Multistep chemical reactions are increasingly seen as important in a growing number of complex biotransformations. Covalently attached prosthetic groups or swinging arms, and their associated protein domains, are essential to the mechanisms of active-site coupling and substrate channeling in a number of the multifunctional enzyme systems responsible. The protein domains, for which the posttranslational machinery in the cell is highly specific, are crucially important, contributing to the processes of molecular recognition that define and protect the substrates and the catalytic intermediates. The domains have novel folds and move by virtue of conformationally flexible linker regions that tether them to other components of their respective multienzyme complexes. Structural and mechanistic imperatives are becoming apparent as the assembly pathways and the coupling of multistep reactions catalyzed by these dauntingly complex molecular machines are unraveled.

Swinging Arms and Swinging Domains in Multifunctional Enzymes: Catalytic Machines for Multistep Reactions

Biological water at the interface of proteins is critical to their equilibrium structures and enzyme function and to phenomena such as molecular recognition and protein-protein interactions. To actually probe the dynamics of water structure at the surface, we must examine the protein itself, without disrupting the native structure, and the ultrafast elementary processes of hydration. Here we report direct study, with femtosecond resolution, of the dynamics of hydration at the surface of the enzyme protein Subtilisin Carlsberg, whose single Trp residue (Trp-113) was used as an intrinsic biological fluorescent probe. For the protein, we observed two well separated dynamical solvation times, 0.8 ps and 38 ps, whereas in bulk water, we obtained 180 fs and 1.1 ps. We also studied a covalently bonded probe at a separation of approx 7 A and observed the near disappearance of the 38-ps component, with solvation being practically complete in (time constant) 1.5 ps. The degree of rigidity of the probe (anisotropy decay) and of the water environment (protein vs. micelle) was also studied. These results show that hydration at the surface is a dynamical process with two general types of trajectories, those that result from weak interactions with the selected surface site, giving rise to bulk-type solvation (approx 1 ps), and those that have a stronger interaction, enough to define a rigid water structure, with a solvation time of 38 ps, much slower than that of the bulk. At a distance of approx 7 A from the surface, essentially all trajectories are bulk-type. The theoretical framework for these observations is discussed.

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Biological water at the protein surface: dynamical solvation probed directly with femtosecond resolution.

In 1970, three laboratories independently made a discovery that, for aromatic fluorophores embedded into different rigid and highly viscous media, the spectroscopic properties do not conform to classical rules. The fluorescence spectra can depend on excitation wavelength, and the excited-state energy transfer, if present, fails at the "red" excitation edge. These red-edge effects were related to the existence of excited-state distribution of fluorophores on their interaction energy with the environment and the slow rate of dielectric relaxation of this environment. In these conditions the site-selection can be provided by variation of the energy of illuminating light quanta, and the behaviour of selected species can be followed as a function of time and other variables. These observations found extensive application in different areas of research: colloid and polymer science, molecular biophysics, photochemistry and photobiology. In particular, they led to the development of very productive methods of studying the dynamics of dielectric relaxations in protein and membranes, using the tryptophan emission and the emission of a variety of probes. These studies were extended to the time domain with the observation of new site-selective effects in emission intensity and anisotropy decays. They stimulated the emergence and development of cryogenic energy-selective and single-molecular techniques that became valuable tools in their own right in chemistry and biophysics research. Site-selection effects were discovered for electron-transfer and proton-transfer reactions if they depended on the dynamics of the environment. This review is focused on the progress in the field of red-edge effects, their applications and prospects.

The red‐edge effects: 30 years of exploration

Proteins are the working chemists of living cells. They are complex macromolecules, which display a rich and sometimes counterintuitive behaviour on many length- and timescales. They contain charged and polar groups, and electrostatic interactions control important aspects of their structure and function. Experiments and computer simulations have been used intensively to probe their electrostatic and dielectric properties. A simple framework to rationalize the results is continuum electrostatics, even though proteins are smaller than the usual, macroscopic length scales of continuum theory. We discuss selected topics, including protein structure, dynamics, and solvation; the dielectric response of proteins at large (macromolecular) and small (atomic) length scales, and the physical and numerical basis of current continuum models of proteins and protein solvation.

