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The Arp2/3 complex, first isolated from Acanthamoeba castellani by affinity chromatography on profilin, consists of seven polypeptides; two actin-related proteins, Arp2 and Arp3; and five apparently novel proteins, p40, p35, p19, p18, and p14 (Machesky et al., 1994). The complex is homogeneous by hydrodynamic criteria with a Stokes' radius of 5.3 nm by gel filtration, sedimentation coefficient of 8.7 S, and molecular mass of 197 kD by analytical ultracentrifugation. The stoichiometry of the subunits is 1:1:1:1:1:1:1, indicating the purified complex contains one copy each of seven polypeptides. In electron micrographs, the complex has a bilobed or horseshoe shape with outer dimensions of approximately 13 x 10 nm, and mathematical models of such a shape and size are consistent with the measured hydrodynamic properties. Chemical cross-linking with a battery of cross-linkers of different spacer arm lengths and chemical reactivities identify the following nearest neighbors within the complex: Arp2 and p40; Arp2 and p35; Arp3 and p35; Arp3 and either p18 or p19; and p19 and p14. By fluorescent antibody staining with anti-p40 and -p35, the complex is concentrated in the cortex of the ameba, especially in linear structures, possibly actin filament bundles, that lie perpendicular to the leading edge. Purified Arp2/3 complex binds actin filaments with a Kd of 2.3 microM and a stoichiometry of approximately one complex molecule per actin monomer. In electron micrographs of negatively stained samples, Arp2/3 complex decorates the sides of actin filaments. EDC/NHS cross-links actin to Arp3, p35, and a low molecular weight subunit, p19, p18, or p14. We propose structural and topological models for the Arp2/3 complex and suggest that affinity for actin filaments accounts for the localization of complex subunits to actin-rich regions of Acanthamoeba.

Covalent labeling of therapeutic drugs and proteins with polyethylene glycol (PEGylation) is an important modification for improving stability, solubility, and half-life. PEGylation alters protein solution behavior through its impact on thermodynamic nonideality by increasing the excluded volume, and on hydrodynamic nonideality by increasing the frictional drag. To understand PEGylation's impact, we investigated the thermodynamic and hydrodynamic properties of a model system consisting of PEGylated human serum albumin derivatives using analytical ultracentrifugation (AUC) and dynamic light scattering (DLS). We constructed PEGylated human serum albumin derivatives of single, linear 5K, 10K, 20K, and 40K PEG chains and a single branched-chain PEG of 40K (2 × 20K). Sedimentation velocity (SV) experiments were analyzed using SEDANAL direct boundary fitting to extract ideal sedimentation coefficients s o , hydrodynamic nonideality k s , and thermodynamic nonideality 2BM 1 SV terms. These quantities allow the determination of the Stokes radius R s , the frictional ratio f/f o , and the swollen or entrained volume V s /v, which measure size, shape, and solvent interaction. We performed sedimentation equilibrium experiments to obtain independent measurements of thermodynamic nonideality 2BM 1 SE . From DLS measurements, we determined the interaction parameter, k D , the concentration dependence of the apparent diffusion coefficient, D, and from extrapolation of D to c = 0 a second estimate of R s . R s values derived from SV and DLS measurements and ensemble model calculations (see complementary study) are then used to show that k s  + k D  = theoretical 2B 22 M 1 . In contrast, experimental BM 1 values from SV and sedimentation equilibrium data collectively allow for similar analysis for protein-PEG conjugates and show that k s  + k D  = 1.02-1.07 ∗ BM 1 , rather than the widely used k s  + k D  = 2BM 1 developed for hard spheres. The random coil behavior of PEG dominates the colloidal properties of PEG-protein conjugates and exceeds the sum of a random coil and hard-sphere volume due to excess entrained water., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The biotech industry has great interest in investigating therapeutic proteins in high concentration environments like human serum. The fluorescence detection system (Aviv-FDS) allows the performance of analytical ultracentrifuge (AUC) sedimentation velocity (SV) experiments in tracer or BOLTS protocols. Here, we compare six pooled human serum samples by AUC SV techniques and demonstrate the potential of this technology for characterizing therapeutic antibodies in serum. Control FDS SV experiments on serum alone reveal a bilirubin-HSA complex whose sedimentation is slowed by solution nonideality and exhibits a Johnston-Ogston (JO) effect due to the presence of high concentrations of IgG. Absorbance SV experiments on diluted serum samples verify the HSA-IgG composition as well as a significant IgM pentamer boundary at 19 s. Alexa-488 labeled Simponi (Golimumab) is used as a tracer to investigate the behavior of a therapeutic monoclonal antibody (mAb) in serum, and the sedimentation behavior of total IgG in serum. Serum dilution experiments allow extrapolation to zero concentration to extract s o , while global direct boundary fitting with SEDANAL verifies the utility of a matrix of self- and cross-term phenomenological nonideality coefficients (k s and BM 1 ) and the source of the JO effect. The best fits include weak reversible association (~ 4 × 10 3  M -1 ) between Simponi and total human IgG. Secondary mAbs to human IgG and IgM verify the formation of a 10.2 s 1:1 complex with human IgG and a 19 s complex with human IgM pentamers. These results demonstrate that FDS AUC allows a range of approaches for investigating therapeutic antibodies in human serum., (© 2023. European Biophysical Societies' Association.)

The Aviv fluorescence detection system (Aviv-FDS) has allowed the performance of sedimentation velocity experiments on therapeutic antibodies in highly concentrated environments like formulation buffers and serum. Methods were implemented in the software package SEDANAL for the analysis of nonideal, weakly associating AUC data acquired on therapeutic antibodies and proteins (Wright et al. Eur Biophys J 47:709-722, 2018, Anal Biochem 550:72-83, 2018). This involved fitting both hydrodynamic, k s , and thermodynamic, BM 1 , nonideality where concentration dependence is expressed as s = s o /(1 + k s c) and D = D o (1 + 2BM 1 c)/(1 + k s c) and s o and D o are values extrapolated to c = 0 (mg/ml). To gain insight into the consequences of these phenomenological parameters, we performed simulations with SEDANAL of a monoclonal antibody as a function of k s (0-100 ml/g) and BM 1 (0-100 ml/g). This provides a visual understanding of the separate and joint impact of k s and BM 1 on the shape of high-concentration sedimentation velocity boundaries and the challenge of their unique determination by finite element methods. In addition, mAbs undergo weak self- and hetero-association (Yang et al. Prot Sci 27:1334-1348, 2018) and thus we have simulated examples of nonideal weak association over a wide range of concentrations (1-120 mg/ml). Here we demonstrate these data are best analyzed by direct boundary global fitting to models that account for k s , BM 1 and weak association. Because a typical clinical dose of mAb is 50-200 mg/ml, these results have relevance for biophysical understanding of concentrated therapeutic proteins.

