Virginia F. Smith

Multistate Equilibrium Unfolding of Escherichia coli Dihydrofolate Reductase: Thermodynamic and Spectroscopic Description of the Native, Intermediate, and Unfolded Ensembles†

The thermodynamic and spectroscopic properties of a cysteine-free variant of Escherichia coli dihydrofolate reductase (AS-DHFR) were investigated using the combined effects of urea and temperature as denaturing agents. Circular dichroism (CD), absorption, and fluorescence spectra were recorded during temperature-induced unfolding at different urea concentrations and during urea-induced unfolding at different temperatures. The first three vectors obtained by singular-value decomposition of each set of unfolding spectra were incorporated into a global analysis of a unique thermodynamic model. Although individual unfolding profiles can be described as a two-state process, a simultaneous fit of 99 vectors requires a three-state model as the minimal scheme to describe the unfolding reaction along both perturbation axes. The model, which involves native (N), intermediate (I), and unfolded (U) states, predicts a maximum apparent stability, DeltaG degrees (NU), of 6 kcal mol(-)(1) at 15 degrees C, an apparent m(NU) value of 2 kcal mol(-)(1) M(-)(1), and an apparent heat capacity change, DeltaC(p)()(-NU), of 2.5 kcal mol(-)(1) K(-)(1). The intermediate species has a maximum stability of approximately 2 kcal mol(-)(1) and a compactness closer to that of the native than to that of the unfolded state. The population of the intermediate is maximal ( approximately 70%) around 50 degrees C and falls below the limits of detection of > or =2 M urea or at temperatures of <35 or >65 degrees C. The fluorescence properties of the equilibrium intermediate resemble those of a transient intermediate detected during refolding from the urea-denatured state, suggesting that a tryptophan-containing hydrophobic cluster in the adenosine-binding domain plays a key role in both the equilibrium and kinetic reactions. The CD spectroscopic properties of the native state reveal the presence of two principal isoforms that differ in ligand binding affinities and in the packing of the adenosine-binding domain. The relative populations of these species change slightly with temperature and do not depend on the urea concentration, implying that the two native isoforms are well-structured and compact. Global analysis of data from multiple spectroscopic probes and several methods of unfolding is a powerful tool for revealing structural and thermodynamic properties of partially and fully folded forms of DHFR.

SecA, the Motor of the Secretion Machine, Binds Diverse Partners on One Interactive Surface

In all living cells, regulated passage across membranes of specific proteins occurs through a universally conserved secretory channel. In bacteria and chloroplasts, the energy for the mechanical work of moving polypeptides through that channel is provided by SecA, a regulated ATPase. Here, we use site-directed spin labeling and electron paramagnetic resonance spectroscopy to identify the interactive surface used by SecA for each of the diverse binding partners encountered during the dynamic cycle of export. Although the binding sites overlap, resolution at the level of aminoacyl side chains allows us to identify contacts that are unique to each partner. Patterns of constraint and mobilization of residues on that interactive surface suggest a conformational change that may underlie the coupling of ATP hydrolysis to precursor translocation.

Bispecific receptor globulins, novel tools for the study of cellular interactions: Preparation and characterization of an E-selectin/P-selectin bispecific receptor globulin

In recent years the functional consequences of receptor/ligand interactions have been studied in vitro and in vivo using monospecific recombinant immunoglobulin fusion proteins (recombinant/receptor globulins, Rg). These proteins are encoded by chimeric genes composed of a DNA fragment encoding the extracellular domain of a cell surface protein grafted onto a DNA fragment encoding immunoglobulin constant domains. In order to extend the range of applications of Rgs we investigated the possibility of preparing bispecific Rgs. These bispecific fusion proteins contain the extracellular domains of two cell surface proteins held together in close proximity by the constant domains of an immunoglobulin. Here we describe the preparation and characterization of a bispecific Rg which contains the extracellular domains of two adhesion molecules expressed by activated vascular endothelial cells, E-selectin and P-selectin. These two proteins play an important role in initiating leukocyte adhesion to the vascular cell wall at sites of inflammation. Binding studies showed that the E-selectin/P-selectin bispecific immunoglobulin fusion protein (ELAM-1/GMP140 Rg) has an enhanced ability to bind to the myeloid cell line HL60 when compared to the monospecific Rg fusion proteins from which it was derived.

Testing the role of chain connectivity on the stability and structure of dihydrofolate reductase from E. coli: fragment complementation and circular permutation reveal stable, alternatively folded forms.

The effects of chain cleavage and circular permutation on the structure, stability, and activity of dihydrofolate reductase (DHFR) from Escherichia coli were investigated by various spectroscopic and biochemical methods. Cleavage of the backbone after position 86 resulted in two fragments, {1–86} and {87–159}, each of which are poorly structured and enzymatically inactive. When combined in a 1 : 1 molar ratio, however, the fragments formed a high‐affinity (Ka = 2.6 × 107 M−1) complex that displays a weakly cooperative urea‐induced unfolding transition at micromolar concentrations. The retention of about 15% of the enzymatic activity of full‐length DHFR is surprising, considering that the secondary structure in the complex is substantially reduced from its wild‐type counterpart. In contrast, a circularly permuted form with its N‐terminus at position 86 has similar overall stability to full‐length DHFR, about 50% of its activity, substantial secondary structure, altered side‐chain packing in the adenosine binding domain, and unfolds via an equilibrium intermediate not observed in the wild‐type protein. After addition of ligand or the tight‐binding inhibitor methotrexate, both the fragment complex and the circular permutant adopt more native‐like secondary and tertiary structures. These results show that changes in the backbone connectivity can produce alternatively folded forms and highlight the importance of protein‐ligand interactions in stabilizing the active site architecture of DHFR.