https://iopscience.iop.org/article/10.1088/0034-4885/66/5/202/pdf

Electrostatics and dynamics of proteins

X-ray diffraction is used to study the binding of xenon and krypton to a variety of crystallised proteins: porcine pancreatic elastase; subtilisin Carlsberg from Bacillus licheniformis; cutinase from Fusarium solani; collagenase from Hypoderma lineatum; hen egg lysozyme, the lipoamide dehydrogenase domain from the outer membrane protein P64k from Neisseria meningitidis; urate-oxidase from Aspergillus flavus, mosquitocidal delta-endotoxin CytB from Bacillus thuringiensis and the ligand-binding domain of the human nuclear retinoid-X receptor RXR-alpha. Under gas pressures ranging from 8 to 20 bar, xenon is able to bind to discrete sites in hydrophobic cavities, ligand and substrate binding pockets, and into the pore of channel-like structures, These xenon complexes can be used to map hydrophobic sites in proteins, or as heavy-atom derivatives in the isomorphous replacement method of structure determination. (C) 1998 Wiley-Liss, Inc.

Exploring hydrophobic sites in proteins with xenon or krypton.

The fluorescent dynamic Stokes shift (FDSS) method has emphasized a time-dependent dipolar relaxation process around the single tryptophan residue (Trp31) in cytidine monophosphate kinase from E. coli (CMPK). This Trp residue, located close to the protein surface in a hydrophobic pocket, is weakly accessible to acrylamide, a water-soluble quencher. It exhibits fluorescence characteristics suitable for a detailed study of dipolar relaxation:  (i) a fluorescence decay almost monoexponential and (ii) a fluorescence emission maximum of 329 nm, in a wavelength range intermediate between those of a completely polar environment and a strongly apolar one. This emission maximum is shifted to 320 nm by decreasing the temperature to 230−240 K with glycerol as cryoprotectant. A time constant (∼100 ps) affected by a negative preexponential, evidenced in the red-edge fluorescence intensity decays, supports the existence of an excited-state reaction. A multiphasic FDSS (with time constants ranging from ∼100 ps to severa...

Nanosecond Fluorescence Dynamic Stokes Shift of Tryptophan in a Protein Matrix

Human recombinant interferon α2 belongs a to family of proteins active against a wide range of viruses. It contains two tryptophan residues located at positions 77 and 141 in the peptide sequence. The fluorescence emission spectrum of these tryptophan residues displays a maximum at 335 nm. The fluorescence intensity decay is described by one broad excited-state-lifetime population centered around a value of 1.7 ns (full width at half maximum, 1.5 ns). These observations suggest that in the native protein, both tryptophan residues emit from similar environments, not directly exposed to the surrounding solvent. The anisotropy decay is essentially biexponential. The correlation-time value characterizing the Brownian rotation of the protein varies linearly with the viscosity/temperature ratio. The calculated hydrodynamic volumes are compatible with the existence of a dimer and a tetramer, at pH 5.5 and 9.4, respectively. Addition of urea at pH 5.5 disrupts the dimer and modifies to some extent the excited-state-lifetime distribution which becomes more heterogeneous. Disulfjtiebond reduction also dissociates the dimer and leads to a highly heterogeneous fluorescence-intensity decay with four excited-state-lifetime populations. An opening of the local structure in the Trp region of the protein is likely to occur in these conditions. The fast-anisotropy-decay components can be due to either fast rotation or energy transfer between the indoles. Close proximity of the two Trp residues (less than 1 nm) is suggested from steady-state and time-resolved fluorescence-anisotropy measurements in vitrified medium [95% (by mass) glycerol at - 38°C]. This suggestion is in agreement with the recently published three-dimensional structure of the homologous protein murine interferon β [Senda, T., Shimazu, T., Matsuda, S. Kawano, G., Shimizu, H., Nakamura, K. T. & Mitsui, Y. (1992) EMBO J. 11, 3193–3201].

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1992.tb17500.x

Time‐resolved fluorescence study of human recombinant interferon α2

Crystallization and preliminary X-ray investigation of a recombinant outer membrane protein from Neisseria meningitidis.

/pdf/x-ray-investigation-of-a-recombinant-outer-membrane-protein-5gdpxloy3e.pdf

Ines M. Li De La Sierra

Papers

Nanosecond Fluorescence Dynamic Stokes Shift of Tryptophan in a Protein Matrix

Time‐resolved fluorescence study of human recombinant interferon α2

Crystallization and preliminary X-ray investigation of a recombinant outer membrane protein from Neisseria meningitidis.

x-ray investigation of a recombinant outer membrane protein from Neisseria meningitidis