Krabbe disease, also known as globoid cell leukodystrophy, is a rare genetic neurodegenerative disease caused by a deficiency of the galactocerebrosidase enzyme. To understand the association status of human beta-galactocerebrosidase (hGALC) in solution, we employed analytical ultracentrifugation (AUC). Our AUC results show that hGALC has a tendency for reversible self-association. Self-association decreases as the concentration of sodium chloride increases from 50 to 500 mM. This indicates that ionic interactions are involved in the association. The association is also dependent on pH, and high order oligomerization decreases as the pH increases from 4.5 to 7.5. Taken together, our results indicate that hGALC has the highest tendency for oligomerization at physiological ionic strength and pH (lysosomal lumen). This is the first report describing the self-associating property of hGALC in solution., Competing Interests: Research was funded by Shire US Inc, a member of the Takeda group of companies. EL, NM, and KT are full-time employees of Takeda Pharmaceutical Company Limited. This does not alter our adherence to PLOS ONE policy on sharing data and materials. EL, NM, and KT are stockholders in Takeda Pharmaceutical Company Limited. NM and KT are authors of patent application of WO2011163651A2.

Brownian dynamics (BD) has been applied as a comprehensive tool to model sedimentation and diffusion of nanoparticles in analytical ultracentrifugation (AUC) experiments. In this article, we extend the BD algorithm by considering space-dependent diffusion and solvent compressibility. With this, the changes in the sedimentation and diffusion coefficient from altered solvent properties at increased pressures are accurately taken into account. Moreover, it is demonstrated how the concept of space-dependent diffusion is employed to describe concentration-dependent sedimentation and diffusion coefficients, in particular, through the Gralen coefficient and the second virial coefficient. The influence of thermodynamic nonideality on diffusional properties can be accurately simulated and agree with well-known evaluation tools. BD simulations for sedimentation equilibrium and sedimentation velocity (SV) AUC experiments including effects of hydrodynamic and thermodynamic nonideality are validated by global evaluation in SEDANAL. The interplay of solvent compressibility and retrieved nonideality parameters can be studied utilizing BD. Finally, the second virial coefficient is determined for lysozyme from SV AUC experiments and BD simulations and compared to membrane osmometry. These results are in line with DLVO theory. In summary, BD simulations are established for the validation of nonideal sedimentation in AUC providing a sound basis for the evaluation of complex interactions even in polydisperse systems.

The goal of this work is to develop a preclinical method for quantitative hydrodynamic and thermodynamic analysis of therapeutic proteins in crowded environments like human serum. The method utilizes tracer amounts of fluorescently labeled monoclonal antibodies and the Aviv AU-FDS optical system. We have performed sedimentation velocity experiments as a function of mAb, human serum albumin and human IgG concentration to extract self- and cross-term hydrodynamic nonideality effects. SV measurements are consistently complicated by weak mAb-mAb and mAb-IgG interactions (Wright et al. in Anal Biochem 550:72-83, 2018). In an attempt to explore different approaches we have investigated measurements of diffusion coefficients by traditional synthetic boundary experiments. Here we present a new technique incorporated into SEDANAL that can globally analyze the full time course of synthetic boundary experiments. This approach also utilizes F-mAb against a high concentration of unlabeled carrier protein (HSA or IgG). In principle both diffusion and sedimentation coefficient information can be extracted including hydrodynamic and thermodynamic nonideality. The method can be performed at a traditional low speed (5-7K rpm) or at high speeds. The high speed method can also be used to measure D and s for small molecules like fluorescein (often contaminants of F-HSA and F-mAb). The advantage of synthetic boundary over the standard sedimentation velocity method is that it allows for higher precision determination of diffusion coefficients. The concentration dependence of D can be corrected for hydrodynamic nonideality effects by plotting D * (1 + k ij c j ) vs total carrier concentration. The slope of the fitted data allows an alternate approach to determine self- and cross-term thermodynamic nonideality. This method can also explore cross-term diffusion coefficient effects. These results are compared to dynamic light scattering approaches which are limited to k D determinations for solutions of pure protein.

The preclinical characterization of biopharmaceuticals seeks to determine the stability, state of aggregation, and interaction of the antibody/drug with other macromolecules in serum. Analytical ultracentrifugation is the best experimental method to understand these factors. Sedimentation velocity experiments using the AU-FDS system were performed in order to quantitatively characterize the nonideality of fluorescently labeled therapeutic antibodies in high concentrations of human serum proteins. The two most ubiquitous serum proteins are human serum albumin, HSA, and γ-globulins, predominantly IgG. Tracer experiments were done pairwise as a function of HSA, IgG, and therapeutic antibody concentration. The sedimentation coefficient for each fluorescently labeled component as a function of the concentration of the unlabeled component yields the hydrodynamic nonideality (k s ). This generates a 3x3 matrix of k s values that describe the nonideality of each pairwise interaction. The k s matrix is validated by fitting both 2:1 mixtures of HSA (1-40 mg/ml) and IgG (0.5-20 mg/ml) as serum mimics, and human serum dilutions (10-100%). The data are well described by SEDANAL global fitting with the k s nonideality matrix. The k s values for antibodies are smaller than expected and appear to be masked by weak association. Global fitting to a k s and K 2 model significantly improves the fits., (Copyright © 2018 Elsevier Inc. All rights reserved.)