Characterization of three areas of interactions stabilizing complexes between SecA and SecB, two proteins involved in protein export

The general secretory, Sec, system translocates precursor polypeptides from the cytosol across the cytoplasmic membrane in Escherichia coli. SecB, a small cytosolic chaperone, captures the precursor polypeptides before they fold and delivers them to the membrane translocon through interactions with SecA. Both SecB and SecA display twofold symmetry and yet the complex between the two is stabilized by contacts that are distributed asymmetrically. Two distinct regions of interaction have been defined previously and here we identify a third. Calorimetric studies of complexes stabilized by different subsets of these interactions were carried out to determine the binding affinities and the thermodynamic parameters that underlie them. We show here that there is no change in affinity when either one of two contact areas out of the three is lacking. This fact and the asymmetry of the binding contacts may be important to the function of the complex in protein export.

SecB: a chaperone from Escherichia coli.

Publisher Summary The chapter presents a study related to SecB, a chaperone from Escherichia coli (E.coli). SecB is a molecular chaperone in E.coli that is dedicated to the facilitation of the export of a number of proteins destined for the periplasmic space or the outer membrane. This role in export is demonstrated in vivo by the accumulation of pulse-labeled precursor species in a strain that is devoid of SecB and in vitro by showing that SecB is required for translocation of precursors into inverted vesicles of cytoplasmic membrane. The binding frame for SecB within two physiologic ligands, maltose-binding protein, and galactose-binding protein has been determined. SecB has the ability to bind selectively and with high affinity to polypeptides that are in a nonnative state. SecB binds precursor polypeptides and maintains them in a state competent for translocation through the cytoplasmic membrane. SecB is purified from strains of E. coli harboring plasmids that contain the secB gene under strong, regulated promoters. The purification involves passage of the sample over a Q-Sepharose ion-exchange column, followed by chromatography using a molecular sieve resin, Sephacryl S-300, and finally a Mono Q ion-exchange column.

Hard sphere sequential impulse model for endoergic reactions: Angular and velocity distributions of the products

Formalism is developed for the scattering of the products of the reaction A+BC → AB+C, produced by the sequence of collisions: A strikes B which then strikes C. The collisions are treated as purely hard sphere impulses except that energy may be absorbed in either collision. Analytical expressions are given for the differential cross sections of the scattered C, which may be converted easily to those of the scattered AB product. Angular‐velocity contour diagrams for C are presented which illustrate the features of the model. Formulas for the total reactive cross sections are also given. Finally, comparisons are made between this model and other widely used models.

The relationship between chain connectivity and domain stability in the equilibrium and kinetic folding mechanisms of dihydrofolate reductase from E.coli

Abstract The role of domains in defining the equilibrium and kinetic folding properties of dihydrofolate reductase (DHFR) from Escherichia coli was probed by examining the thermodynamic and kinetic properties of a set of variants in which the chain connectivity in the discontinuous loop domain (DLD) and the adenosine-binding domain (ABD) was altered by permutation. To test the concept that chain cleavage can selectively destabilize the domain in which the N- and C-termini are resident, permutations were introduced at one position within the ABD, one within the DLD and one at a boundary between the domains. The results demonstrated that a continuous ABD is required for a stable thermal intermediate and a continuous DLD is required for a stable urea intermediate. The permutation at the domain interface had both a thermal and urea intermediate. Strikingly, the observable kinetic folding responses of all three permuted proteins were very similar to the wild-type protein. These results demonstrate a crucial role for stable domains in defining the energy surface for the equilibrium folding reaction of DHFR. If domain connectivity affects the kinetic mechanism, the effects must occur in the sub-millisecond time range.

Single-Molecule Peptide–Lipid Affinity Assay Reveals Interplay between Solution Structure and Partitioning

Interactions between short protein segments and phospholipid bilayers dictate fundamental aspects of cellular activity and have important applications in biotechnology. Yet, the lack of a suitable methodology for directly probing these interactions has hindered the mechanistic understanding. We developed a precision atomic force microscopy-based single-molecule force spectroscopy assay and probed partitioning into lipid bilayers by measuring the mechanical force experienced by a peptide. Protein segments were constructed from the peripheral membrane protein SecA, a key ATPase in bacterial secretion. We focused on the first 10 amino-terminal residues of SecA (SecA2-11) that are lipophilic. In addition to the core SecA2-11 sequence, constructs with nearly identical chemical composition but with differing geometry were used: two copies of SecA2-11 linked in series and two copies SecA2-11 linked in parallel. Lipid bilayer partitioning interactions of peptides with differing structures were distinguished. To model the energetic landscape, a theory of diffusive barrier crossing was extended to incorporate a superposition of potential barriers with variable weights. Analysis revealed two dissociation pathways for the core SecA2-11 sequence with well-separated intrinsic dissociation rates. Molecular dynamics simulations showed that the three peptides had significant conformational differences in solution that correlated well with the measured variations in the propensity to partition into the bilayer. The methodology is generalizable and can be applied to other peptide and lipid species.

Variable mortality from the 1918–1919 influenza pandemic during military training

During the 1918-1919 pandemic, influenza mortality widely varied across populations and locations. Records of U.S. military members in mobilization camps (n = 40), military academies, and officer training schools were examined to document differences in influenza experiences during the fall 1918. During the fall-winter 1918-1919, mortality percentages were higher among soldiers in U.S. Army mobilization camps (0.34-4.3%) than among officer trainees (0-1.0%). Susceptibility to infection and clinical expressions of 1918 pandemic influenza varied largely based on host epidemiological characteristics rather than the inherent virulence of the virus.