The tryptophan residues of aspartate transcarbamylase: site-directed mutagenesis and time-resolved fluorescence spectroscopy.

Aspartate transcarbamylase (EC 2.1.3.2) contains two tryptophan residues in position 209 and 284 of the catalytic chains (c) and no such chromophore in the regulatory chains (r). Thus, as a dodecamer [(c3)2(r2)3] the native enzyme molecule contains 12 tryptophan residues. The present study of the regulatory conformational changes in this enzyme is based on the fluorescence properties of these intrinsic probes. Site-directed mutagenesis was used in order to differentiate the respective contributions of the two tryptophans to the fluorescence properties of the enzyme and to identify the mobility of their environment in the course of the different regulatory processes. Each of these tryptophan residues gives two independent fluorescence decays, suggesting that the catalytic subunit exists in two slightly different conformational states. The binding of the substrate analog N-phosphonacetyl-L-aspartate promotes the same fluorescence signal whether or not the catalytic subunits are associated with the regulatory subunits, suggesting that the substrate-induced conformational change of the catalytic subunit is the essential trigger for the quaternary structure transition involved in cooperativity. The binding of the substrate analog affects mostly the environment of tryptophan 284, while the binding of the activator ATP affects mostly the environment of tryptophan 209, confirming that this activator acts through a mechanism different from that involved in homotropic cooperativity.     

Time resolved spectroscopy of the tryptophyl fluorescence of the E. coli LAC repressor.

A new crystal form of a mitogenic lectin from pea seeds ($\text{Pisum sativum}$) has been obtained which is suitable for high resolution structural work. The crystals are orthorhombic, space group P2₁2₁2₁, with unit cell dimensions: $\text{a}$ = 64.2Å, $\text{b}$ = 72. 7Å, $\text{c}$ = 108. 3Å. The asymmetric unit contains one protein molecule.     

Pulse fluorimetry study of energy transfers between tryptophan residues and NADPH in beef liver glutamate dehydrogenase complexes

A method is proposed to determine the rates of singlet energy transfers in an array of chromophores containing a finite number of donors and fluorescent acceptors. This method is based on measurements of transfer efficiency coupled with pulse fluorimetry. Three classes of donors can be distinguished which differ in their energy transfer rate. The rates of the first, the second and the third class are respectively greater than, of the order of, and smaller than the emission rate. The method is applied to the study of the energy transfers from tryptophan residues to NADPH, in ternary and quaternary glutamate dehydrogenase complexes. Practically, all these tryptophan residues belong to the first class. They can be divided into two subclasses having different transfer rate values. The distances between these residues and the NADPH site are of the order of 2.5 nm. In addition, the ligand binding induces a protein conformational change, leading to a fluorescence quenching of the tryptophanyl emission.     

Conformational fluctuations and protein reactivity. Determination of the rate-constant spectrum and consequences in elementary biochemical processes

When a protein's active site happens to be strongly coupled with the protein structure, the rate constant of the reaction may eventually be modulated by the conformational fluctuations. Evidence for this effect has long been provided by extensive flash photolysis investigations of liganded hemoproteins and more recently of the non-heme respiratory protein hemerythrin in hydro-organic solvents. Within a given protein conformational substate, an elementary reaction step is characterized by one single free energy barrier and by a first-order rate constant, k, which changes with temperature according to an Arrhenius law. At physiological temperature and low viscosity, ultrafast conformational relaxation causes efficient averaging of the reaction rates and the protein displays exponential kinetics with an average rate constant (k). Under sufficiently general conditions, it can be shown that (k) also follows a simple Arrhenius law with 'effective' values of the pre-exponential factor Aeff and activation enthalpy Heff. It is found that Aeff strongly depends on the overall shape of the rate constant distribution and that Heff actually corresponds to the lower limit of the enthalpy of activation, i.e. the value associated with the highest possible reaction rate. The underlying distribution of rate constants can be reconstructed from a set of experiments in which the kinetics depart from an exponential, i.e. at low temperature and high viscosity. The most probable distribution of exponentials consistent with the observed kinetics of the geminate recombinations of oxygen with photodissociated hemerythrin has been determined by using a new approach, known as the maximum entropy method. The results are consistent with a single pre-exponential value and a distributed enthalpy spectrum. As expected, Heff does not coincide either with the most probable nor with the average value of the enthalpy. The most salient findings are that the probability for any protein molecule to have an enthalpy of activation equal to the effective value Heff vanishes and that Aeff differs by nearly three orders of magnitude from the true value A0. Biochemical reaction rates are actually average values, since protein reactions are measured under physiological conditions, where conformational relaxation is always fast. Our understanding of the significance of Aeff and Heff is therefore entirely dependent on the knowledge of the distribution function of the rate constants. In particular, enthalpy and entropy terms of similar reactions performed by different proteins cannot be compared as long as the distribution of the rate constants remains unknown.     

