Thomas C. Irving

The In Situ Supermolecular Structure of Type I Collagen

BACKGROUND The proteins belonging to the collagen family are ubiquitous throughout the animal kingdom. The most abundant collagen, type I, readily forms fibrils that convey the principal mechanical support and structural organization in the extracellular matrix of connective tissues such as bone, skin, tendon, and vasculature. An understanding of the molecular arrangement of collagen in fibrils is essential since it relates molecular interactions to the mechanical strength of fibrous tissues and may reveal the underlying molecular pathology of numerous connective tissue diseases. RESULTS Using synchrotron radiation, we have conducted a study of the native fibril structure at anisotropic resolution (5.4 A axial and 10 A lateral). The intensities of the tendon X-ray diffraction pattern that arise from the lateral packing (three-dimensional arrangement) of collagen molecules were measured by using a method analogous to Rietveld methods in powder crystallography and to the separation of closely spaced peaks in Laue diffraction patterns. These were then used to determine the packing structure of collagen by MIR. CONCLUSIONS Our electron density map is the first obtained from a natural fiber using these techniques (more commonly applied to single crystal crystallography). It reveals the three-dimensional molecular packing arrangement of type I collagen and conclusively proves that the molecules are arranged on a quasihexagonal lattice. The molecular segments that contain the telopeptides (central to the function of collagen fibrils in health and disease) have been identified, revealing that they form a corrugated arrangement of crosslinked molecules that strengthen and stabilize the native fibril.

Skeletal muscle performance determined by modulation of number of myosin motors rather than motor force or stroke size.

Skeletal muscle can bear a high load at constant length, or shorten rapidly when the load is low. This force-velocity relationship is the primary determinant of muscle performance in vivo. Here we exploited the quasi-crystalline order of myosin II motors in muscle filaments to determine the molecular basis of this relationship by X-ray interference and mechanical measurements on intact single cells. We found that, during muscle shortening at a wide range of velocities, individual myosin motors maintain a force of about 6 pN while pulling an actin filament through a 6 nm stroke, then quickly detach when the motor reaches a critical conformation. Thus we show that the force-velocity relationship is primarily a result of a reduction in the number of motors attached to actin in each filament in proportion to the filament load. These results explain muscle performance and efficiency in terms of the molecular mechanism of the myosin motor.

Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development

Phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) and myosin binding protein‐C (cMyBP‐C) by protein kinase A (PKA) independently accelerate the kinetics of force development in ventricular myocardium. However, while MLCK treatment has been shown to increase the Ca2+ sensitivity of force (pCa50), PKA treatment has been shown to decrease pCa50, presumably due to cardiac troponin I phosphorylation. Further, MLCK treatment increases Ca2+‐independent force and maximum Ca2+‐activated force, whereas PKA treatment has no effect on either force. To investigate the structural basis underlying the kinase‐specific differential effects on steady‐state force, we used synchrotron low‐angle X‐ray diffraction to compare equatorial intensity ratios (I1,1/I1,0) to assess the proximity of myosin cross‐bridge mass relative to actin and to compare lattice spacings (d1,0) to assess the inter‐thick filament spacing in skinned myocardium following treatment with either MLCK or PKA. As we showed previously, PKA phosphorylation of cMyBP‐C increases I1,1/I1,0 and, as hypothesized, treatment with MLCK also increased I1,1/I1,0, which can explain the accelerated rates of force development during activation. Importantly, interfilament spacing was reduced by ∼2 nm (Δ 3.5%) with MLCK treatment, but did not change with PKA treatment. Thus, RLC or cMyBP‐C phosphorylation increases the proximity of cross‐bridges to actin, but only RLC phosphorylation affects lattice spacing, which suggests that RLC and cMyBP‐C modulate the kinetics of force development by similar structural mechanisms; however, the effect of RLC phosphorylation to increase the Ca2+ sensitivity of force is mediated by a distinct mechanism, most probably involving changes in interfilament spacing.

Shape of tropoelastin, the highly extensible protein that controls human tissue elasticity

Elastin enables the reversible deformation of elastic tissues and can withstand decades of repetitive forces. Tropoelastin is the soluble precursor to elastin, the main elastic protein found in mammals. Little is known of the shape and mechanism of assembly of tropoelastin as its unique composition and propensity to self-associate has hampered structural studies. In this study, we solve the nanostructure of full-length and corresponding overlapping fragments of tropoelastin using small angle X-ray and neutron scattering, allowing us to identify discrete regions of the molecule. Tropoelastin is an asymmetric coil, with a protruding foot that encompasses the C-terminal cell interaction motif. We show that individual tropoelastin molecules are highly extensible yet elastic without hysteresis to perform as highly efficient molecular nanosprings. Our findings shed light on how biology uses this single protein to build durable elastic structures that allow for cell attachment to an appended foot. We present a unique model for head-to-tail assembly which allows for the propagation of the molecule’s asymmetric coil through a stacked spring design.

