Red blood cell adhesion on a solid/liquid interface

Red blood cells (RBCs), previously fixed with glutaraldehyde, adhere to glass slides coated with fibrinogen. The RBC deposition process on the horizontal glass surface is investigated by analyzing the relative surface covered by the RBCs, as well as the variance of this surface coverage, as a function of the concentration of particles. This study is performed by optical microscopy and image analysis. A model, derived from the classical random sequential adsorption model, has been developed to account for the experimental results. This model highlights the strong influence of the hydrodynamic interactions during the deposition process.

Free energy of burying hydrophobic residues in the interface between protein subunits

We have obtained an experimental estimate of the free energy change associated with variations at the interface between protein subunits, a subject that has raised considerable interest since the concept of accessible surface area was introduced by Lee and Richards [Lee, B. & Richards, F. M. (1971) J. Mol. Biol. 55, 379–400]. We determined by analytical ultracentrifugation the dimer–tetramer equilibrium constant of five single and three double mutants of human Hb. One mutation is at the stationary α1β1 interface, and all of the others are at the sliding α1β2 interface where cleavage of the tetramer into dimers and ligand-linked allosteric changes are known to occur. A surprisingly good linear correlation between the change in the free energy of association of the mutants and the change in buried hydrophobic surface area was obtained, after corrections for the energetic cost of losing steric complementarity at the αβ dimer interface. The slope yields an interface stabilization free energy of −15 ± 1.2 cal/mol upon burial of 1 Å2 of hydrophobic surface, in very good agreement with the theoretical estimate given by Eisenberg and McLachlan [Eisenberg, D. & McLachlan, A. D. (1986) Nature (London) 319, 199–203].

Binding site of brefeldin A at the interface between the small G protein ADP-ribosylation factor 1 (ARF1) and the nucleotide-exchange factor Sec7 domain

Sec7 domains (Sec7d) catalyze the exchange of guanine nucleotide on ARFs. Recent studies indicated that brefeldin A (BFA) inhibits Sec7d-catalyzed nucleotide exchange on ARF1 in an uncompetitive manner by trapping an early intermediate of the reaction: a complex between GDP-bound ARF1 and Sec7d. Using 3H-labeled BFA, we show that BFA binds to neither isolated Sec7d nor isolated ARF1–GDP, but binds to the transitory Sec7d–ARF1–GDP complex and stabilizes it. Two pairs of residues at positions 190–191 and 198–208 (Arno numbering) in Sec7d contribute equally to the stability of BFA binding, which is also sensitive to mutation of H80 in ARF1. The catalytic glutamic (E156) residue of Sec7d is not necessary for BFA binding. In contrast, BFA does not bind to the intermediate catalytic complex between nucleotide-free ARF1 and Sec7d. These results suggest that, on initial docking steps between ARF1–GDP and Sec7d, BFA inserts like a wedge between the switch II region of ARF1–GDP and a surface encompassing residues 190–208, at the border of the characteristic hydrophobic groove of Sec7d. Bound BFA would prevent the switch regions of ARF1–GDP from reorganizing and forming tighter contacts with Sec7d and thereby would maintain the bound GDP of ARF1 at a distance from the catalytic glutamic finger of Sec7d.

Visualizing the dynamics of T cell activation: Intracellular adhesion molecule 1 migrates rapidly to the T cell/B cell interface and acts to sustain calcium levels

T cell recognition typically involves both the engagement of a specific T cell receptor with a peptide/major histocompatibility complex (MHC) and a number of accessory interactions. One of the most important interactions is between the integrin lymphocyte function-associated antigen 1 (LFA-1) on the T cell and intracellular adhesion molecule 1 (ICAM-1) on an antigen-presenting cell. By using fluorescence video microscopy and an ICAM-1 fused to a green fluorescent protein, we find that the elevation of intracellular calcium in the T cell that is characteristic of activation is followed almost immediately by the rapid accumulation of ICAM-1 on a B cell at a tight interface between the two cells. This increased density of ICAM-1 correlates with the sustained elevation of intracellular calcium in the T cell, known to be critical for activation. The use of peptide/MHC complexes and ICAM-1 on a supported lipid bilayer to stimulate T cells also indicates a major role for ICAM-1/LFA-1 in T cell activation but, surprisingly, not for adhesion, as even in the absence of ICAM-1 the morphological changes and adhesive characteristics of an activated T cell are seen in this system. We suggest that T cell antigen receptor-mediated recognition of a very small number of MHC/peptide complexes could trigger LFA-1/ICAM-1 clustering and avidity regulation, thus amplifying and stabilizing the production of second messengers.

