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In vitro, calmodulin (CaM) and S100A1 activate the skeletal muscle ryanodine receptor ion channel (RyR1) at submicromolar Ca2+ concentrations, whereas at micromolar Ca2+ concentrations, CaM inhibits RyR1. One amino acid substitution (RyR1-L3625D) has previously been demonstrated to impair CaM binding and regulation of RyR1. Here we show that the RyR1-L3625D substitution also abolishes S100A1 binding. To determine the physiological relevance of these findings, mutant mice were generated with the RyR1-L3625D substitution in exon 74, which encodes the CaM and S100A1 binding domain of RyR1. Homozygous mutant mice (Ryr1D/D) were viable and appeared normal. However, single RyR1 channel recordings from Ryr1D/D mice exhibited impaired activation by CaM and S100A1 and impaired CaCaM inhibition. Isolated flexor digitorum brevis muscle fibers from Ryr1D/D mice had depressed Ca2+ transients when stimulated by a single action potential. However, during repetitive stimulation, the mutant fibers demonstrated greater relative summation of the Ca2+ transients. Consistently, in vivo stimulation of tibialis anterior muscles in Ryr1D/D mice demonstrated reduced twitch force in response to a single action potential, but greater summation of force during high-frequency stimulation. During repetitive stimulation, Ryr1D/D fibers exhibited slowed inactivation of sarcoplasmic reticulum Ca2+ release flux, consistent with increased summation of the Ca2+ transient and contractile force. Peak Ca2+ release flux was suppressed at all voltages in voltage-clamped Ryr1D/D fibers. The results suggest that the RyR1-L3625D mutation removes both an early activating effect of S100A1 and CaM and delayed suppressing effect of CaCaM on RyR1 Ca2+ release, providing new insights into CaM and S100A1 regulation of skeletal muscle excitation-contraction coupling. [ABSTRACT FROM AUTHOR]

Internal ionizable groups in proteins are relatively rare but they are essential for catalysis and energy transduction. To examine molecular determinants of their unusual and functionally important properties, we engineered 25 variants of staphylococcal nuclease with lysine residues at internal positions. Nineteen of the Lys residues have depressed pKa values, some as low as 5.3, and 20 titrate without triggering any detectable conformational reorganization. Apparently, simply by being buried in the protein interior, these Lys residues acquired pKa values comparable to those of naturally occurring internal ionizable groups involved in catalysis and biological H+ transport. The pKa values of some of the internal Lys residues were affected by interactions with surface carboxylic groups. The apparent polarizability reported by the pKa values varied significantly from location to location inside the protein. These data will enable an unprecedented examination of the positional dependence of the dielectric response of a protein. This study also shows that the ability of proteins to withstand the presence of charges in their hydrophobic interior is a fundamental property inherent to all stable proteins, not a specialized adaptation unique to proteins that evolved to depend on internal charges for function. [ABSTRACT FROM AUTHOR]

Internal ionizable groups are quite rare in water-soluble globular proteins. Presumably, this reflects the incompatibility between charges and the hydrophobic environment in the protein interior. Here we show that proteins can have an inherently high tolerance for internal ionizable groups. The 25 internal positions in staphylococcal nuclease were substituted one at a time with Lys, Glu, or Asp without abolishing enzymatic activity and without detectable changes in the conformation of the protein. Similar results with substitutions of 6 randomly chosen internal positions in ribonuclease H with Lys and Glu suggest that the ability of proteins to tolerate internal ionizable groups might be a property common to many proteins. Eighty-six of the 87 substitutions made were destabilizing, but in all but one case the proteins remained in the native state at neutral pH. By comparing the stability of each variant protein at two different pH values it was established that the pK a values of most of the internal ionizable groups are shifted; many of the internal ionizable groups are probably neutral at physiological pH values. These studies demonstrate that special structural adaptations are not needed for ionizable groups to exist stably in the hydrophobic interior of proteins. The studies suggest that enzymes and other proteins that use internal ionizable groups for functional purposes could have evolved through the random accumulation of mutations that introduced ionizable groups at internal positions, followed by evolutionary adaptation and optimization to modulate stability, dynamics, and other factors necessary for function. [ABSTRACT FROM AUTHOR]

Abstract: The pK a values of internal ionizable groups are usually very different from the normal pK a values of ionizable groups in water. To examine the molecular determinants of pK a values of internal groups, we compared the properties of Lys, Asp, and Glu at internal position 38 in staphylococcal nuclease. Lys38 titrates with a normal or elevated pK a, whereas Asp38 and Glu38 titrate with elevated pK a values of 7.0 and 7.2, respectively. In the structure of the L38K variant, the buried amino group of the Lys38 side chain makes an ion pair with Glu122, whereas in the structure of the L38E variant, the buried carboxyl group of Glu38 interacts with two backbone amides and has several nearby carboxyl oxygen atoms. Previously, we showed that the pK a of Lys38 is normal owing to structural reorganization and water penetration concomitant with ionization of the Lys side chain. In contrast, the pK a values of Asp38 and Glu38 are perturbed significantly owing to an imbalance between favorable polar interactions and unfavorable contributions from dehydration and from Coulomb interactions with surface carboxylic groups. Their ionization is also coupled to subtle structural reorganization. These results illustrate the complex interplay between local polarity, Coulomb interactions, and structural reorganization as determinants of pK a values of internal groups in proteins. This study suggests that improvements to computational methods for pK a calculations will require explicit treatment of the conformational reorganization that can occur when internal groups ionize. [Copyright &y& Elsevier]

In vitro, calmodulin (CaM) and S100A1 activate the skeletal muscle ryanodine receptor ion channel (RyR1) at submicromolar Ca(2+) concentrations, whereas at micromolar Ca(2+) concentrations, CaM inhibits RyR1. One amino acid substitution (RyR1-L3625D) has previously been demonstrated to impair CaM binding and regulation of RyR1. Here we show that the RyR1-L3625D substitution also abolishes S100A1 binding. To determine the physiological relevance of these findings, mutant mice were generated with the RyR1-L3625D substitution in exon 74, which encodes the CaM and S100A1 binding domain of RyR1. Homozygous mutant mice (Ryr1(D/D)) were viable and appeared normal. However, single RyR1 channel recordings from Ryr1(D/D) mice exhibited impaired activation by CaM and S100A1 and impaired CaCaM inhibition. Isolated flexor digitorum brevis muscle fibers from Ryr1(D/D) mice had depressed Ca(2+) transients when stimulated by a single action potential. However, during repetitive stimulation, the mutant fibers demonstrated greater relative summation of the Ca(2+) transients. Consistently, in vivo stimulation of tibialis anterior muscles in Ryr1(D/D) mice demonstrated reduced twitch force in response to a single action potential, but greater summation of force during high-frequency stimulation. During repetitive stimulation, Ryr1(D/D) fibers exhibited slowed inactivation of sarcoplasmic reticulum Ca(2+) release flux, consistent with increased summation of the Ca(2+) transient and contractile force. Peak Ca(2+) release flux was suppressed at all voltages in voltage-clamped Ryr1(D/D) fibers. The results suggest that the RyR1-L3625D mutation removes both an early activating effect of S100A1 and CaM and delayed suppressing effect of CaCaM on RyR1 Ca(2+) release, providing new insights into CaM and S100A1 regulation of skeletal muscle excitation-contraction coupling.