This work explores the heterogeneity of aggregation of polyglutamine fusion constructs in crude extracts of transgenic Caenorhabditis elegans animals. The work takes advantage of the recent technical advances in fluorescence detection for the analytical ultracentrifuge. Further, new sedimentation velocity methods, such as the multi-speed method for data capture and wide distribution analysis for data analysis, are applied to improve the resolution of the measures of heterogeneity over a wide range of sizes. The focus here is to test the ability to measure sedimentation of polyglutamine aggregates in complex mixtures as a prelude to future studies that will explore the effects of genetic manipulation and environment on aggregation and toxicity. Using sedimentation velocity methods, we can detect a wide range of aggregates, ranging from robust analysis of the monomer species through an intermediate and quite heterogeneous population of oligomeric species, and all the way up to detecting species that likely represent intact inclusion bodies based on comparison to an analysis of fluorescent puncta in living worms by confocal microscopy. Our results support the hypothesis that misfolding of expanded polyglutamine tracts into insoluble aggregates involves transitions through a number of stable intermediate structures, a model that accounts for how an aggregation pathway can lead to intermediates that can have varying toxic or protective attributes. An understanding of the details of intermediate and large-scale aggregation for polyglutamine sequences, as found in neurodegenerative diseases such as Huntington's Disease, will help to more precisely identify which aggregated species may be involved in toxicity and disease., (© 2015 The Protein Society.)

Scaffold proteins bind to and functionally link protein members of signaling pathways. Interaction of the scaffold proteins, myosin phosphatase target subunit (MYPT1) and myosin phosphatase-RhoA interacting protein (MRIP), causes co-localization of myosin phosphatase and RhoA to actomyosin. To examine biophysical properties of interaction of MYPT1 with MRIP, we employed analytical ultracentrifugation and surface plasmon resonance. In regard to MRIP, its residues 724-837 are sufficient for the MYPT1/MRIP interaction. Moreover, MRIP binds to MYPT1 as either a monomer or a dimer. With respect to MYPT1, its leucine repeat region, LR (residues 991-1030) is sufficient to account for the MYPT1/MRIP interaction. Furthermore, point mutations that replace glutamic acids 998-1000 within LR reduced the binding affinity toward MRIP. This suggests that the glutamic acids of MYPT1 play an important role in the interaction.

Analytical ultracentrifugation (AUC) has proven to be a powerful tool for the study of particle size distributions, particle shapes, and interactions with high accuracy and unrevealed resolution. In this work we show how the analysis of sedimentation velocity data from the AUC equipped with a multiwavelength detector (MWL) can be used to gain an even deeper understanding of colloidal and macromolecular mixtures. New data evaluation routines have been integrated in the software SEDANAL to allow for the handling of MWL data. This opens up a variety of new possibilities because spectroscopic information becomes available for individual components in mixtures at the same time using MWL-AUC. For systems of known optical properties information on the hydrodynamic properties of the individual components in a mixture becomes accessible. For the first time, the determination of individual extinction spectra of components in mixtures is demonstrated via MWL evaluation of sedimentation velocity data. In our paper we first provide the informational background for the data analysis and expose the accessible parameters of our methodology. We further demonstrate the data evaluation by means of simulated data. Finally, we give two examples which are highly relevant in the field of nanotechnology using colored silica and gold nanoparticles of different size and extinction properties.

Here we give an overview of the history of sedimentation velocity analysis focusing on seminal and fundamental contributions that derived from early ultracentrifugation studies. We introduce the concepts of nonequilibrium thermodynamics and outline the derivation of the Svedberg and the Lamm equations and the requirements for including both hydrodynamic and thermodynamic nonideality. We introduce the phenomenological equations for coupled flows as developed from the principles of nonequilibrium or irreversible thermodynamics and derive a form of the Lamm equation that incorporates cross-diffusion coefficients and coupled gradient terms. We give an historical overview of solutions to the Lamm equation including Fujita-MacCosham solutions and Claverie finite-element numerical solutions and discuss the software that have implemented these solutions. We discuss the three major optical systems (absorbance, interference, and fluorescence) and recently developed multiwavelength systems. We also suggest a number of experimental practices and guidelines for optimizing the determination of s and D and discuss the appropriate centerpiece components and their utility. This chapter complements other recent reviews submitted by the authors (Correia, Lyons, Sherwood, & Stafford, 2015; Stafford, 2015) and should be considered an effort to revive the importance of irreversible thermodynamics in the understanding and analysis of sedimentation velocity ultracentrifugation data., (© 2015 Elsevier Inc. All rights reserved.)

Oxidation of actin monomer (G-actin) with copper o-phenanthroline resulted in a rapid, high yield of disulfide cross-linked dimer. The cross-link is due to an intermolecular disulfide bond between actin Cys374 of each molecule, resulting in a tail-to-tail, i.e., antiparallel, actin dimer. Analytical ultracentrifugation profiles of G-actin can be ascribed to the existence of actin monomers with very little, if any, dimer. Thus, actin dimers are not energetically favorable, indicating that cross-linked dimers are formed during random diffusional collisions. On the other hand, a similar oxidation of actin polymer (F-actin) resulted in a much lower yield of the cross-linked actin dimer that showed no sign of leveling off. Therefore, it is proposed that the cross-linked dimer from actin polymer is due to collisional complexes of actin monomers that are in equilibrium with the polymer during actin treadmilling. These results account for the reported observation that during the early stages of actin polymerization (where the actin monomer concentration is high) cross-linked antiparallel actin dimers are formed in relatively high yield whereas none are formed at later stages of polymerization. These findings raise questions concerning the validity of the antiparallel actin dimer model of in vitro actin polymerization that is based on the assumption that the ability to form cross-linked actin dimers implies the existence of stable dimers.

The SLIP1-SLBP complex activates translation of replication-dependent histone mRNAs. In this report, we describe how the activity of the SLIP1-SLBP complex is modulated by phosphorylation and oligomerization. Biophysical characterization of the free proteins shows that whereas SLIP1 is a homodimer that does not bind RNA, human SLBP is an intrinsically disordered protein that is phosphorylated at 23 Ser/Thr sites when expressed in a eukaryotic expression system such as baculovirus. The bacterially expressed unphosphorylated SLIP1-SLBP complex forms a 2:2 high-affinity (K(D) < 0.9 nM) heterotetramer that is also incapable of binding histone mRNA. In contrast, phosphorylated SLBP from baculovirus has a weak affinity (K(D) ~3 μM) for SLIP1. Sequential binding of phosphorylated SLBP to the histone mRNA stem-loop motif followed by association with SLIP1 is required to form an "active" ternary complex. Phosphorylation of SLBP at Thr171 promotes dissociation of the heterotetramer to the SLIP1-SLBP heterodimer. Using alanine scanning mutagenesis, we demonstrate that the binding site on SLIP1 for SLBP lies close to the dimer interface. A single-point mutant near the SLIP1 homodimer interface abolished interaction with SLBP in vitro and reduced the abundance of histone mRNA in vivo. On the basis of these biophysical studies, we propose that oligomerization and SLBP phosphorylation may regulate the SLBP-SLIP1 complex in vivo. SLIP1 may act to sequester SLBP in vivo, protecting it from proteolytic degradation as an inactive heterotetramer, or alternatively, formation of the SLIP1-SLBP heterotetramer may facilitate removal of SLBP from the histone mRNA prior to histone mRNA degradation.

