Direct detection of the myosin super-relaxed state and interacting-heads motif in solution

Abstract

The interacting-heads motif (IHM) is a structure of myosin that has been proposed to modulate cardiac output by occluding myosin molecules from undergoing the force-generating cycle. It is hypothesized to be the structural basis for the super-relaxed state (SRX), a low-ATPase kinetic state thought to be cardioprotective. The goal of the present study was to test this hypothesis by determining directly and quantitatively the fractions of myosin in the IHM and SRX under the same conditions in solution. To detect the structural IHM, we used time-resolved fluorescence resonance energy transfer to quantitate two distinct populations. One population was observed at a center distance of 2.0 nm, whereas the other was not detectable by fluorescence resonance energy transfer, implying a distance greater than 4 nm. We confirmed the IHM assignment to the 2.0-nm population by applying the same cross-linking protocol used previously to image the IHM by electron microscopy. Under the same conditions, we also measured the fraction of myosin in the SRX using stopped-flow kinetics. Our results show that the populations of SRX and IHM myosin were similar, a that to and is proposed to by the SRX and we that a greater the SRX than the IHM We that the IHM structure is not to the SRX kinetic The interacting-heads motif (IHM) is a structure of myosin that has been proposed to modulate cardiac output by occluding myosin molecules from undergoing the force-generating cycle. It is hypothesized to be the structural basis for the super-relaxed state (SRX), a low-ATPase kinetic state thought to be cardioprotective. The goal of the present study was to test this hypothesis by determining directly and quantitatively the fractions of myosin in the IHM and SRX under the same conditions in solution. To detect the structural IHM, we used time-resolved fluorescence resonance energy transfer to quantitate two distinct populations. One population was observed at a center distance of 2.0 nm, whereas the other was not detectable by fluorescence resonance energy transfer, implying a distance greater than 4 nm. We confirmed the IHM assignment to the 2.0-nm population by applying the same cross-linking protocol used previously to image the IHM by electron microscopy. Under the same conditions, we also measured the fraction of myosin in the SRX using stopped-flow kinetics. Our results show that the populations of SRX and IHM myosin were similar, a that to and is proposed to by the SRX and we that a greater the SRX than the IHM We that the IHM structure is not to the SRX kinetic is a that the of structural is by and the It of a of two the and and myosin to of the of the The from the to two a and a that and of the of the has to in myosin two It is proposed that the structural basis for this state is the interacting-heads motif (IHM) of a myosin in the motif has been a of myosin the of from a myosin interacting-heads motif and and for myosin in for the IHM has been in of myosin from electron motif has been a of myosin the of and for myosin in structure of cardiac myosin structure in and has been observed by by is by in myosin not the state of myosin cardiac and fluorescence is a in of the of in the IHM, the of the myosin of the the of the motif has been a of myosin the of of in the IHM is the of the is proposed to the of the to the The and the motif of myosin The myosin and the basis of by that in this the of the for the IHM The myosin and the basis of by The present study a of the of the that the the and other that the has been hypothesized that the IHM is the structural basis for the super-relaxed state (SRX), a state by than and to than is for of the myosin molecules in state of cardiac myosin in the is a in in SRX is in the in fluorescence that is from and by in a in the is to the SRX state state of cardiac myosin in the kinetic has also been observed in myosin molecules and state of cardiac It is that the SRX kinetic state to the IHM structural this has not been directly the same under this we used fluorescence resonance energy transfer the two of myosin to detect the IHM structural state directly in and directly the fraction of myosin molecules in SRX measured by under to time-resolved distinct populations of to a distinct of To that is the IHM, we used a that is to the the IHM and we the and the structural were by a that has for and has been to the SRX population state of cardiac the state of myosin and the of of from myosin molecules to the state of we that by the fraction of myosin in We also a of to the SRX population to the the IHM and SRX state of cardiac myosin in the of cardiac myosin was and the of the by the and the in state of cardiac To the the of to was to detectable not the and the interacting-heads and for and and of from of the and in the fraction of the population in image the state of fluorescence of was by a of the were by a to the whereas the to a distance a center of 2.0 a of We that myosin molecules the a distance that is to detect and that of the myosin molecules in the IHM state To that we the IHM, we used a of myosin to the population of IHM SRX population the of IHM myosin by test the of IHM, was a used to the IHM in electron motif has been a of myosin the of We that a a was also in the same to a fraction and a fraction in we that the fraction in from to The myosin molecules in is the to we the myosin molecules in the to not the test the SRX and the IHM the same myosin was under conditions to the The of were previously to the SRX population measured by stopped-flow state of cardiac the SRX and the IHM in the not at to super-relaxed not