RESUMEN
A wide range of de novo protein structure designs have been achieved, but the complexity of naturally occurring protein structures is still far beyond these designs. Here, to expand the diversity and complexity of de novo designed protein structures, we sought to develop a method for designing 'difficult-to-describe' α-helical protein structures composed of irregularly aligned α-helices like globins. Backbone structure libraries consisting of a myriad of α-helical structures with five or six helices were generated by combining 18 helix-loop-helix motifs and canonical α-helices, and five distinct topologies were selected for de novo design. The designs were found to be monomeric with high thermal stability in solution and fold into the target topologies with atomic accuracy. This study demonstrated that complicated α-helical proteins are created using typical building blocks. The method we developed will enable us to explore the universe of protein structures for designing novel functional proteins.
Asunto(s)
Pliegue de Proteína , Proteínas , Proteínas/química , Estructura Secundaria de Proteína , Conformación Proteica en Hélice alfaRESUMEN
A fundamental question in protein evolution is whether nature has exhaustively sampled nearly all possible protein folds throughout evolution, or whether a large fraction of the possible folds remains unexplored. To address this question, we defined a set of rules for ß-sheet topology to predict novel αß-folds and carried out a systematic de novo protein design exploration of the novel αß-folds predicted by the rules. The designs for all eight of the predicted novel αß-folds with a four-stranded ß-sheet, including a knot-forming one, folded into structures close to the design models. Further, the rules predicted more than 10,000 novel αß-folds with five- to eight-stranded ß-sheets; this number far exceeds the number of αß-folds observed in nature so far. This result suggests that a vast number of αß-folds are possible, but have not emerged or have become extinct due to evolutionary bias.
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Pliegue de Proteína , Proteínas , Estructura Secundaria de Proteína , Proteínas/química , Conformación Proteica en Lámina betaRESUMEN
Allostery produces concerted functions of protein complexes by orchestrating the cooperative work between the constituent subunits. Here we describe an approach to create artificial allosteric sites in protein complexes. Certain protein complexes contain subunits with pseudo-active sites, which are believed to have lost functions during evolution. Our hypothesis is that allosteric sites in such protein complexes can be created by restoring the lost functions of pseudo-active sites. We used computational design to restore the lost ATP-binding ability of the pseudo-active site in the B subunit of a rotary molecular motor, V1-ATPase. Single-molecule experiments with X-ray crystallography analyses revealed that binding of ATP to the designed allosteric site boosts this V1's activity compared with the wild-type, and the rotation rate can be tuned by modulating ATP's binding affinity. Pseudo-active sites are widespread in nature, and our approach shows promise as a means of programming allosteric control over concerted functions of protein complexes.
Asunto(s)
ATPasas de Translocación de Protón Vacuolares , Dominio Catalítico , Sitio Alostérico , Modelos Moleculares , ATPasas de Translocación de Protón Vacuolares/química , Adenosina Trifosfato/química , Sitios de UniónRESUMEN
G-protein coupled receptors (GPCRs) are known for their low stability and large conformational changes upon transitions between multiple states. A widely used method for stabilizing these receptors is to make chimeric receptors by fusing soluble proteins (i.e., fusion partner proteins) into the intracellular loop 3 (ICL3) connecting the transmembrane helices 5 and 6 (TM5 and TM6). However, this fusion approach requires experimental trial and error to identify appropriate soluble proteins, residue positions, and linker lengths for making the fusion. Moreover, this approach has not provided state-targeting stabilization of GPCRs. Here, to rationally stabilize a class A GPCR, adenosine A2A receptor (A2AR) in a target state, we carried out the custom-made de novo design of α-helical fusion partner proteins, which can fix the conformation of TM5 and TM6 to that in an inactive state of A2AR through straight helical connections without any kinks or intervening loops. The chimeric A2AR fused with one of the designs (FiX1) exhibited increased thermal stability. Moreover, compared with the wild type, the binding affinity of the chimera against the agonist NECA was significantly decreased, whereas that against the inverse agonist ZM241385 was similar, indicating that the inactive state was selectively stabilized. Our strategy contributes to the rational state-targeting stabilization of GPCRs.