The vav exchange factor is an essential regulator in actin-dependent receptor translocation to the lymphocyte–antigen-presenting cell interface

During the interaction of a T cell with an antigen-presenting cell (APC), several receptor ligand pairs, including the T cell receptor (TCR)/major histocompatibility complex (MHC), accumulate at the T cell/APC interface in defined geometrical patterns. This accumulation depends on a movement of the T cell cortical actin cytoskeleton toward the interface. Here we study the involvement of the guanine nucleotide exchange factor vav in this process. We crossed 129 vav−/− mice with B10/BR 5C.C7 TCR transgenic mice and used peptide-loaded APCs to stimulate T cells from the offspring. We found that the accumulation of TCR/MHC at the T cell/APC interface and the T cell actin cytoskeleton rearrangement were clearly defective in these vav+/− mice. A comparable defect in superantigen-mediated T cell activation of T cells from non-TCR transgenic 129 mice was also observed, although in this case it was more apparent in vav−/− mice. These data indicate that vav is an essential regulator of cytoskeletal rearrangements during T cell activation.

Redesigning an FKBP–ligand interface to generate chemical dimerizers with novel specificity

FKBP ligand homodimers can be used to activate signaling events inside cells and animals that have been engineered to express fusions between appropriate signaling domains and FKBP. However, use of these dimerizers in vivo is potentially limited by ligand binding to endogenous FKBP. We have designed ligands that bind specifically to a mutated FKBP over the wild-type protein by remodeling an FKBP-ligand interface to introduce a specificity binding pocket. A compound bearing an ethyl substituent in place of a carbonyl group exhibited sub-nanomolar affinity and 1,000-fold selectivity for a mutant FKBP with a compensating truncation of a phenylalanine residue. Structural and functional analysis of the new pocket showed that recognition is surprisingly relaxed, with the modified ligand only partially filling the engineered cavity. We incorporated the specificity pocket into a fusion protein containing FKBP and the intracellular domain of the Fas receptor. Cells expressing this modified chimeric protein potently underwent apoptosis in response to AP1903, a homodimer of the modified ligand, both in culture and when implanted into mice. Remodeled dimerizers such as AP1903 are ideal reagents for controlling the activities of cells that have been modified by gene therapy procedures, without interference from endogenous FKBP.

Identification of protein oligomerization states by analysis of interface conservation

The discrimination of true oligomeric protein–protein contacts from nonspecific crystal contacts remains problematic. Criteria that have been used previously base the assignment of oligomeric state on consideration of the area of the interface and/or the results of scoring functions based on statistical potentials. Both techniques have a high success rate but fail in more than 10% of cases. More importantly, the oligomeric states of several proteins are incorrectly assigned by both methods. Here we test the hypothesis that true oligomeric contacts should be identifiable on the basis of an increased degree of conservation of the residues involved in the interface. By quantifying the degree of conservation of the interface and comparing it with that of the remainder of the protein surface, we develop a new criterion that provides a highly effective complement to existing methods.