the from of and in of to to be not from fluorescence resonance energy image a of the the population than is a used to It has been to myosin and electron has that cardiac myosin the cross-linking of was in the IHM state state of cardiac this we that a of the from to the IHM is by the was to and by of than in a a of and of The to a a in the population in The myosin the same to in the population in the myosin in the was to the population in was not from the IHM population also the SRX is of greater the SRX a in a in super-relaxed state population in of of of myosin 4 and fluorescence resonance energy image of cardiac to a of motif has been a of myosin the of we directly observed the IHM of using and We that of the myosin molecules in the IHM structural state The SRX measured by has been to from to in myosin of state of cardiac the state of myosin and the of of from myosin molecules to and the of We that of the myosin was SRX in the stopped-flow is the of the IHM myosin in the we to the SRX population state of cardiac not results that the IHM is not for the of the distance in of the motif of of from a myosin interacting-heads motif and The myosin and the basis of by results from of cardiac myosin and is nm, is than the center distance by the of is that of the that this than the of the this is not results from of cardiac myosin and the also to this were to the of myosin this that of the myosin molecules of at 4 by The myosin and the basis of by the distance to the IHM by myosin a used to the IHM structure in myosin for electron motif has been a of myosin the of We that and the IHM this we a of myosin that not the of the that is hypothesized to the be to to the IHM in also that the of to myosin of cardiac myosin of the myosin and for of we hypothesized that the of myosin in the SRX is of the cycle. Our and that the fraction of the SRX population by of myosin state of cardiac the state of myosin and the of of from myosin molecules to we observed that not the of the IHM state by study the structural state of IHM under conditions to used previously to detect results the hypothesis that is a the IHM structural state and the SRX Our results to structural to the IHM and the other from the we that not cardiac myosin in the in cardiac a of that myosin by the and in the of cardiac myosin of the myosin The of also the to Our results show that the structure to that the population of myosin in SRX that in the is a population of SRX myosin thought to be that not the IHM myosin was from from in the of the cardiac myosin of the of myosin to and in of and a of The was at 4 was in the of the cardiac myosin myosin was to in and for at by of by by were for cardiac a at was in and by by in the of the cardiac myosin We the of a at 4 and by The was in and at was by and was confirmed using and were 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and fluorescence was measured be to is a that the of structural is by and the It of a of two the and and myosin to of the of the The from the to two a and a that and The of the of the has to in myosin two It is proposed that the structural basis for this state is the interacting-heads motif (IHM) of a myosin in the motif has been a of myosin the of from a myosin interacting-heads motif and and for myosin in for the IHM has been in of myosin from electron motif has been a of myosin the of and for myosin in structure of cardiac myosin structure in and has been observed by by is by in myosin not the state of myosin cardiac and fluorescence is a in of the of in the IHM, the of the myosin of the the of the motif has been a of myosin the of of in the IHM is the of the is proposed to the of the to the The and the motif of myosin The myosin and the basis of by that in this the of the for the IHM The myosin and the basis of by The present study a of the of the that the the and other that the It has been hypothesized that the IHM is the structural basis for the super-relaxed state (SRX), a state by than and to than is for of the myosin molecules in state of cardiac myosin in the is a in in SRX is in the in fluorescence that is from and by in a in the is to the SRX state state of cardiac myosin in the kinetic has also been observed in myosin molecules and state of cardiac It is that the SRX kinetic state to the IHM structural this has not been directly the same under To this we used fluorescence resonance energy transfer the two of myosin to detect the IHM structural state directly in and directly the fraction of myosin molecules in SRX measured by under to time-resolved distinct populations of to a distinct of To that is the IHM, we used a that is to the To the IHM and we the and the structural were by a that has for and has been to the SRX population state of cardiac the state of myosin and the of of from myosin molecules to the state of we that by the fraction of myosin in We also a of to the SRX population to the the IHM and SRX state of cardiac myosin in the of cardiac myosin was and the of the by the and the in state of cardiac To the the of to was to detectable not the and the state of fluorescence of was by a of the were by a to the whereas the to a distance a center of 2.0 a of We that myosin molecules the a distance that is to detect and that of the myosin molecules in the IHM state To that we the IHM, we used a of myosin to the population of IHM SRX population the of IHM myosin by test the of IHM, was a used to the IHM in electron motif has been a of myosin the of We that a a was also in the same to a fraction and a fraction in we that the fraction in from to The myosin molecules in is the to we the myosin molecules in the to not the test the SRX and the IHM the same myosin was under conditions to the