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Agonistas del Receptor de Adenosina A2/metabolismo , Proteínas/metabolismo , Receptor de Adenosina A2A/química , Receptor de Adenosina A2A/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Adenosina/metabolismo , Agonistas del Receptor de Adenosina A2/química , Humanos , Ligandos , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación Proteica en Hélice alfa , Proteínas/química , Proteínas Recombinantes de Fusión/químicaRESUMEN
A wide range of de novo design of αß-proteins has been achieved based on the design rules, which describe secondary structure lengths and loop torsion patterns favorable for design target topologies. This paper proposes design rules for register shifts in ßαß-motifs, which have not been reported previously, but are necessary for determining a target structure of de novo design of αß-proteins. By analyzing naturally occurring protein structures in a database, we found preferences for register shifts in ßαß-motifs, and derived the following empirical rules: (1) register shifts must not be negative regardless of torsion types for a constituent loop in ßαß-motifs; (2) preferred register shifts strongly depend on the loop torsion types. To explain these empirical rules by physical interactions, we conducted physics-based simulations for systems mimicking a ßαß-motif that contains the most frequently observed loop type in the database. We performed an exhaustive conformational sampling of the loop region, imposing the exclusion volume and hydrogen bond satisfaction condition. The distributions of register shifts obtained from the simulations agreed well with those of the database analysis, indicating that the empirical rules are a consequence of physical interactions, rather than an evolutionary sampling bias. Our proposed design rules will serve as a guide to making appropriate target structures for the de novo design of αß-proteins.
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Secuencias de Aminoácidos , Proteínas/química , Simulación por Computador , Bases de Datos de Proteínas , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Péptidos/química , Unión Proteica , Estructura Secundaria de Proteína , Estadística como AsuntoRESUMEN
We previously elucidated principles for designing ideal proteins with completely consistent local and non-local interactions which have enabled the design of a wide range of new αß-proteins with four or fewer ß-strands. The principles relate local backbone structures to supersecondary-structure packing arrangements of α-helices and ß-strands. Here, we test the generality of the principles by employing them to design larger proteins with five- and six- stranded ß-sheets flanked by α-helices. The initial designs were monomeric in solution with high thermal stability, and the nuclear magnetic resonance (NMR) structure of one was close to the design model, but for two others the order of strands in the ß-sheet was swapped. Investigation into the origins of this strand swapping suggested that the global structures of the design models were more strained than the NMR structures. We incorporated explicit consideration of global backbone strain into the design methodology, and succeeded in designing proteins with the intended unswapped strand arrangements. These results illustrate the value of experimental structure determination in guiding improvement of de novo design, and the importance of consistency between local, supersecondary, and global tertiary interactions in determining protein topology. The augmented set of principles should inform the design of larger functional proteins.
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Ingeniería de Proteínas/métodos , Proteínas/química , Dicroismo Circular , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas/genéticaRESUMEN
Protein design provides a stringent test for our understanding of protein folding. We previously described principles for designing ideal protein structures stabilized by consistent local and nonlocal interactions, based on a set of rules relating local backbone structures to tertiary packing motifs. The principles have made possible the design of protein structures having various topologies with high thermal stability. Whereas nonlocal interactions such as tight hydrophobic core packing have traditionally been considered to be crucial for protein folding and stability, the rules proposed by our previous studies suggest the importance of local backbone structures to protein folding. In this study, we investigated the robustness of folding of de novo designed proteins to the reduction of the hydrophobic core, by extensive mutation of large hydrophobic residues (Leu, Ile) to smaller ones (Val) for one of the designs. Surprisingly, even after 10 Leu and Ile residues were mutated to Val, this mutant with the core mostly filled with Val was found to not be in a molten globule state and fold into the same backbone structure as the original design, with high stability. These results indicate the importance of local backbone structures to the folding ability and high thermal stability of designed proteins and suggest a method for engineering thermally stabilized natural proteins.
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Conformación Proteica , Ingeniería de Proteínas , Pliegue de Proteína , Proteínas/ultraestructura , Secuencia de Aminoácidos/genética , Sustitución de Aminoácidos/genética , Interacciones Hidrofóbicas e Hidrofílicas , Mutación/genética , Estabilidad Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas/química , Proteínas/genética , TermodinámicaRESUMEN
Here we developed an orange light-absorbing chromoprotein named ShadowR as a novel acceptor for performing fluorescence lifetime imaging microscopy-based Förster resonance energy transfer (FLIM-FRET) measurement in living cells. ShadowR was generated by replacing hydrophobic amino acids located at the surface of the chromoprotein Ultramarine with hydrophilic amino acids in order to reduce non-specific interactions with cytosolic proteins. Similar to Ultramarine, ShadowR shows high absorption capacity and no fluorescence. However, it exhibits reduced non-specific binding to cytosolic proteins and is highly expressed in HeLa cells. Using tandem constructs and a LOVTRAP system, we showed that ShadowR can be used as a FRET acceptor in combination with donor mRuby2 or mScarlet in HeLa cells. Thus, ShadowR is a useful, novel FLIM-FRET acceptor.