Conformational dynamics and intersubunit energy transfer in wild-type and mutant lipoamide dehydrogenase from Azotobacter vinelandii. A multidimensional time-resolved polarized fluorescence study.

Time-resolved fluorescence and fluorescence anisotropy data surfaces of flavin adenine dinucleotide bound to lipoamide dehydrogenase from Azotobacter vinelandii in 80% glycerol have been obtained by variation of excitation energy and temperature between 203 and 303 K. The fluorescence kinetics of a deletion mutant lacking 14 COOH-terminal amino acids were compared with the wild-type enzyme to study a possible interaction of the COOH-terminal tail with the active site of the enzyme. The flavin adenine dinucleotide fluorescence in both proteins exhibits a bimodal lifetime distribution as recovered by the maximum entropy method of data analysis. The difference in standard enthalpy and entropy of associated conformational substates was retrieved from the fractional contributions of the two lifetime classes. Activation energies of thermal quenching were obtained that confirm that the isoalloxazines in the deletion mutant are solvent accessible in contrast to the wild-type enzyme. Red-edge spectroscopy in conjunction with variation of temperature provides the necessary experimental axes to interpret the fluorescence depolarization in terms of intersubunit energy transfer rather than reorientational dynamics of the flavins. The results can be explained by a compartmental model that describes the anisotropy decay of a binary, inhomogeneously broadened, homoenergy transfer system. By using this model in a global analysis of the fluorescence anisotropy decay surface, the distance between and relative orientation of the two isoalloxazine rings are elucidated. For the wild-type enzyme, this geometrical information is in agreement with crystallographic data of the A. vinelandii enzyme, whereas the mutual orientation of the subunits in the deletion mutant is slightly altered. In addition, the ambiguity in the direction of the emission transition moment in the isoalloxazine ring is solved. The anisotropy decay parameters also provide information on electronic and dipolar relaxational properties of the flavin active site. The local environment of the prosthetic groups in the deletion mutant of the A. vinelandii enzyme is highly inhomogeneous, and a transition from slow to rapid dipolar relaxation is observed over the measured temperature range. In the highly homogeneous active site of the wild-type enzyme, dipolar relaxation is slowed down beyond the time scale of fluorescence emission at any temperature studied. Our results are in favor of a COOH-terminal polypeptide interacting with the active site, thereby shielding the isoalloxazines from the solvent. This biological system forms a very appropriate tool to test the validity of photophysical models describing homoenergy transfer.     

Tryptophan residues at subunit interfaces used as fluorescence probes to investigate homotropic and heterotropic regulation of aspartate transcarbamylase

The homotropic and heterotropic interactions in Escherichia coli aspartate transcarbamylase (EC 2.1.3.2) are accompanied by various structure modifications. The large quaternary structure change associated with the T to R transition, promoted by substrate binding, is accompanied by different local conformational changes. These tertiary structure modifications can be monitored by fluorescence spectroscopy, after introduction of a tryptophan fluorescence probe at the site of investigation. To relate unambiguously the fluorescence signals to structure changes in a particular region, both naturally occurring Trp residues in positions 209c and 284c of the catalytic chains were previously substituted with Phe residues. The regions of interest were the so-called 240's loop at position Tyr240c, which undergoes a large conformational change upon substrate binding, and the interface between the catalytic and regulatory chains in positions Asn153r and Phe145r supposed to play a role in the different regulatory processes. Each of these tryptophan residues presents a complex fluorescence decay with three to four independent lifetimes, suggesting that the holoenzyme exists in slightly different conformational states. The bisubstrate analogue N-phosphonacetyl-L-aspartate affects mostly the environment of tryptophans at position 240c and 145r, and the fluorescence signals were related to ligand binding and the quaternary structure transition, respectively. The binding of the nucleotide activator ATP slightly affects the distribution of the conformational substates as probed by tryptophan residues at position 240c and 145r, whereas the inhibitor CTP modifies the position of the C-terminal residues as reflected by the fluorescence properties of Trp153r. These results are discussed in correlation with earlier mutagenesis studies and mechanisms of the enzyme allosteric regulation.     

