynamics-function relationships in Asian elephant (Elephas maximus) myoglobin. An optical spectroscopy and flash photolysis study on functionally important motions.

In this work we report the thermal behavior (10-300 K) of the Soret band lineshape of deoxy and carbonmonoxy derivatives of Asian elephant (Elephas maximus) and horse myoglobins together with their carbon monoxide recombination kinetics after flash photolysis; the results are compared to analogous data relative to sperm whale myoglobin. The Soret band profile is modeled as a Voigt function that accounts for the coupling with high and low frequency vibrational modes, while inhomogeneous broadening is taken into account with suitable distributions of purely electronic transition frequencies. This analysis makes it possible to isolate the various contributions to the overall lineshape that; in turn, give information on structural and dynamic properties of the systems studied. The optical spectroscopy data point out sizable differences between elephant myoglobin on one hand and horse and sperm whale myoglobins on the other. These differences, more pronounced in deoxy derivatives, involve both the structure and dynamics of the heme pocket; in particular, elephant myoglobin appears to be characterized by larger anharmonic contributions to soft modes than the other two proteins. Flash photolysis data are analyzed as sums of kinetic processes with temperature-dependent fractional amplitudes, characterized by discrete pre-exponentials and either discrete or distributed activation enthalpies. In the whole temperature range investigated the behavior of elephant myoglobin appears to be more complex than that of horse and sperm whale myoglobins, which is in agreement with the increased anharmonic contributions to soft modes found in the former protein. Thus, to satisfactorily fit the time courses for CO recombination to elephant myoglobin five distinct processes are needed, only one of which is populated over the whole temperature range investigated. The remarkable convergence and complementarity between optical spectroscopy and flash photolysis data confirms the utility of combining these two experimental techniques in order to gain new and deeper insights into the functional relevance of protein fluctuations.