It has recently been reported that tropomyosin exists exclusively as a dimer in physiological salt conditions. It is shown in the present work using analytical ultracentrifugation that, on the contrary, tropomyosin is in equilibrium between monomer, dimer and tetramer with a weak tendency to dimerize and tetramerize. Such a finding has consequences for the assembly of the tropomyosin-actin complex., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The self-association of human interferon-α2b (hIFN-α2b), albinterferon-α2b (a recombinant protein with human serum albumin and hIFN-α2b peptides fused together in a single polypeptide chain), and Pegasys (PEGylated hIFN-α2a) was characterized by analytical ultracentrifugation analyses. By examining the apparent sedimentation coefficient distribution profiles of each protein at different concentrations, it was concluded that the above three proteins are self-associating in albinterferon-α2b formulation buffer. By model fitting of sedimentation data using SEDANAL software, the stoichiometry and equilibrium constants of the self-association of these proteins were characterized. The self-association of hIFN-α2b results in the formation of stable dimers, fast-reversible tetramers, octamers, and hexadecamers. In contrast, although both albinterferon-α2b and Pegasys are self-associated, their self-association stoichiometries are significantly different from that of hIFN-α2b. The self-association of albinterferon-α2b results in the formation of reversible dimers and trimers, whereas the self-association of Pegasys gives only reversible dimers. The self-association behaviors of hIFN-α2b and albinterferon-α2b involves attractive electrostatic forces, which can be suppressed to a negligible level in low pH (pH 4.0-4.5) and high salt concentration (400 mM NaCl) buffer, allowing quantification of their size variant contents by sedimentation velocity analysis., (Copyright © 2011 Wiley-Liss, Inc.)

For 25 years, the Gibbs Conference on Biothermodynamics has focused on the use of thermodynamics to extract information about the mechanism and regulation of biological processes. This includes the determination of equilibrium constants for macromolecular interactions by high precision physical measurements. These approaches further reveal thermodynamic linkages to ligand binding events. Analytical ultracentrifugation has been a fundamental technique in the determination of macromolecular reaction stoichiometry and energetics for 85 years. This approach is highly amenable to the extraction of thermodynamic couplings to small molecule binding in the overall reaction pathway. In the 1980s this approach was extended to the use of sedimentation velocity techniques, primarily by the analysis of tubulin-drug interactions by Na and Timasheff. This transport method necessarily incorporates the complexity of both hydrodynamic and thermodynamic nonideality. The advent of modern computational methods in the last 20 years has subsequently made the analysis of sedimentation velocity data for interacting systems more robust and rigorous. Here we review three examples where sedimentation velocity has been useful at extracting thermodynamic information about reaction stoichiometry and energetics. Approaches to extract linkage to small molecule binding and the influence of hydrodynamic nonideality are emphasized. These methods are shown to also apply to the collection of fluorescence data with the new Aviv FDS., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Myosin VIIa is crucial in hearing and visual processes. We examined the kinetic and association properties of the baculovirus expressed, truncated mouse myosin VIIa construct containing the head, all 5IQ motifs and the putative coiled coil domain (myosin VIIa-5IQ). The construct appears to be monomeric as determined by analytical ultracentrifugation experiments, and only single headed molecules were detected by negative stain electron microscopy. The relatively high basal steady-state rate of 0.18 s(-1) is activated by actin only by ∼3.5-fold resulting in a V(max) of 0.7 s(-1) and a K(ATPase) of 11.5 μM. There is no single rate-limiting step of the ATP hydrolysis cycle. The ATP hydrolysis step (M·T M·D·P) is slow (12 s(-1)) and the equilibrium constant (K(H)) of 1 suggests significant reversal of hydrolysis. In the presence of actin ADP dissociates with a rate constant of 1.2 s(-1). Phosphate dissociation is relatively fast (>12 s(-1)), but the maximal rate could not be experimentally obtained at actin concentrations ≤ 50 μM because of the weak binding of the myosin VIIa-ADP-P(i) complex to actin. At higher actin concentrations the rate of attached hydrolysis (0.4 s(-1)) becomes significant and partially rate-limiting. Our findings suggest that the myosin VIIa is a "slow", monomeric molecular motor with a duty ratio of 0.6.

The ability to obtain a sedimentation coefficient distribution as the run proceeds, and to get an early idea of the quality of a particular sample, has not been made available in real-time during the run in any of the existing software packages. It is desirable on many occasions to be able to see the number of components present in a sample at an early stage of the run. The ability to ascertain the extent of heterogeneity of sample would help enormously to reduce the amount of time that is necessary to obtain that information. Most software packages currently available require that the run be completed before analysis is carried out or at least some of the early scans analyzed off-line to determine if the run should continue. A software package called SEDVIEW has been developed by us to allow early analysis in real-time.