The of were previously to the SRX population measured by stopped-flow state of cardiac the SRX and the IHM in the not at to super-relaxed not the from of and in of to to be not from fluorescence resonance energy image a of the the population than is a used to It has been to myosin and electron has that cardiac myosin the cross-linking of was in the IHM state state of cardiac this we that a of the from to the IHM is by the was to and by of than in a a of and of The to a a in the population in The myosin the same to in the population in the myosin in the was to the population in was not from the IHM population also the SRX is of greater the SRX a in a in super-relaxed state population in of of of myosin 4 and fluorescence resonance energy image myosin in the of cardiac myosin was and the of the by the and the in state of cardiac To the the of to was to detectable not the and cardiac myosin was and the of the by the and the in state of cardiac To the the of to was to detectable not the and the state of fluorescence of was by a of the were by a to the whereas the to a distance a center of 2.0 a of We that myosin molecules the a distance that is to detect and that of the myosin molecules in the IHM state To that we the IHM, we used a of myosin to the population of IHM SRX fluorescence of was by a of the were by a to the whereas the to a distance a center of 2.0 a of We that myosin molecules the a distance that is to detect and that of the myosin molecules in the IHM state To that we the IHM, we used a of myosin to the population of IHM SRX The population the of IHM myosin by test the of IHM, was a used to the IHM in electron motif has been a of myosin the of We that a a was also in the same to a fraction and a fraction in we that the fraction in from to The myosin molecules in is the to we the myosin molecules in the To test the of IHM, was a used to the IHM in electron motif has been a of myosin the of We that a a was also in the same to a fraction and a fraction in we that the fraction in from to The myosin molecules in is the to we the myosin molecules in the to not the test the SRX and the IHM the same myosin was under conditions to the The of were previously to the SRX population measured by stopped-flow state of cardiac the SRX and the IHM in the not at To test the SRX and the IHM the same myosin was under conditions to the The of were previously to the SRX population measured by stopped-flow state of cardiac the SRX and the IHM in the not at a of the the population than is a used to It has been to myosin and electron has that cardiac myosin the cross-linking of was in the IHM state state of cardiac this we that a of the from to the IHM is by the was to and by of than in a a of and of The to a a in the population in The myosin the same to in the population in the myosin in the was to the population in was not from the IHM population also the SRX is of greater the SRX is a used to It has been to myosin and electron has that cardiac myosin the cross-linking of was in the IHM state state of cardiac this we that a of the from to the IHM is by the was to and by of than in a a of and of The to a a in the population in The myosin the same to in the population in the myosin in the was to the population in was not from the IHM population also the SRX is of greater the SRX of cardiac to a of motif has been a of myosin the of we directly observed the IHM of using and We that of the myosin molecules in the IHM structural state The SRX measured by has been to from to in myosin of state of cardiac the state of myosin and the of of from myosin molecules to and the of We that of the myosin was SRX in the stopped-flow is the of the IHM myosin in the we to the SRX population state of cardiac not results that the IHM is not for the of the distance in of the motif of of from a myosin interacting-heads motif and The myosin and the basis of by results from of cardiac myosin and is nm, is than the center distance by the of is that of the that this than the of the this is not results from of cardiac myosin and the also to this were to the of myosin this that of the myosin molecules of at 4 by The myosin and the basis of by the distance to the IHM by myosin a used to the IHM structure in myosin for electron motif has been a of myosin the of We that and the IHM this we a of myosin that not the of the that is hypothesized to the be to to the IHM in also that the of to myosin of cardiac myosin of the myosin and for of we hypothesized that the of myosin in the SRX is of the cycle. Our and that the fraction of the SRX population by of myosin state of cardiac the state of myosin and the of of from myosin molecules to we observed that not the of the IHM state by study the structural state of IHM under conditions to used previously to detect results the hypothesis that is a the IHM structural state and the SRX Our results to structural to the IHM and the other from the we that not cardiac myosin in the in cardiac a of that myosin by the and in the of cardiac myosin of the myosin The of also the to Our results show that the structure to that the population of myosin in SRX that in the is a population of SRX myosin thought to be that not the IHM of cardiac to a of motif has been a of myosin the of we directly observed the IHM of using and We that of the myosin molecules in the IHM structural state The SRX measured by has been to from to in myosin of state of cardiac the state of myosin and the of of from myosin molecules to and the of We that of the myosin was SRX in the stopped-flow is the of the IHM myosin in the we to the SRX population state of cardiac not results that the IHM is not for the of the The distance in of the motif of of from a myosin