BimD/SPO76 is at the interface of cell cycle progression, chromosome morphogenesis, and recombination

BIMD of Aspergillus nidulans belongs to a highly conserved protein family implicated, in filamentous fungi, in sister-chromatid cohesion and DNA repair. We show here that BIMD is chromosome associated at all stages, except from late prophase through anaphase, during mitosis and meiosis, and is involved in several aspects of both programs. First, bimD+ function must be executed during S through M. Second, in bimD6 germlings, mitotic nuclear divisions and overall cellular program occur more rapidly than in wild type. Thus, BIMD, an abundant chromosomal protein, is a negative regulator of normal cell cycle progression. Third, bimD6 reduces the level of mitotic interhomolog recombination but does not alter the ratio between crossover and noncrossover outcomes. Moreover, bimD6 is normal for intrachromosomal recombination. Therefore, BIMD is probably not involved in the enzymology of recombinational repair per se. Finally, during meiosis, staining of the Sordaria ortholog Spo76p delineates robust chromosomal axes, whereas BIMD stains all chromatin. SPO76 and bimD are functional homologs with respect to their roles in mitotic chromosome metabolism but not in meiosis. We propose that BIMD exerts its diverse influences on cell cycle progression as well as chromosome morphogenesis and recombination by modulating chromosome structure.

Energy-driven subunit rotation at the interface between subunit a and the c oligomer in the FO sector of Escherichia coli ATP synthase

Subunit rotation within the F1 catalytic sector of the ATP synthase has been well documented, identifying the synthase as the smallest known rotary motor. In the membrane-embedded FO sector, it is thought that proton transport occurs at a rotor/stator interface between the oligomeric ring of c subunits (rotor) and the single-copy a subunit (stator). Here we report evidence for an energy-dependent rotation at this interface. FOF1 was expressed with a pair of substituted cysteines positioned to allow an intersubunit disulfide crosslink between subunit a and a c subunit [aN214C/cM65C; Jiang, W. & Fillingame, R. H. (1998) Proc. Natl. Acad. Sci. USA 95, 6607–6612]. Membranes were treated with N,N′-dicyclohexyl-[14C]carbodiimide to radiolabel the D61 residue on less than 20% of the c subunits. After oxidation to form an a–c crosslink, the c subunit properly aligned to crosslink to subunit a was found to contain very little 14C label relative to other members of the c ring. However, exposure to MgATP before oxidation significantly increased the radiolabel in the a–c crosslink, indicating that a different c subunit was now aligned with subunit a. This increase was not induced by exposure to MgADP/Pi. Furthermore, preincubation with MgADP and azide to inhibit F1 or with high concentrations of N,N′-dicyclohexylcarbodiimide to label most c subunits prevented the ATP effect. These results provide evidence for an energy-dependent rotation of the c ring relative to subunit a.

Packing at the protein-water interface.

We have determined the packing efficiency at the protein-water interface by calculating the volumes of atoms on the protein surface and nearby water molecules in 22 crystal structures. We find that an atom on the protein surface occupies, on average, a volume approximately 7% larger than an atom of equivalent chemical type in the protein core. In these calculations, larger volumes result from voids between atoms and thus imply a looser or less efficient packing. We further find that the volumes of individual atoms are not related to their chemical type but rather to their structural location. More exposed atoms have larger volumes. Moreover, the packing around atoms in locally concave, grooved regions of protein surfaces is looser than that around atoms in locally convex, ridge regions. This as a direct manifestation of surface curvature-dependent hydration. The net volume increase for atoms on the protein surface is compensated by volume decreases in water molecules near the surface. These waters occupy volumes smaller than those in the bulk solvent by up to 20%; the precise amount of this decrease is directly related to the extent of contact with the protein.

Interface between culturally based preferences and genetic preferences: female mate choice in Poecilia reticulata.

The relative contribution of genetic and socio-cultural factors in the shaping of behavior is of fundamental importance to biologists and social scientists, yet it has proven to be extremely difficult to study in a controlled, experimental fashion. Here I describe experiments that examined the strength of genetic and cultural (imitative) factors in determining female mate choice in the guppy, Poecilia reticulata. Female guppies from the Paria River in Trinidad have a genetic, heritable preference for the amount of orange body color possessed by males. Female guppies will, however, also copy (imitate) the mate choice of other females in that when two males are matched for orange color, an "observer" female will copy the mate choice of another ("model") female. Three treatments were undertaken in which males differed by an average of 12%, 24%, or 40% of the total orange body color. In all cases, observer females viewed a model female prefer the less colorful male. When males differed by 12% or 24%, observer females preferred the less colorful male and thus copied the mate choice of others, despite a strong heritable preference for orange body color in males. When males differed by 40% orange body color, however, observer females preferred the more colorful male and did not copy the mate choice of the other female. In this system, then, imitation can "override" genetic preferences when the difference between orange body color in males is small or moderate, but genetic factors block out imitation effects when the difference in orange body color in males is large. This experiment provides the first attempt to experimentally examine the relative strength of cultural and genetic preferences for a particular trait and suggests that these two factors moderate one another in shaping social behavior.