Investigation of the effect of high hydrostatic pressure on proteins and lipidic membranes by dynamic fluorescence spectroscopy

Dynamic fluorescence spectroscopy brings new insight into the functional and structural changes of biological molecules under moderate and high hydrostatic pressure. The principles of time-resolved fluorescence methods are briefly described and the resulting type of information is summarized. A first set of selected applications of the use of dynamic fluorescence on pressure effects on proteins in terms of denaturation, ternary and quaternary structure, aggregation and also interaction with DNA are presented. A second set of applications is devoted to the effect of pressure and of cholesterol on lateral heterogeneity of lipidic membranes.     

Behaviour of bovine phosphatidylethanolamine-binding protein with model membranes. Evidence of affinity for negatively charged membranes.

The ability of phosphatidylethanolamine-binding protein (PEBP) to bind membranes was tested by using small and large unilamellar vesicles and monolayers composed of l-alpha-1,2-dimyristoylphosphatidylcholine, l-alpha-1,2-dimyristoylphosphatidylglycerol and l-alpha-1,2-dimyristoylphosphatidylethanolamine. PEBP only bound to model membranes containing l-alpha-1,2-dimyristoylphosphatidylglycerol; the interaction was primarily due to electrostatic forces between the basic protein and the acidic phospholipids. Further experiments indicated that the interaction was not dependent on the length and unsaturation of the phospholipid acyl chains and was not modified by the presence of cholesterol in the membrane. PEBP affinity for negatively charged membranes is puzzling considering the previous identification of the protein as a phosphatidylethanolamine-binding protein, and suggests that the association of PEBP with phospholipid membranes is driven by a mechanism other than its binding to solubilized phosphatidylethanolamine. An explanation was suggested by its three-dimensional structure: a small cavity at the protein surface has been reported to be the binding site of the polar head of phosphatidylethanolamine, while the N-terminal and C-terminal parts of PEBP, exposed at the protein surface, appear to be involved in the interaction with membranes. To test this hypothesis, we synthesized the two PEBP terminal regions and tested them with model membranes in parallel with the whole protein. Both peptides displayed the same behaviour as whole PEBP, indicating that they could participate in the binding of PEBP to membranes. Our results strongly suggest that PEBP directly interacts with negatively charged membrane microdomains in living cells.     

Writing In and Reading ICU Diaries: Qualitative Study of Families' Experience in the ICU

Purpose

Keeping an ICU patient diary has been reported to benefit the patient''s recovery. Here, we investigated the families'' experience with reading and writing in patient ICU diaries kept by both the family and the staff.

Methods

We conducted a qualitative study involving 32 semi-structured in-depth interviews of relatives of 26 patients (34% of all family members who visited patients) who met our ICU-diary criterion, i.e., ventilation for longer than 48 hours. Grounded theory was used to conceptualise the interview data via a three-step coding process (open coding, axial coding, and selective coding).

Results

Communicative, emotional, and humanising experiences emerged from our data. First, family members used the diaries to access, understand, and assimilate the medical information written in the diaries by staff members, and then to share this information with other family members. Second, the diaries enabled family members to maintain a connection with the patient by documenting their presence and expressing their love and affection. Additionally, families confided in the diaries to maintain hope. Finally, family members felt the diaries humanized the medical staff and patient.

Conclusions

Our findings indicate positive effects of diaries on family members. The diaries served as a powerful tool to deliver holistic patient- and family-centered care despite the potentially dehumanising ICU environment. The diaries made the family members aware of their valuable role in caring for the patient and enhanced their access to and comprehension of medical information. Diaries may play a major role in improving the well-being of ICU-patient families.