ABSTRACT
Protein structural plasticity is critical to many biological activities and accurate determination of its temporal and spatial fluctuations is challenging and difficult. Here, we report our extensive characterization of global flexibility of a globular heme protein of myoglobin using resonance energy transfer as a molecular ruler. With site-directed mutagenesis, we use a tryptophan scan to examine local structural fluctuations from B to H helices utilizing 10 tryptophan-heme energy transfer pairs with femtosecond resolution. We observed ultrafast resonance energy transfer dynamics by following a nearly single exponential behavior in 10-100 ps, strongly indicating that the globular structure of myoglobin is relatively rigid, with no observable static or slow dynamic conformational heterogeneity. The observation is against our molecular dynamics simulations, which show large local fluctuations and give multiple exponential energy transfer behaviors, suggesting too flexible of the global structure and thus raising a serious issue of the force fields used in simulations. Finally, these ultrafast energy transfer dynamics all occur on the similar time scales of local environmental relaxations (solvation), leading to nonexponential processes caused by energy relaxations, not structural fluctuations. Our analyses of such processes reveal an intrinsic compressed- and/or stretched-exponential behaviors and elucidate the nature of inherent nonequilibrium of ultrafast resonance energy transfer in proteins. This new concept of compressed nonequilibrium transfer dynamics should be applied to all protein studies by time-resolved Förster resonance energy transfer (FRET).
Subject(s)
Heme/chemistry , Myoglobin/chemistry , Tryptophan/chemistry , Fluorescence Resonance Energy Transfer , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Myoglobin/genetics , Pliability , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Solutions , Spectrometry, Fluorescence , Thermodynamics , Time Factors , Tryptophan/geneticsABSTRACT
Flavoproteins are unique redox coenzymes, and the dynamic solvation at their function sites is critical to the understanding of their electron-transfer properties. Here, we report our complete characterization of the function-site solvation of holoflavodoxin in three redox states and of the binding-site solvation of apoflavodoxin. Using intrinsic flavin cofactor and tryptophan residue as the local optical probes with two site-specific mutations, we observed distinct ultrafast solvation dynamics at the function site in the three states and at the related recognition site of the cofactor, ranging from a few to hundreds of picoseconds. The initial ultrafast motion in 1-2.6 ps reflects the local water-network relaxation around the shallow, solvent-exposed function site. The second relaxation in 20-40 ps results from the coupled local water-protein fluctuation. The third dynamics in hundreds of picoseconds is from the intrinsic fluctuation of the loose loops flanking the cofactor at the function site. These solvation dynamics with different amplitudes well correlate with the redox states from the oxidized form, to the more rigid semiquinone and to the much looser hydroquinone. This observation of the redox control of local protein conformation plasticity and water network flexibility is significant, and such an intimate relationship is essential to the biological function of interprotein electron transfer.
Subject(s)
Flavodoxin/chemistry , Thermodynamics , Crystallography, X-Ray , Flavodoxin/isolation & purification , Models, Molecular , Oxidation-Reduction , Solvents/chemistryABSTRACT
We report here our systematic characterization of resonance energy transfer between intrinsic tryptophan and the prosthetic heme group in myoglobin in order to develop a novel energy-transfer pair as a molecular ruler in heme proteins to study local conformation fluctuations. With site-directed mutagenesis, we designed four tryptophan mutants along the A-helix of myoglobin and each mutant contains only a single tryptophan-heme energy-transfer pair. With femtosecond resolution, we observed, even at separation distances of 15-25 A, ultrafast energy transfer in tens to hundreds of picoseconds. On these time scales, the donor and acceptor cannot be randomized and the orientation factor in Forster energy transfer is highly restricted. Thus, direct measurement of the orientation-factor changes at different mutation sites reveals relative local structure flexibility and conformation fluctuations as particularly demonstrated here for positions of tryptophan 7 and 14. More importantly, the local environment relaxation occurs on the similar time scales of the energy transfer dynamics, resulting in a nonequilibrium dynamic process. With femtosecond- and wavelength-resolved fluorescence dynamics, we are able to determine the time scales of such nonequilibrium energy-transfer dynamics and elucidate the mechanism of the nonexponential energy-transfer dynamics caused by local dynamic heterogeneity and/or local environment relaxation.
Subject(s)
Energy Transfer , Myoglobin/chemistry , Animals , Heme/chemistry , Kinetics , Molecular Dynamics Simulation , Movement , Mutation , Myoglobin/genetics , Myoglobin/metabolism , Protein Engineering , Protein Structure, Secondary , Quantum Theory , Spectrometry, Fluorescence , Temperature , TryptophanABSTRACT
We report here our systematic studies of the heme dynamics and induced protein conformational relaxations in two redox states of ferric and ferrous cytochrome c upon femtosecond excitation. With a wide range of probing wavelengths from the visible to the UV and a site-directed mutation we unambiguously determined that the protein dynamics in the two states are drastically different. For the ferrous state the heme transforms from 6-fold to 5-fold coordination with ultrafast ligand dissociation in less than 100 fs, followed by vibrational cooling within several picoseconds, but then recombining back to its original 6-fold coordination in 7 ps. Such impulsive bond breaking and late rebinding generate proteinquakes and strongly perturb the local heme site and shake global protein conformation, which were found to completely recover in 13 and 42 ps, respectively. For the ferric state the heme however maintains its 6-fold coordination. The dynamics mainly occur at the local site, including ultrafast internal conversion in hundreds of femtoseconds, vibrational cooling on the similar picosecond time scale, and complete ground-state recovery in 10 ps, and no global conformation relaxation was observed.
Subject(s)
Cytochromes c/chemistry , Animals , Ferric Compounds/chemistry , Ferrous Compounds/chemistry , Heme/chemistry , Horses , Models, Molecular , Mutagenesis, Site-Directed , Oxidation-Reduction , Phenylalanine/chemistry , Photolysis , Protein Conformation , Spectrophotometry, Ultraviolet , Thermodynamics , Tryptophan/chemistryABSTRACT
Making complicated data easier to understand has always been a challenge. Four types of visualization applications (CAD, generalized, specialized, and custom) have successfully been used by automotive manufacturers such as General Motors to help meet this goal. Here are some ways that common processes can be developed for all types of visualization.