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Fenómenos Biofísicos , Fluorescencia , Proteínas Luminiscentes/química , Microscopía Fluorescente/métodos , Transferencia Resonante de Energía de Fluorescencia , Expresión Génica/genética , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Unión Proteica/genéticaRESUMEN
The successful de novo design of proteins can provide insights into the physical chemical basis of stability, the role of evolution in constraining amino acid sequences, and the production of customizable platforms for engineering applications. Previous guanidine hydrochloride (GdnHCl; an ionic denaturant) experiments of a designed, naturally occurring ßα fold, Di-III_14, revealed a cooperative, two-state unfolding transition and a modest stability. Continuous-flow mixing experiments in our laboratory revealed a simple two-state reaction in the microsecond to millisecond time range and consistent with the thermodynamic results. In striking contrast, the protein remains folded up to 9.25 M in urea, a neutral denaturant, and hydrogen exchange (HDX) NMR analysis in water revealed the presence of numerous high-energy states that interconvert on a time scale greater than seconds. The complex protection pattern for HDX corresponds closely with a pair of electrostatic networks on the surface and an extensive network of hydrophobic side chains in the interior of the protein. Mutational analysis showed that electrostatic and hydrophobic networks contribute to the resistance to urea denaturation for the WT protein; remarkably, single charge reversals on the protein surface restore the expected urea sensitivity. The roughness of the energy surface reflects the densely packed hydrophobic core; the removal of only two methyl groups eliminates the high-energy states and creates a smooth surface. The design of a very stable ßα fold containing electrostatic and hydrophobic networks has created a complex energy surface rarely observed in natural proteins.
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Guanidina/química , Pliegue de Proteína , Urea/química , Interacciones Hidrofóbicas e Hidrofílicas , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Electricidad EstáticaRESUMEN
Protein design holds promise for applications such as the control of cells, therapeutics, new enzymes and protein-based materials. Recently, there has been progress in rational design of protein molecules, and a lot of attempts have been made to create proteins with functions of our interests. The key to the progress is the development of methods for controlling desired protein tertiary structures with atomic-level accuracy. A theory for protein folding, the consistency principle, proposed by Nobuhiro Go in 1983, was a compass for the development. Anfinsen hypothesized that proteins fold into the free energy minimum structures, but Go further considered that local and non-local interactions in the free energy minimum structures are consistent with each other. Guided by the principle, we proposed a set of rules for designing ideal protein structures stabilized by consistent local and non-local interactions. The rules made possible designs of amino acid sequences with funnel-shaped energy landscapes toward our desired target structures. So far, various protein structures have been created using the rules, which demonstrates significance of our rules as intended. In this review, we briefly describe how the consistency principle impacts on our efforts for developing the design technology.
RESUMEN
Integrin αß heterodimer cell surface receptors mediate adhesive interactions that provide traction for cell migration. Here, we test whether the integrin, when engaged to an extracellular ligand and the cytoskeleton, adopts a specific orientation dictated by the direction of actin flow on the surface of migrating cells. We insert GFP into the rigid, ligand-binding head of the integrin, model with Rosetta the orientation of GFP and its transition dipole relative to the integrin head, and measure orientation with fluorescence polarization microscopy. Cytoskeleton and ligand-bound integrins orient in the same direction as retrograde actin flow with their cytoskeleton-binding ß-subunits tilted by applied force. The measurements demonstrate that intracellular forces can orient cell surface integrins and support a molecular model of integrin activation by cytoskeletal force. Our results place atomic, Å-scale structures of cell surface receptors in the context of functional and cellular, µm-scale measurements.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Membrana Celular/metabolismo , Movimiento Celular , Leucocitos/metabolismo , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Secuencia de Aminoácidos , Polarización de Fluorescencia/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Células Jurkat , Leucocitos/citología , Antígeno-1 Asociado a Función de Linfocito/genética , Microscopía Fluorescente/métodos , Unión Proteica , Homología de Secuencia de AminoácidoRESUMEN
Integrins are transmembrane receptors that, upon activation, bind extracellular ligands and link them to the actin filament (F-actin) cytoskeleton to mediate cell adhesion and migration. Cytoskeletal forces in migrating cells generated by polymerization- or contractility-driven "retrograde flow" of F-actin from the cell leading edge have been hypothesized to mediate integrin activation for ligand binding. This predicts that these forces should align and orient activated, ligand-bound integrins at the leading edge. Here, polarization-sensitive fluorescence microscopy of GFP-αVß3 integrins in fibroblasts shows that integrins are coaligned in a specific orientation within focal adhesions (FAs) in a manner dependent on binding immobilized ligand and a talin-mediated linkage to the F-actin cytoskeleton. These findings, together with Rosetta modeling, suggest that integrins in FA are coaligned and may be highly tilted by cytoskeletal forces. Thus, the F-actin cytoskeleton sculpts an anisotropic molecular scaffold in FAs, and this feature may underlie the ability of migrating cells to sense directional extracellular cues.
