RESUMEN
The force required to rupture the streptavidin-biotin complex was calculated here by computer simulations. The computed force agrees well with that obtained by recent single molecule atomic force microscope experiments. These simulations suggest a detailed multiple-pathway rupture mechanism involving five major unbinding steps. Binding forces and specificity are attributed to a hydrogen bond network between the biotin ligand and residues within the binding pocket of streptavidin. During rupture, additional water bridges substantially enhance the stability of the complex and even dominate the binding interactions. In contrast, steric restraints do not appear to contribute to the binding forces, although conformational motions were observed.
Asunto(s)
Proteínas Bacterianas/química , Biotina/química , Simulación por Computador , Modelos Químicos , Fenómenos Químicos , Química Física , Enlace de Hidrógeno , Ligandos , Microscopía de Fuerza Atómica , Conformación Molecular , Conformación Proteica , Estreptavidina , TermodinámicaRESUMEN
Infrared spectroscopic measurements of hydration of the stratum corneum before and after stripping the skin five and ten times with scotch tape are reported. Investigations on 64 healthy persons show that for individuals over 45 and under 15 years of age the range of the measurement values is strikingly larger than for persons in the age group 15 - 45 years of age. A small, but not significant reduction in the measurement values is found in females as compared to males. A comparison of the clinically unaffected skin of atopic dermatitis patients with the skin of normal persons points to an increase in the hydration of the stratum corneum of atopic dermatitis patients, which is especially evident in patients who show clinical evidence of "dry" and rough skin.
Asunto(s)
Agua Corporal/análisis , Dermatitis Atópica/metabolismo , Piel/análisis , Adolescente , Adulto , Anciano , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Enfermedades de la Piel/metabolismo , Espectrofotometría InfrarrojaAsunto(s)
Alcoholismo/fisiopatología , Pulmón/fisiopatología , Adulto , Alcoholismo/sangre , Alcoholismo/complicaciones , Bronquitis/inducido químicamente , Bronquitis/etiología , Monóxido de Carbono , Susceptibilidad a Enfermedades , Disnea/inducido químicamente , Disnea/etiología , Etanol/efectos adversos , Femenino , Humanos , Cirrosis Hepática/complicaciones , Pulmón/efectos de los fármacos , Enfermedades Pulmonares/complicaciones , Enfermedades Pulmonares Obstructivas/inducido químicamente , Enfermedades Pulmonares Obstructivas/etiología , Masculino , Persona de Mediana Edad , Capacidad de Difusión Pulmonar , Pruebas de Función Respiratoria , Albúmina Sérica/análisis , Fumar/complicaciones , Espirometría , Capacidad VitalRESUMEN
Unbinding of a spin-labeled dinitrophenyl (DNP) hapten from the monoclonal antibody AN02 F(ab) fragment has been studied by force probe molecular dynamics (FPMD) simulations. In our nanosecond simulations, unbinding was enforced by pulling the hapten molecule out of the binding pocket. Detailed inspection of the FPMD trajectories revealed a large heterogeneity of enforced unbinding pathways and a correspondingly large flexibility of the binding pocket region, which exhibited induced fit motions. Principal component analyses were used to estimate the resulting entropic contribution of approximately 6 kcal/mol to the AN02/DNP-hapten bond. This large contribution may explain the surprisingly large effect on binding kinetics found for mutation sites that are not directly involved in binding. We propose that such "entropic control" optimizes the binding kinetics of antibodies. Additional FPMD simulations of two point mutants in the light chain, Y33F and I96K, provided further support for a large flexibility of the binding pocket. Unbinding forces were found to be unchanged for these two mutants. Structural analysis of the FPMD simulations suggests that, in contrast to free energies of unbinding, the effect of mutations on unbinding forces is generally nonadditive.
Asunto(s)
Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/metabolismo , Simulación por Computador , Entropía , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Complejo Antígeno-Anticuerpo/genética , Sitios de Unión , Cristalografía por Rayos X , Haptenos/química , Haptenos/metabolismo , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/metabolismo , Cadenas Ligeras de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/metabolismo , Cinética , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica , Solventes , Marcadores de Spin , Electricidad EstáticaRESUMEN
Recent advances in atomic force microscopy, biomembrane force probe experiments, and optical tweezers allow one to measure the response of single molecules to mechanical stress with high precision. Such experiments, due to limited spatial resolution, typically access only one single force value in a continuous force profile that characterizes the molecular response along a reaction coordinate. We develop a theory that allows one to reconstruct force profiles from force spectra obtained from measurements at varying loading rates, without requiring increased resolution. We show that spectra obtained from measurements with different spring constants contain complementary information.
RESUMEN
Recent developments in piconewton instrumentation allow the manipulation of single molecules and measurements of intermolecular as well as intramolecular forces. Dextran filaments linked to a gold surface were probed with the atomic force microscope tip by vertical stretching. At low forces the deformation of dextran was found to be dominated by entropic forces and can be described by the Langevin function with a 6 angstrom Kuhn length. At elevated forces the strand elongation was governed by a twist of bond angles. At higher forces the dextran filaments underwent a distinct conformational change. The polymer stiffened and the segment elasticity was dominated by the bending of bond angles. The conformational change was found to be reversible and was corroborated by molecular dynamics calculations.