Daniel N. Marti

Calculation of protein ionization equilibria with conformational sampling: pKa of a model leucine zipper, GCN4 and barnase †

The use of conformational ensembles provided by nuclear magnetic resonance (NMR) experiments or generated by molecular dynamics (MD) simulations has been regarded as a useful approach to account for protein motions in the context of pKa calculations, yet the idea has been tested occasionally. This is the first report of systematic comparison of pKa estimates computed from long multiple MD simulations and NMR ensembles. As model systems, a synthetic leucine zipper, the naturally occurring coiled coil GCN4, and barnase were used. A variety of conformational averaging and titration curve‐averaging techniques, or combination thereof, was adopted and/or modified to investigate the effect of extensive global conformational sampling on the accuracy of pKa calculations. Clustering of coordinates is proposed as an approach to reduce the vast diversity of MD ensembles to a few structures representative of the average electrostatic properties of the system in solution. Remarkable improvement of the accuracy of pKa predictions was achieved by the use of multiple MD simulations. By using multiple trajectories the absolute error in pKa predictions for the model leucine zipper was reduced to as low as approximately 0.25 pKa units. The validity, advantages, and limitations of explicit conformational sampling by MD, compared with the use of an average structure and a high internal protein dielectric value as means to improve the accuracy of pKa calculations, are discussed. Proteins 2002;46:41–60.

Electrostatic interactions in leucine zippers: thermodynamic analysis of the contributions of Glu and His residues and the effect of mutating salt bridges.

Electrostatic interactions play a complex role in stabilizing proteins. Here, we present a rigorous thermodynamic analysis of the contribution of individual Glu and His residues to the relative pH-dependent stability of the designed disulfide-linked leucine zipper AB(SS). The contribution of an ionized side-chain to the pH-dependent stability is related to the shift of the pK(a) induced by folding of the coiled coil structure. pK(a)(F) values of ten Glu and two His side-chains in folded AB(SS) and the corresponding pK(a)(U) values in unfolded peptides with partial sequences of AB(SS) were determined by 1H NMR spectroscopy: of four Glu residues not involved in ion pairing, two are destabilizing (-5.6 kJ mol(-1)) and two are interacting with the positive alpha-helix dipoles and are thus stabilizing (+3.8 kJ mol(-1)) in charged form. The two His residues positioned in the C-terminal moiety of AB(SS) interact with the negative alpha-helix dipoles resulting in net stabilization of the coiled coil conformation carrying charged His (-2.6 kJ mol(-1)). Of the six Glu residues involved in inter-helical salt bridges, three are destabilizing and three are stabilizing in charged form, the net contribution of salt-bridged Glu side-chains being destabilizing (-1.1 kJ mol(-1)). The sum of the individual contributions of protonated Glu and His to the higher stability of AB(SS) at acidic pH (-5.4 kJ mol(-1)) agrees with the difference in stability determined by thermal unfolding at pH 8 and pH 2 (-5.3 kJ mol(-1)). To confirm salt bridge formation, the positive charge of the basic partner residue of one stabilizing and one destabilizing Glu was removed by isosteric mutations (Lys-->norleucine, Arg-->norvaline). Both mutations destabilize the coiled coil conformation at neutral pH and increase the pK(a) of the formerly ion-paired Glu side-chain, verifying the formation of a salt bridge even in the case where a charged side-chain is destabilizing. Because removing charges by a double mutation cycle mainly discloses the immediate charge-charge effect, mutational analysis tends to overestimate the overall energetic contribution of salt bridges to protein stability.

Is There a Relationship between Children's Mental Disorders and Their Ideal Family Constructs?

Psychiatric outpatients (N = 72) and nonclinical children (N = 72) from 1st to 12th grades portrayed their families as they wished they were (ideal representation), using the Family System Test (FAST), a figure placement technique that focuses on cohesion and hierarchy. Results showed that constructs of ideal family relations were related to age group (1st to 3rd graders versus 4th to 12th graders) and to clinical status (clinical versus nonclinical) but not to the type of mental disorder. First, younger children portrayed less cohesion than did older ones. Second, clinical respondents were less likely to represent cohesive family patterns and were more likely to report ideal family situations as special events. Results are discussed from a developmental as well as from a family systems perspective.

EVALUATION OF FAMILY STRUCTURES WITH FIGURE PLACEMENT TECHNIQUES

The relevance of figure placement techniques for analyzing family cohesion and hierarchy structures in various settings is described and the completion of further studies including these instruments is suggested.

Family System Test (FAST) : The relevance of parental family constructs for clinical intervention

On the basis of theFamily System Test (FAST), family representations of parents with healthy and biopsychosocially distressed children were compared, and family structure changes in the context of interventions on the parent-level were described. Results indicated that parental family constructs are related to offsprings health and clinical intervention. Parents of psychiatric patients showed their families to have low cohesion, and hierarchically unclear generational boundaries (i.e. unbalanced structure). As had been their wish at therapy onset, the family patterns they indicated after completion of treatment were balanced.