Myosin VI contains an inserted sequence that is unique among myosin superfamily members and has been suggested to be a determinant of the reverse directionality and unusual motility of the motor. It is thought that each head of a two-headed myosin VI molecule binds one calmodulin (CaM) by means of a single ''IQ motif''. Using truncations of the myosin VI protein and electrospray ionization(ESI)-MS, we demonstrate that in fact each myosin VI head binds two CaMs. One CaM binds to a conventional IQ motif either with or without calcium and likely plays a regulatory role when calcium binds to its N-terminal lobe. The second CaM binds to a unique insertion between the converter region and IQ motif. This unusual CaM-binding site normally binds CaM with four Ca 2؉ and can bind only if the C-terminal lobe of CaM is occupied by calcium. Regions of the MD outside of the insert peptide contribute to the Ca 2؉ -CaM binding, as truncations that eliminate elements of the MD alter CaM binding and allow calcium dissociation. We suggest that the Ca 2؉ -CaM bound to the unique insert represents a structural CaM, and not a calcium sensor or regulatory component of the motor. This structure is likely an integral part of the myosin VI ''converter'' region and repositions the myosin VI ''lever arm'' to allow reverse direction (minus-end) motility on actin.M yosin VI was the first myosin demonstrated to move toward the pointed (minus) end of an actin filament (1). This discovery was based on the hypothesis that reverse direction would require a redesign of the domain of myosin, known as the converter, that rotates on changes in the state of the nucleotide binding pocket and actin-myosin interface. The converter is attached directly to what is known as the myosin ''lever arm,'' which is made up of a variable number of ''IQ motifs'' that are binding sites for calmodulin (CaM) or specialized myosin light chains (2). This lever arm effectively amplifies the movements of the converter, and thus the step size of most myosins is thought to be a function of lever arm length (3-5). Myosin VI contains a unique insertion between the converter and its single IQ motif (6). Cryo-electron microscopy revealed that the myosin VI lever arm points toward the pointed (minus) end of the actin filament, and motility assays showed that the motor moves toward the minus end of the actin filament (1). Based on this finding, it was suggested that this unique insert is involved in altering the converter to reposition the lever arm.Since the lever arm region of each head of the myosin VI dimer consists of a single IQ motif, it has been assumed that the lever arm contains one CaM. Because, for both myosin II and myosin V, the length of the lever arm has been shown to correspond to the step size associated with a single ATPase cycle (5,7,8), myosin VI would be expected to have a small (Ϸ5 nm) step size. However, single molecule experiments with optical tweezers revealed that myosin VI has a broad distribution of step sizes centered on 30 nm (9), which is not consist...

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Numerous putative post-translational modifications may induce variations of monoclonal antibodies charge distribution that can potentially affect their biological activity. The characterization and the monitoring of these charge variants are critical quality requirements to ensure stability and process consistency. Charge variants are usually characterized by preparative ion exchange chromatography, collection of fractions and subsequent reverse-phase liquid chromatography with mass spectrometry analysis. While this process can be automatized by on-line two-dimensional chromatography, it remains often complex and time consuming. For this reason, a straightforward on-line charge variant analysis method is highly desirable and analytical laboratories are actively pursuing efforts to overcome this challenge. In this study, a mixed mode ion exchange chromatographic method using volatile salts and coupled on-line to native mass spectrometry was developed in association with a middle-up approach for a detailed characterization of monoclonal antibodies charge variants. An aged monoclonal antibody, presenting a complex charge variant profile was successfully investigated by this methodology as a case study. Results demonstrate that deamidation of the heavy chain was the major degradation pathway after long-term storage at 5°C while oxidation was rather low. The method was also very useful to identify all the clipped forms of the antibody.

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