Titin Isoform Variance and Length Dependence of Activation in Skinned Bovine Cardiac Muscle

We have explored the role of the giant elastic protein titin in the Frank‐Starling mechanism of the heart by measuring the sarcomere length (SL) dependence of activation in skinned cardiac muscles with different titin‐based passive stiffness characteristics. We studied muscle from the bovine left ventricle (BLV), which expresses a high level of a stiff titin isoform, and muscle from the bovine left atrium (BLA), which expresses more compliant titin isoforms. Passive tension was also varied in each muscle type by manipulating the pre‐history of stretch prior to activation. We found that the SL‐dependent increases in Ca2+ sensitivity and maximal Ca2+‐activated tension were markedly more pronounced when titin‐based passive tension was high. Small‐angle X‐ray diffraction experiments revealed that the SL dependence of reduction of interfilament lattice spacing is greater in BLV than in BLA and that the lattice spacing is coupled with titin‐based passive tension. These results support the notion that titin‐based passive tension promotes actomyosin interaction by reducing the lattice spacing. This work indicates that titin may be a factor involved in the Frank‐Starling mechanism of the heart by promoting actomyosin interaction in response to stretch.

Length-dependent activation in three striated muscle types of the rat.

The process whereby sarcomere length modulates the sensitivity of the myofilaments to Ca2+ is termed length‐dependent activation. Length‐dependent activation is a property of all striated muscles, yet the relative extent of length‐dependent activation between skeletal muscle and cardiac muscle is unclear. Although length‐dependent activation may be greater in fast skeletal muscle (FSM) than in slow skeletal muscle (SSM), there has not been a well controlled comparison of length‐dependent activation between skeletal muscle and cardiac muscle (CM). Accordingly, we measured sarcomere length‐dependent properties in skinned soleus (SSM), psoas (FSM) and ventricular trabeculae (CM) of the rat under carefully controlled conditions. The free Ca2+‐force relationship was determined at sarcomere lengths (SL) of 1.95 μm, 2.10 μm and 2.25 μm and fitted to a modified Hill equation. FSM and SSM were more sensitive to Ca2+ than CM. Length‐dependent activation was ordered as CM > FSM > SSM. Cooperativity as measured by the Hill coefficient of the Ca2+‐force relationship was not significantly different between CM and FSM, both of which exhibited greater cooperativity than SSM. SL did not significantly alter this parameter in each muscle type. To establish whether the observed differences can be explained by alterations in interfilament spacing, we measured myofilament lattice spacing (LS) by synchrotron X‐ray diffraction in relaxed, skinned muscle preparations. LS was inversely proportional to SL for each muscle type. The slope of the SL‐LS relationship, however, was not significantly different between striated muscle types. We conclude that (1) length‐dependent activation differs among the three types of striated muscle and (2) these differences in the length‐dependent properties among the striated muscle types may not solely be explained by the differences in the response of interfilament spacing to changes in muscle length in relaxed, skinned isolated muscle preparations.

Improved fitting of solution X-ray scattering data to macromolecular structures and structural ensembles by explicit water modeling.

A new procedure, AXES, is introduced for fitting small-angle X-ray scattering (SAXS) data to macromolecular structures and ensembles of structures. By using explicit water models to account for the effect of solvent, and by restricting the adjustable fitting parameters to those that dominate experimental uncertainties, including sample/buffer rescaling, detector dark current, and, within a narrow range, hydration layer density, superior fits between experimental high resolution structures and SAXS data are obtained. AXES results are found to be more discriminating than standard Crysol fitting of SAXS data when evaluating poorly or incorrectly modeled protein structures. AXES results for ensembles of structures previously generated for ubiquitin show improved fits over fitting of the individual members of these ensembles, indicating these ensembles capture the dynamic behavior of proteins in solution.

Frank-Starling law of the heart and the cellular mechanisms of length-dependent activation.