Smooth muscle myosin light chain kinase (smMLCK) is a calcium-calmodulin complex-dependent enzyme that activates contraction of smooth muscle. The polypeptide chain of rabbit uterine smMLCK (Swiss-Prot entry P29294) contains the catalytic/regulatory domain, three immunoglobulin-related motifs (Ig), one fibronectin-related motif (Fn3), a repetitive, proline-rich segment (PEVK), and, at the N-terminus, a unique F-actin-binding domain. We have evaluated the spatial arrangement of these domains in a recombinant 125 kDa full-length smMLCK and its two catalytically active C-terminal fragments (77 kDa, residues 461-1147, and 61 kDa, residues 461-1002). Electron microscopic images of smMLCK cross-linked to F-actin show particles at variable distances (11-55 nm) from the filament, suggesting that a well-structured C-terminal segment of smMLCK is connected to the actin-binding domain by a long, flexible tether. We have used structural homology and molecular dynamics methods to construct various all-atom representation models of smMLCK and its two fragments. The theoretical sedimentation coefficients computed with HYDROPRO were compared with those determined by sedimentation velocity. We found agreement between the predicted and observed sedimentation coefficients for models in which the independently folded catalytic domain, Fn3, and Ig domains are aligned consecutively on the long axis of the molecule. The PEVK segment is modeled as an extensible linker that enables smMLCK to remain bound to F-actin and simultaneously activate the myosin heads of adjacent myosin filaments at a distance of >or=40 nm. The structural properties of smMLCK may contribute to the elasticity of smooth muscle cells.

The shape and solution properties of fibrinogen are affected by the location of the C-terminal portion of the Aalpha chains, which is presently still controversial. We have measured the hydrodynamic properties of a human fibrinogen fraction with these appendages mostly intact, of chicken fibrinogen, where they lack 11 characteristic 13-amino acids repeats, and of human fragment X, a plasmin early degradation product in which they have been removed. The human fibrinogen/fragment X samples were extensively characterized by SDS-PAGE/Western blotting and mass spectrometry, allowing their composition to be precisely determined. The solution properties of all samples were then investigated by analytical ultracentrifugation and size-exclusion HPLC coupled with multi-angle light scattering and differential pressure viscometry detectors. The measured parameters suggest that the extra repeats have little influence on the overall fibrinogen conformation, while a significant change is brought about by the removal of the C-terminal portion of the Aalpha chains beyond residue Aalpha200., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

The molecular chaperone Hsp27 exists as a distribution of large oligomers that are disassembled by phosphorylation at Ser-15, -78, and -82. It is controversial whether the unphosphorylated Hsp27 or the widely used triple Ser-to-Asp phospho-mimic mutant is the more active molecular chaperone in vitro. This question was investigated here by correlating chaperone activity, as measured by the aggregation of reduced insulin or alpha-lactalbumin, with Hsp27 self-association as monitored by analytical ultracentrifugation. Furthermore, because the phospho-mimic is generally assumed to reproduce the phosphorylated molecule, the size and chaperone activity of phosphorylated Hsp27 were compared with that of the phospho-mimic. Hsp27 was triply phosphorylated by MAPKAP-2 kinase, and phosphorylation was tracked by urea-PAGE. An increasing degree of suppression of insulin or alpha-lactalbumin aggregation correlated with a decreasing Hsp27 self-association, which was the least for phosphorylated Hsp27 followed by the mimic followed by the unphosphorylated protein. It was also found that Hsp27 added to pre-aggregated insulin did not reverse aggregation but did inhibit these aggregates from assembling into even larger aggregates. This chaperone activity appears to be independent of Hsp27 phosphorylation. In conclusion, the most active chaperone of insulin and alpha-lactalbumin was the Hsp27 (elongated) dimer, the smallest Hsp27 subunit observed under physiological conditions. Next, the Hsp27 phospho-mimic is only a partial mimic of phosphorylated Hsp27, both in self-association and in chaperone function. Finally, the efficient inhibition of insulin aggregation by Hsp27 dimer led to the proposal of two models for this chaperone activity.

It has been known for some time that slow kinetics will distort the shape of a reversible reaction boundary. Here we present a tutorial on direct boundary fitting of sedimentation velocity data for a monomer-dimer system that exhibits kinetic effects. Previous analysis of a monomer-dimer system suggested that rapid reaction behavior will persist until the relaxation time of the system exceeds 100 s (reviewed in Kegeles and Cann, 1978). Utilizing a kinetic integrator feature in Sedanal (Stafford and Sherwood, 2004), we can now fit for the k(off) values and measure the uncertainty at the 95% confidence interval. For the monomer-dimer system the range of well determined k(off) values is limited to 0.005 to 10(-5) s(-1) corresponding to relaxation times (at a loading concentration of the Kd) of approximately 70 to approximately 33,000 s. For shorter relaxation times the system is fast and only the equilibrium constant K but not k(off) can be uniquely determined. For longer relaxation times the system is irreversibly slow, and assuming the system was at initial equilibrium before the start of the run, only the equilibrium constant K but not k(off) can be uniquely determined.

We have previously presented a tutorial on direct boundary fitting of sedimentation velocity data for kinetically mediated monomer-dimer systems [Correia and Stafford, 2009]. We emphasized the ability of Sedanal to fit for the k(off) values and measure their uncertainty at the 95% confidence interval. We concluded for a monomer-dimer system the range of well-determined k(off) values is limited to 0.005-10(-5) s(-1) corresponding to relaxation times of approximately 70 to approximately 33,000 s. More complicated reaction schemes introduce the potential complexity of low concentrations of an intermediate that may also influence the kinetic behavior during sedimentation. This can be seen in a cooperative ABCD system (A+B --> C; B+C --> D) where C, the 1:1 complex, is sparsely populated (K(1)=10(4) M(-1), K(2)=10(8) M(-1)). Under these conditions a k(1,off)<0.01 s(-1) produces slow kinetic features. The low concentration of species C contributes to this effect while still allowing the accurate estimation of k(1,off) (although k(2,off) can readily compensate and contribute to the kinetics). More complex reactions involving concerted assembly or cooperative ring formation with low concentrations of intermediate species also display kinetic effects due to a slow flux of material through the sparsely populated intermediate states. This produces a kinetically limited reaction boundary that produces partial resolution of individual species during sedimentation. Cooperativity of ring formation drives the reaction and thus separation of these two effects, kinetics and energetics, can be challenging. This situation is experimentally exhibited by systems that form large oligomers or rings and may especially contribute to formation of micelles and various protein aggregation diseases including formation of beta-amyloid and tau aggregates. Simulations, quantitative parameter estimation by direct boundary fitting and diagnostic features for these systems are presented with an emphasis on the features available in Sedanal to simulate and analyze kinetically mediated systems.