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Actinas/metabolismo , Citoesqueleto/metabolismo , Embrión de Mamíferos/metabolismo , Fibroblastos/metabolismo , Adhesiones Focales/metabolismo , Integrina alfaVbeta3/metabolismo , Actinas/genética , Animales , Línea Celular , Movimiento Celular/fisiología , Citoesqueleto/genética , Embrión de Mamíferos/citología , Fibroblastos/citología , Adhesiones Focales/genética , Integrina alfaVbeta3/genética , RatonesRESUMEN
We have previously shown that monomeric globular αß-proteins can be designed de novo with considerable control over topology, size, and shape. In this paper, we investigate the design of cyclic homo-oligomers from these starting points. We experimented with both keeping the original monomer backbones fixed during the cyclic docking and design process, and allowing the backbone of the monomer to conform to that of adjacent subunits in the homo-oligomer. The latter flexible backbone protocol generated designs with shape complementarity approaching that of native homo-oligomers, but experimental characterization showed that the fixed backbone designs were more stable and less aggregation prone. Designed C2 oligomers with ß-strand backbone interactions were structurally confirmed through x-ray crystallography and small-angle X-ray scattering (SAXS). In contrast, C3-C5 designed homo-oligomers with primarily nonpolar residues at interfaces all formed a range of oligomeric states. Taken together, our results suggest that for homo-oligomers formed from globular building blocks, improved structural specificity will be better achieved using monomers with increased shape complementarity and with more polar interfaces.
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Ingeniería de Proteínas , Subunidades de Proteína/química , Proteínas/química , Cristalografía por Rayos X , Interacciones Hidrofóbicas e Hidrofílicas , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Multimerización de Proteína , Dispersión del Ángulo Pequeño , Electricidad Estática , Difracción de Rayos XRESUMEN
We recently described general principles for designing ideal protein structures stabilized by completely consistent local and nonlocal interactions. The principles relate secondary structure patterns to tertiary packing motifs and enable design of different protein topologies. To achieve fine control over protein shape and size within a particular topology, we have extended the design rules by systematically analyzing the codependencies between the lengths and packing geometry of successive secondary structure elements and the backbone torsion angles of the loop linking them. We demonstrate the control afforded by the resulting extended rule set by designing a series of proteins with the same fold but considerable variation in secondary structure length, loop geometry, ß-strand registry, and overall shape. Solution NMR structures of four designed proteins for two different folds show that protein shape and size can be precisely controlled within a given protein fold. These extended design principles provide the foundation for custom design of protein structures performing desired functions.
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Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas/química , Secuencia de Aminoácidos , Diseño Asistido por Computadora , Cristalografía por Rayos X , Espectroscopía de Resonancia Magnética/métodos , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Ingeniería de Proteínas/métodos , Proteínas/clasificación , Proteínas/genética , Reproducibilidad de los Resultados , SolucionesRESUMEN
Protein-based hydrogels usually do not exhibit high stretchability or toughness, significantly limiting the scope of their potential biomedical applications. Here we report the engineering of a chemically cross-linked, highly elastic and tough protein hydrogel using a mechanically extremely labile, de novo-designed protein that assumes the classical ferredoxin-like fold structure. Due to the low mechanical stability of the ferredoxin-like fold structure, swelling of hydrogels causes a significant fraction of the folded domains to unfold. Subsequent collapse and aggregation of unfolded ferredoxin-like domains leads to intertwining of physically and chemically cross-linked networks, entailing hydrogels with unusual physical and mechanical properties: a negative swelling ratio, high stretchability and toughness. These hydrogels can withstand an average strain of 450% before breaking and show massive energy dissipation. Upon relaxation, refolding of the ferredoxin-like domains enables the hydrogel to recover its massive hysteresis. This novel biomaterial may expand the scope of hydrogel applications in tissue engineering.