About a century ago, Otto Frank in Germany and Ernest Starling in England reported on the relationship between the extent of ventricular filling and pump function of the heart, a phenomenon collectively referred to as FrankStarlings Law of the Heart. Franks experiments employed the isolated frog heart and suggested that maximum ventricular pressure critically depends on whether the heart is operating under ejecting or isovolumic conditions [10]. That is, ejection appeared to deactivate cardiac contraction. Starlings experiments [31], using a isolated canine heart-lung preparation, showed that cardiac output was directly proportional to filling pressure and, thus, ventricular volume and independent of peripheral resistance: “... the larger the diastolic volume of the heart... the greater is the energy of its contraction.” More recently, Suga and Sagawa proposed a time-varying elastance model to describe ventricular contractile function [35]. These investigators demonstrated, in the isolated canine heart, a direct and proportional relationship between end-systolic pressure and end-systolic volume; this phenomenon has since been confirmed in many species, including human. As illustrated in Fig. 1A, the end-systolic pressure-volume relationship was found to be independent of loading conditions, but critically dependent on contractile state (dashed line). Apart from predicting ventricular stroke volume under a variety of loading conditions, the time varying elastance model similarly provides a framework to predict myocardial oxygen consumption [35]. Both Frank and Starling understood that their data bore a direct similarity to the variation of active force development in skeletal muscle as function of muscle length [3], thereby linking ventricular function to a fundamental property of cardiac muscle. Indeed, twitch force in isolated cardiac muscle has been shown to be directly proportional to systolic sarcomere length [21]. Furthermore, the shape of the relationship is modulated by contractile state such that more force is generated at a given sarcomere length when contractile activation is elevated (illustrated in Fig. 1B) [21]. What cellular mechanisms might underlie this phenomenon? At first sight, it may appear logical to suggest that variation of contractile filament overlap underlies the Frank-Starling Law of the Heart (described further below and in Fig. 2). However, the relationship between contractile force and sarcomere length is far too steep and to variable between contractile states (Fig. 1B) to be solely explained by such a simple mechanism [1,20, 21]. The contractile apparatus is activated by calcium ions that are released by the sarcoplasmic reticulum upon activation. Early experiments by Fabiato suggested that the released amount of this activator calcium varied with sarcomere length [8], but these results have not since been confirmed. More recent experiments [7,17, 20] clearly demonstrated that it is the level of activation of the cardiac contractile apparatus itself that is sensitive to changes in sarcomere length (Fig. 1C). These experiments were performed on chemically permeabilized (skinned) isolated cardiac muscle, a preparation that allows direct access to the contractile apparatus such that steady state force can be measured as function of activator calcium concentration. Thus, as is illustrated in Fig. 1C, there is a direct proportionality between sarcomere length and the sensitivity to calcium ions of the cardiac sarcomere, such that more force is generated at a given concentration of activator calcium as sarcomere length is increased (the curves are shifted to the left on the [activator calcium] axes at higher sarcomere length). Hence, it can be said that the sarcomere possesses a length dependent activation property. It should be noted that intact twitching J.P. Konhilas · P.P. de Tombe ()) Department of Physiology and Biophysics, and Cardiovascular Sciences Program, College of Medicine, University of Illinois at Chicago, Chicago, IL, 60612, USA e-mail: pdetombe@uic.edu Tel.: +1-312-3550259 Fax: +1-312-3550261

Protein Kinase A–Mediated Phosphorylation of cMyBP-C Increases Proximity of Myosin Heads to Actin in Resting Myocardium

Protein kinase A-mediated (PKA) phosphorylation of cardiac myosin binding protein C (cMyBP-C) accelerates the kinetics of cross-bridge cycling and may relieve the tether-like constraint of myosin heads imposed by cMyBP-C. We favor a mechanism in which cMyBP-C modulates cross-bridge cycling kinetics by regulating the proximity and interaction of myosin and actin. To test this idea, we used synchrotron low-angle x-ray diffraction to measure interthick filament lattice spacing and the equatorial intensity ratio, I11/I10, in skinned trabeculae isolated from wild-type and cMyBP-C null (cMyBP-C−/−) mice. In wild-type myocardium, PKA treatment appeared to result in radial or azimuthal displacement of cross-bridges away from the thick filaments as indicated by an increase (approximately 50%) in I11/I10 (0.22±0.03 versus 0.33±0.03). Conversely, PKA treatment did not affect cross-bridge disposition in mice lacking cMyBP-C, because there was no difference in I11/I10 between untreated and PKA-treated cMyBP-C−/− myocardium (0.40±0.06 versus 0.42±0.05). Although lattice spacing did not change after treatment in wild-type (45.68±0.84 nm versus 45.64±0.64 nm), treatment of cMyBP-C−/− myocardium increased lattice spacing (46.80±0.92 nm versus 49.61±0.59 nm). This result is consistent with the idea that the myofilament lattice expands after PKA phosphorylation of cardiac troponin I, and when present, cMyBP-C, may stabilize the lattice. These data support our hypothesis that tethering of cross-bridges by cMyBP-C is relieved by phosphorylation of PKA sites in cMyBP-C, thereby increasing the proximity of cross-bridges to actin and increasing the probability of interaction with actin on contraction.

Structure of Flexible Filamentous Plant Viruses

ABSTRACT Flexible filamentous viruses make up a large fraction of the known plant viruses, but in comparison with those of other viruses, very little is known about their structures. We have used fiber diffraction, cryo-electron microscopy, and scanning transmission electron microscopy to determine the symmetry of a potyvirus, soybean mosaic virus; to confirm the symmetry of a potexvirus, potato virus X; and to determine the low-resolution structures of both viruses. We conclude that these viruses and, by implication, most or all flexible filamentous plant viruses share a common coat protein fold and helical symmetry, with slightly less than 9 subunits per helical turn.