All biological processes involve molecular interactions that result in either binding, self-association, or hetero-associations of one form or another. It is important to understand that no interactions are completely all-or-none. Some approach all-or-none only when there is strong positive cooperativity. Examples will be given of typical biomolecular interactions and their expected dependence on concentration, in order to point out the relatively wide range of concentration over which these types of phenomena take place. This chapter is concerned both with the binding of low-molecular-weight ligands to macromolecules as well as interactions between macromolecules using analytical ultracentrifugation (AUC) as a tool for measuring association properties of these systems. The theory of sedimentation of both ideal and nonideal interacting and noninteracting systems is discussed. Examples are given of each type of system along with a discussion of how each type of system can be analyzed. Several methods of data analysis are discussed.

The giant protein titin has important roles in muscle sarcomere integrity, elasticity and contractile activity. The key role in elasticity was highlighted in recent years by single-molecule mechanical studies, which showed a direct relationship between the non-uniform structure of titin and the hierarchical mechanism of its force-extension behavior. Further advances in understanding mechanisms controlling sarcomere structure and elasticity require detailed knowledge of titin arrangement and interactions in situ. Here we present data on the structure and self-interactive properties of an approximately 290 kDa ( approximately 100 nm long) tryptic fragment from the I-band part of titin that is extensible in situ. The fragment includes the conserved 'distal' tandem Ig segment of the molecule and forms side-by-side oligomers with distinctive 4 nm cross-striations. Comparisons between these oligomers and the end filaments seen at the tips of native thick filaments indicate identical structure. This shows that end-filaments are formed by the elastic parts of six titin molecules connecting each end of the thick filament to the Z-line. Self-association of elastic titin into stiff end-filaments adds a further hierarchical level in the mechanism of titin extensibility in muscle cells. Self-association of this part of titin may be required to prevent interference of the individual flexible molecules with myosin cross-bridges interacting with actin.

Radiolabeled single-chain Fv (sFv) molecules display highly specific tumor retention in the severe combined immunodeficient (SCID) mouse model; however, the absolute quantity of sFv retained in the tumors is diminished by the rapid renal elimination resulting from the small size of the sFv molecules (Mr 27,000) and by dissociation of the monovalent sFv from tumor-associated antigen. We previously reported significant improvement in tumor retention without a loss of targeting specificity on converting monovalent sFv into divalent [(sFv')2] dimers, linked by a disulfide bond between COOH-terminal cysteinyl peptides engineered into the sFv'. However, our data for enhanced dimer localization in tumors could not distinguish between the contributions of enhanced avidity and increased systemic retention associated with the larger size of 54 kDa [(sFv')2] dimers relative to 27-kDa sFv. In this investigation, we have compared tumor targeting of divalent anti-c-erbB-2/HER2/neu 741F8-1 (sFv')2 homodimers with monovalent 741F8/26-10 (sFv')2 heterodimers (Mr 54,000) and 741F8 sFv monomers (741F8 sFv has binding specificity for erbB-2/HER2/neu and 26-10 sFv specificity for digoxin and related cardiac glycosides). These studies allowed us to distinguish the dominant effect of valency over molecular weight in accounting for the superior tumor retention of 741F8-1 (sFv')2 homodimers. Each of the radioiodinated species was administered i.v. to SCID mice bearing SK-OV-3 human tumor xenografts and tumor localization at 24 hours post i.v. injection was determined for 125I-741F8-1 (sFv')2 (3.57 %ID/g), 125I-741F8/26-10 (sFv')2 (1.13 %ID/g), and 125I-741F8-1 sFv (1.25 %ID/g). These findings substantiate that the improved tumor retention of (sFv')2 homodimers over sFv monomers results from the availability of dual binding sites rather than from the slower systemic clearance of homodimers.

E3-19K is a type I membrane glycoprotein expressed by adenoviruses (Ads) to modulate host antiviral immune responses. We have developed an expression system for the endoplasmic reticulum lumenal domain (residues 1 to 100) of Ad type 2 E3-19K tagged with a C-terminal His6 sequence in baculovirus-infected insect cells. In this system, recombinant E3-19K is secreted into the culture medium. A characterization of soluble E3-19K by analytical ultracentrifugation and circular dichroism showed that the protein is monomeric and adopts a stable and correctly folded tertiary structure. Using a gel mobility shift assay and analytical ultracentrifugation, we showed that soluble E3-19K associates with soluble peptide-filled and peptide-deficient HLA-A*1101 molecules. This is the first example of a viral immunomodulatory protein that interacts with conformationally distinct forms of class I major histocompatibility complex molecules. The E3-19K/HLA-A*1101 complexes formed in a 1:1 stoichiometry with equilibrium dissociation constants (Kd) of 50 +/- 10 nM for peptide-filled molecules and of about 10 microM for peptide-deficient molecules. A temperature-dependent proteolysis study revealed that the association of E3-19K with peptide-deficient HLA-A*1101 molecules stabilizes the binding groove. Importantly, our studies showed that peptide-deficient HLA-A*1101 molecules sequestered by E3-19K are capable of binding antigenic peptides and maturing into peptide-filled molecules. This firmly establishes that E3-19K does not block binding of antigenic peptides. Together, our results suggest that Ads have evolved to exploit the late and early stages of the class I antigen presentation pathway.

Myosin 10 contains a region of predicted coiled coil 120 residues long. However, the highly charged nature and pattern of charges in the proximal 36 residues appear incompatible with coiled-coil formation. Circular dichroism, NMR, and analytical ultracentrifugation show that a synthesized peptide containing this region forms a stable single alpha-helix (SAH) domain in solution and does not dimerize to form a coiled coil even at millimolar concentrations. Additionally, electron microscopy of a recombinant myosin 10 containing the motor, the three calmodulin binding domains, and the full-length predicted coiled coil showed that it was mostly monomeric at physiological protein concentration. In dimers the molecules were joined only at their extreme distal ends, and no coiled-coil tail was visible. Furthermore, the neck lengths of both monomers and dimers were much longer than expected from the number of calmodulin binding domains. In contrast, micrographs of myosin 5 heavy meromyosin obtained under the same conditions clearly showed a coiled-coil tail, and the necks were the predicted length. Thus the predicted coiled coil of myosin 10 forms a novel elongated structure in which the proximal region is a SAH domain and the distal region is a SAH domain (or has an unknown extended structure) that dimerizes only at its end. Sequence comparisons show that similar structures may exist in the predicted coiled-coil domains of myosins 6 and 7a and MyoM and could function to increase the size of the working stroke.