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Hidrogeles/química , Desplegamiento Proteico , Proteínas/química , Materiales Biocompatibles/química , Dicroismo Circular , Cisteína/química , Elasticidad , Ferredoxinas/química , Pinzas Ópticas , Ingeniería de Proteínas , Estrés Mecánico , Resistencia a la Tracción , Ingeniería de TejidosRESUMEN
Unlike random heteropolymers, natural proteins fold into unique ordered structures. Understanding how these are encoded in amino-acid sequences is complicated by energetically unfavourable non-ideal features--for example kinked α-helices, bulged ß-strands, strained loops and buried polar groups--that arise in proteins from evolutionary selection for biological function or from neutral drift. Here we describe an approach to designing ideal protein structures stabilized by completely consistent local and non-local interactions. The approach is based on a set of rules relating secondary structure patterns to protein tertiary motifs, which make possible the design of funnel-shaped protein folding energy landscapes leading into the target folded state. Guided by these rules, we designed sequences predicted to fold into ideal protein structures consisting of α-helices, ß-strands and minimal loops. Designs for five different topologies were found to be monomeric and very stable and to adopt structures in solution nearly identical to the computational models. These results illuminate how the folding funnels of natural proteins arise and provide the foundation for engineering a new generation of functional proteins free from natural evolution.
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Simulación por Computador , Modelos Moleculares , Pliegue de Proteína , Estabilidad Proteica , Proteínas/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , TermodinámicaRESUMEN
Macromolecular modeling and design are increasingly useful in basic research, biotechnology, and teaching. However, the absence of a user-friendly modeling framework that provides access to a wide range of modeling capabilities is hampering the wider adoption of computational methods by non-experts. RosettaScripts is an XML-like language for specifying modeling tasks in the Rosetta framework. RosettaScripts provides access to protocol-level functionalities, such as rigid-body docking and sequence redesign, and allows fast testing and deployment of complex protocols without need for modifying or recompiling the underlying C++ code. We illustrate these capabilities with RosettaScripts protocols for the stabilization of proteins, the generation of computationally constrained libraries for experimental selection of higher-affinity binding proteins, loop remodeling, small-molecule ligand docking, design of ligand-binding proteins, and specificity redesign in DNA-binding proteins.
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Modelos Moleculares , Lenguajes de Programación , Programas Informáticos , Interfaz Usuario-Computador , Proteínas de Unión al ADN/química , Ligandos , Conformación ProteicaRESUMEN
Protein-design methodology can now generate models of protein structures and interfaces with computed energies in the range of those of naturally occurring structures. Comparison of the properties of native structures and complexes to isoenergetic design models can provide insight into the properties of the former that reflect selection pressure for factors beyond the energy of the native state. We report here that sidechains in native structures and interfaces are significantly more constrained than designed interfaces and structures with equal computed binding energy or stability, which may reflect selection against potentially deleterious non-native interactions.
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Conformación Proteica , Proteínas/química , Simulación por Computador , Bases de Datos de Proteínas , Unión Proteica , Proteínas/genética , TermodinámicaRESUMEN
For simulating proteins at work in millisecond time scale or longer, we develop a coarse-grained (CG) molecular dynamics (MD) method and software, CafeMol. At the resolution of one-particle-per-residue, CafeMol equips four structure-based protein models: (1) the off-lattice Go model, (2) the atomic interaction based CG model for native state and folding dynamics, (3) the multiple-basin model for conformational change dynamics, and (4) the elastic network model for quasiharmonic fluctuations around the native structure. Ligands can be treated either explicitly or implicitly. For mimicking functional motions of proteins driven by some external force, CafeMol has various and flexible means to "switch" the energy functions that induce active motions of the proteins. CafeMol can do parallel computation with modest sized PC clusters. We describe CafeMol methods and illustrate it with several examples, such as rotary motions of F1-ATPase and drug exports from a transporter. The CafeMol source code is available at www.cafemol.org .
RESUMEN
Hexameric ring-shaped AAA+ molecular motors have a key function of active translocation of a macromolecular chain through the central pore. By performing multiscale molecular dynamics (MD) simulations, we revealed that HslU, a AAA+ motor in a bacterial homologue of eukaryotic proteasome, translocates its substrate polypeptide via paddling mechanism during ATP-driven cyclic conformational changes. First, fully atomistic MD simulations showed that the HslU pore grips the threaded signal peptide by the highly conserved Tyr-91 and Val-92 firmly in the closed form and loosely in the open form of the HslU. The grip depended on the substrate sequence. These features were fed into a coarse-grained MD, and conformational transitions of HslU upon ATP cycles were simulated. The simulations exhibited stochastic unidirectional translocation of a polypeptide. This unidirectional translocation is attributed to paddling motions of Tyr-91s between the open and the closed forms: downward motions of Tyr-91s with gripping the substrate and upward motions with slipping on it. The paddling motions were caused by the difference between the characteristic time scales of the pore-radius change and the up-down displacements of Tyr-91s. Computational experiments on mutations at the pore and the substrate were in accord with several experiments.