interacting-heads motif and The myosin and the basis of by results from of cardiac myosin and is nm, is than the center distance by the of is that of the that this than the of the this is not results from of cardiac myosin and the also to this We were to the of myosin this that of the myosin molecules of at 4 by The myosin and the basis of by We the distance to the IHM by myosin a used to the IHM structure in myosin for electron motif has been a of myosin the of We that and the IHM this we a of myosin that not the of the that is hypothesized to the be to to the IHM in also that the of to myosin of cardiac myosin of the myosin and for of we hypothesized that the of myosin in the SRX is of the cycle. Our and that the fraction of the SRX population by of myosin state of cardiac the state of myosin and the of of from myosin molecules to we observed that not the of the IHM state by study the structural state of IHM under conditions to used previously to detect results the hypothesis that is a the IHM structural state and the SRX Our results to structural to the IHM and the other from the we that not cardiac myosin in the in cardiac a of that myosin by the and in the of cardiac myosin of the myosin The of also the to Our results show that the structure to that the population of myosin in SRX that in the is a population of SRX myosin thought to be that not the IHM myosin was from from in the of the cardiac myosin of the of myosin to and in of and a of The was at 4 was in the of the cardiac myosin myosin was to in and for at by of by by were for cardiac a at was in and by by in the of the cardiac myosin We the of a at 4 and by The was in and at was by and was confirmed using and were using to protocol The was a a of and to the is from to was by myosin and to myosin was 4 of in for the and and for at the to from The of of the the myosin was to in to for at by of and was by and confirmed by to were measured by a was using a and a of were measured and and at distinct were was at the and for in and were a for The was from were by fluorescence by to a the of the cardiac myosin The fluorescence be by a of is the fluorescence of the were the of the and and in and the were the The of was by to from to the to The and was to structural the of and center distance and of The that was The was also to a in the fraction of the population in is to the fraction of in of cardiac myosin was measured using at in and in a The and The of to was measured a by at for The was from the by and by the of in were at in 4 and fluorescence fluorescence was stopped-flow of and were at a and and were a The for this is were and for under was at and in fluorescence nm, was measured in a by a was for and fluorescence was measured myosin was from from in the of the cardiac myosin of the of myosin to and in of and a of The was at 4 was in the of the cardiac myosin myosin was to in and for at by of by by were for cardiac a at was in and by by in the of the cardiac myosin We the of a at 4 and by The was in and at was by and was confirmed using and were using to protocol The was a a of and to the is from to was by myosin and to myosin was 4 of in for the and and for at the to from The of of the the myosin was to in to for at by of and was by and confirmed by to myosin was from from in the of the cardiac myosin of the of myosin to and in of and a of The was at 4 was in the of the cardiac myosin myosin was to in and for at by of by by were for cardiac a at was in and by by in the of the cardiac myosin We the of a at 4 and by The was in and at was by and was confirmed using and were using to protocol The was a a of and to the is from to was by myosin and to myosin was 4 of in for the and and for at the to from The of of the the myosin was to in to for at by of and was by and confirmed by to were measured by a was using a and a of were measured and and at distinct were was at the and for in and were a for The was from were by fluorescence by to a the of the cardiac myosin The fluorescence be by a of is the fluorescence of the were the of the and and in and the were the The of was by to from to the to The and was to structural the of and center distance and of The that was The was also to a in the fraction of the population in is to the fraction of in were measured by a was using a and a of were measured and and at distinct were was at the and for in and were a for The was from were by fluorescence by to a the of the cardiac myosin The fluorescence be by a of is the fluorescence of the were the of the and and in and the were the The of was by to from to the to The and was to structural the of and center distance and of The that was The was also to a in the fraction of the population in is to the fraction of in of cardiac myosin was measured using at in and in a The and The of to was measured a by at for The was from the by and by the of in The of cardiac myosin was measured using at in and in a The and The of to was measured a by at for The was from the by and by the of in were at in 4 and fluorescence fluorescence was stopped-flow of and were at a and and were a The for this is were and for under were at in 4 and fluorescence fluorescence was stopped-flow of and were at a and and were a The for this is were and for under was at and in fluorescence nm, was measured in a by a was for and fluorescence was measured was at and in fluorescence nm, was measured in a by a was for and fluorescence was measured be to be to and been and by the of in this other that of to the of this were at the of to the of of for of the stopped-flow and the of for and We and for We for and was by to was by to The is the of the and not the of the of

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