We have previously identified evolutionarily conserved heptad hydrophobic repeat (HR) domains in all isoprotein members of troponin T (TnT) and troponin I (TnI), two subunits of the Ca(2+)-regulatory troponin complex. Our suggestion that the HR domains are involved in the formation of a coiled-coil heterodimer of TnT and TnI has been recently confirmed by the crystal structure of the core domain of the human cardiac troponin complex. Here we studied a series of recombinant deletion mutants of the fast skeletal TnT to determine the minimal sequence required for stable coiled-coil formation with the HR domain of the fast skeletal TnI. Using circular dichroism spectroscopy, we measured the alpha helical content of the coiled-coil formed by the various TnT peptides with TnI HR domain. Sedimentation equilibrium experiments confirmed that the individual peptides of TnT were monomeric but formed heterodimers when mixed with HR domain of TnI. Isothermal titration calorimetry was then used to directly measure the affinity of the TnT peptides for the TnI HR domain. Surprisingly we found that the HR regions alone of the fast skeletal TnT and TnI, as defined earlier, were insufficient to form a coiled-coil. Furthermore we showed that an additional 14 amino acid residues N-terminal to the conserved HR region (TnT residues 165-178) are essential for the stable coiled-coil formation. We discuss the implication of our finding in the fast skeletal troponin isoform in the light of the crystal structure of the cardiac isoform.

The class I myosin, Myo1b, is a calmodulin- and actin-associated molecular motor widely expressed in mammalian tissues. Analytical ultracentrifugation studies indicate that Myo1b purified from rat liver has a Stokes radius of 6.7 nm and a sedimentation coefficient, s(20,w), of 7.0 S with a predicted molar mass of 213 kg/mol. These results indicate that Myo1b is monomeric and consists primarily of a splice variant having five associated calmodulins. Molecular modeling based on the analytical ultracentrifugation studies are supported by electron microscopy studies that depict Myo1b as a single-headed, tadpole-shaped molecule with outer dimensions of 27.9 x 4.0 nm. Above a certain Myo1b/actin ratio, Myo1b bundles actin filaments presumably by virtue of a second actin-binding site. These studies provide new information regarding the oligomeric state and morphology of Myo1b and support a model in which Myo1b cross-links actin through a cryptic actin-binding site.

DnaK, the prokaryotic Hsp70 molecular chaperone, requires the nucleotide exchange factor and heat shock protein GrpE to release ADP. GrpE and DnaK are tightly associated molecules with an extensive protein-protein interface, and in the absence of ADP, the dissociation constant for GrpE and DnaK is in the low nanomolar range. GrpE reduces the affinity of DnaK for ADP, and the reciprocal linkage is also true: ADP reduces the affinity of DnaK for GrpE. The energetic contributions of GrpE side-chains to GrpE-DnaK binding were probed by alanine-scanning mutagenesis. Sedimentation velocity (SV) analytical ultracentrifugation (AUC) was used to measure the equilibrium constants (Keq) for GrpE binding to the ATPase domain of DnaK in the presence of ADP. ADP-bound DnaK is the natural target of GrpE, and the addition of ADP (final concentration of 5 microM) to the preformed GrpE-DnaK(ATPase) complexes allowed the equilibrium association constants to be brought into an experimentally accessible range. Under these experimental conditions, the substitution of one single GrpE amino acid residue, arginine 183 with alanine, resulted in a GrpE-DnaK(ATPase) complex that was weakly associated (Keq =9.4 x 10(4) M). This residue has been previously shown to be part of a thermodynamic linkage between two structural domains of GrpE: the thermosensing long helices and the C-terminal beta-domains. Several other GrpE side-chains were found to have a significant change in the free energy of binding (DeltaDeltaG approximately 1.5 to 1.7 kcal mol(-1)), compared to wild-type GrpE.DnaK(ATPase) in the same experimental conditions. Overall, the strong interactions between GrpE and DnaK appear to be dominated by electrostatics, not unlike barnase and barstar, another well-characterized protein-protein interaction. GrpE, an inherent thermosensor, exhibits non-Arrhenius behavior with respect to its nucleotide exchange function at bacterial heat shock temperatures, and mutation of several solvent-exposed side-chains located along the thermosensing indicated that these residues are indeed important for GrpE-DnaK interactions.

The thiol-disulfide oxidoreductase ERp57 is a soluble protein of the endoplasmic reticulum and the closest known homologue of protein disulfide isomerase. The protein interacts with the two lectin chaperones calnexin and calreticulin and thereby promotes the oxidative folding of newly synthesized glycoproteins. Here we have characterized several fundamental structural and functional properties of ERp57 in vitro, such as the domain organization, shape, redox potential, and the ability to catalyze different thiol-disulfide exchange reactions. Like protein disulfide isomerase, we find ERp57 to be comprised of four structural domains. The protein has an elongated shape of 3.4 +/- 0.1 nm in diameter and 16.8 +/- 0.5 nm in length. The two redox-active a and a' domains were determined to have redox potentials of -0.167 and -0.156 V, respectively. Furthermore, ERp57 was shown to efficiently catalyze disulfide reduction, disulfide isomerization, and dithiol oxidation in substrate proteins. The implications of these findings for the function of the protein in vivo are discussed.

For nearly 50 years David Yphantis has helped advance analytical ultracentrifugation, promoted rigor in the thermodynamic analysis of biochemical data and encouraged students and colleagues to look for the deepest possible understanding of science. This article, written by five of Dave's students, presents some of the impressions he has made over the years.