Protein-protein interaction: a genetic selection for compensating mutations at the barnase-barstar interface.

Barnase and barstar are trivial names of the extracellular RNase and its intracellular inhibitor produced by Bacillus amyloliquefaciens. Inhibition involves the formation of a very tight one-to-one complex of the two proteins. With the crystallographic solution of the structure of the barnase-barstar complex and the development of methods for measuring the free energy of binding, the pair can be used to study protein-protein recognition in detail. In this report, we describe the isolation of suppressor mutations in barstar that compensate for the loss in interaction energy caused by a mutation in barnase. Our suppressor search is based on in vivo selection for barstar variants that are able to protect host cells against the RNAse activity of those barnase mutants not properly inhibited by wild-type barstar. This approach utilizes a plasmid system in which barnase expression is tightly controlled to keep the mutant barnase gene silent. When expression of barnase is turned on, failure to form a complex between the mutant barnase and barstar has a lethal effect on host cells unless overcome by substitution of the wild-type barstar by a functional suppressor derivative. A set of barstar suppressors has been identified for barnase mutants with substitutions in two amino acid positions (residues 102 and 59), which are critically involved in both RNase activity and barstar binding. The mutations selected as suppressors could not have been predicted on the basis of the known protein structures. The single barstar mutation with the highest information content for inhibition of barnase (H102K) has the substitution Y30W. The reduction in binding caused by the R59E mutation in barnase can be partly reversed by changing Glu-76 of barstar, which forms a salt bridge with the Arg-59 in the wild-type complex, to arginine, thus completing an interchange of the two charges.

The roles of language processing in a spoken language interface.

This paper provides an overview of the colloquium's discussion session on natural language understanding, which followed presentations by M. Bates [Bates, M. (1995) Proc. Natl. Acad. Sci. USA 92, 9977-9982] and R. C. Moore [Moore, R. C. (1995) Proc. Natl. Acad. Sci. USA 92, 9983-9988]. The paper reviews the dual role of language processing in providing understanding of the spoken input and an additional source of constraint in the recognition process. To date, language processing has successfully provided understanding but has provided only limited (and computationally expensive) constraint. As a result, most current systems use a loosely coupled, unidirectional interface, such as N-best or a word network, with natural language constraints as a postprocess, to filter or resort the recognizer output. However, the level of discourse context provides significant constraint on what people can talk about and how things can be referred to; when the system becomes an active participant, it can influence this order. But sources of discourse constraint have not been extensively explored, in part because these effects can only be seen by studying systems in the context of their use in interactive problem solving. This paper argues that we need to study interactive systems to understand what kinds of applications are appropriate for the current state of technology and how the technology can move from the laboratory toward real applications.

Commercial applications of speech interface technology: an industry at the threshold.

Speech interface technology, which includes automatic speech recognition, synthetic speech, and natural language processing, is beginning to have a significant impact on business and personal computer use. Today, powerful and inexpensive microprocessors and improved algorithms are driving commercial applications in computer command, consumer, data entry, speech-to-text, telephone, and voice verification. Robust speaker-independent recognition systems for command and navigation in personal computers are now available; telephone-based transaction and database inquiry systems using both speech synthesis and recognition are coming into use. Large-vocabulary speech interface systems for document creation and read-aloud proofing are expanding beyond niche markets. Today's applications represent a small preview of a rich future for speech interface technology that will eventually replace keyboards with microphones and loud-speakers to give easy accessibility to increasingly intelligent machines.