Analysis of sedimentation velocity data for indefinite self-associating systems is often achieved by fitting of weight average sedimentation coefficients (s(20,w)) However, this method discriminates poorly between alternative models of association and is biased by the presence of inactive monomers and irreversible aggregates. Therefore, a more robust method for extracting the binding constants for indefinite self-associating systems has been developed. This approach utilizes a set of fitting routines (SedAnal) that perform global non-linear least squares fits of up to 10 sedimentation velocity experiments, corresponding to different loading concentrations, by a combination of finite element simulations and a fitting algorithm that uses a simplex convergence routine to search parameter space. Indefinite self-association is analyzed with the software program isodesfitter, which incorporates user provided functions for sedimentation coefficients as a function of the degree of polymerization for spherical, linear and helical polymer models. The computer program hydro was used to generate the sedimentation coefficient values for the linear and helical polymer assembly mechanisms. Since this curve fitting method directly fits the shape of the sedimenting boundary, it is in principle very sensitive to alternative models and the presence of species not participating in the reaction. This approach is compared with traditional fitting of weight average data and applied to the initial stages of Mg(2+)-induced tubulin self-associating into small curved polymers, and vinblastine-induced tubulin spiral formation. The appropriate use and limitations of the methods are discussed.

A method is described for the sedimentation velocity analysis of solutions composed of macromolecular solutes of widely disparate size. In sedimentation velocity experiments, usually a single rotor speed is chosen for the entire run, and consequently, the range of observable sedimentation coefficients can be severely limited. This limitation can be removed if the speed is varied during the run, starting with a relatively low speed so that the largest particles can be easily observed. The speed is increased during the run until full speed is attained and the run continued at full speed until the smallest species of interest have cleared the solution. The method, called wide distribution analysis, is based on the method developed originally by Yphantis and co-workers (Proc. Natl. Acad. Sci. USA 78 (1981) 1431) and on the time derivative method of Stafford (Anal. Biochem. 203 (1992) 295), essentially eliminating both the time-independent and radially-independent noise thereby improving the precision, especially for interference optics. An algorithm for analysis of data from both absorbance and interference optics and experimental protocols compatible with the Beckman XL-I Analytical Ultracentrifuge are presented. With these protocols an extremely wide range of sedimentation coefficients from approximately 1.0 to 250000 S can be accommodated in a single multi-speed run.

Analytical ultracentrifugation (AUC) has played and will continue to play an important role in the investigation of protein-protein, protein-DNA and protein-ligand interactions. A major advantage of AUC over other methods is that it allows the analysis of systems free in solution in nearly any buffer without worry about spurious interactions with a supporting matrix. Large amounts of high-quality data can be acquired in relatively short times. Advances in software for the treatment of AUC data over the last decade have eliminated many of the tedious aspects of AUC data analysis, allowing relatively rapid analysis of complicated systems that were previously unapproachable. A software package called sedanal is described that can perform global fits to AUC sedimentation velocity data obtained for both interacting and non-interacting, macromolecular multi-species, multi-component systems, by combining data from multiple runs over a range of sample concentrations and component ratios. Interaction parameters include both forward and reverse rate constants, or equilibrium constants, for each reaction, as well as concentration dependence of both sedimentation and diffusion coefficients. sedanal fits to time-difference data to eliminate time-independent systematic errors inherent in AUC data. The sedanal software package is based on the use of finite-element numerical solutions of the Lamm equation.

We have found that myosin V, an important actin-based vesicle transporter, has a folded conformation that is coupled to inhibition of its enzymatic activity in the absence of cargo and Ca(2+). In the absence of Ca(2+) where the actin-activated MgATPase activity is low, purified brain myosin V sediments in the analytical ultracentrifuge at 14 S as opposed to 11 S in the presence of Ca(2+) where the activity is high. At high ionic strength it sediments at 10 S independent of Ca(2+), and its regulation is poor. These data are consistent with myosin V having a compact, inactive conformation in the absence of Ca(2+) and an extended conformation in the presence of Ca(2+) or high ionic strength. Electron microscopy reveals that in the absence of Ca(2+) the heads and tail are both folded to give a triangular shape, very different from the extended appearance of myosin V at high ionic strength. A recombinant myosin V heavy meromyosin fragment that is missing the distal portion of the tail domain is not regulated by calcium and has only a small change in sedimentation coefficient, which is in the opposite direction to that seen with intact myosin V. Electron microscopy shows that its heads are extended even in the absence of calcium. These data suggest that interaction between the motor and cargo binding domains may be a general mechanism for shutting down motor protein activity and thereby regulating the active movement of vesicles in cells.

GrpE is the nucleotide exchange factor for the Escherichia coli molecular chaperone DnaK, the bacterial homologue of Hsp70. In the temperature range of the bacterial heat shock response, the long helices of GrpE undergo a helix-to-coil transition, and GrpE exhibits non-Arrhenius behavior with respect to its nucleotide exchange function. It is hypothesized that GrpE acts as a thermosensor and that unwinding of the long helices of E. coli GrpE reduces its activity as a nucleotide exchange factor. In turn, it was proposed that temperature-dependent down-regulation of the activity of GrpE may increase the time in which DnaK binds its substrates at higher temperatures. A combination of thermodynamic and hydrodynamic techniques, in concert with the luciferase refolding assay, were used to characterize a molecular mechanism in which the long helices of GrpE are thermodynamically linked with the beta-domains via an intramolecular contact between Phe86 and Arg183. These "thermosensing" long helices were found to be necessary for full activity as a nucleotide exchange factor in the luciferase refolding assay. Point mutations in the beta-domains and in the long helices of GrpE destabilized the beta-domains. Engineered disulfide bonds in the long helices alternately stabilized the long helices and the four-helix bundle. This allowed the previously reported 75 degrees C thermal transition seen in the excess heat capacity function as monitored by differential scanning calorimetry to be further characterized. The observed thermal transition represents the unfolding of the four-helix bundle and the beta-domains. The thermal transitions for these two domains are superimposed but are not thermodynamically linked.

Heavy meromyosin from scallop (scHMM) striated muscle is regulated by calcium binding to the essential light chain. The regulation can be modeled with a calcium-dependent equilibrium between on and off scHMM conformations. The observed rate constant for mant-ADP dissociation from scHMM is calcium dependent, and we show here that it can be used to define the equilibrium constant (K(eq)) between on and off conformations. The data show that K(eq) is markedly ionic strength dependent, with high salt (>/=200 mM) abolishing the off state even in the absence of calcium and low salt (<50 mM) favoring the off state even in the presence of calcium. Debye-Huckel plots of the equilibrium constant (K(eq)) for the on and off forms gave parallel slopes (5.94 +/- 0.33 and 6.36 +/- 0.17 M(-0.5)) in the presence and absence of calcium. The presence of an equilibrium mixture of two conformations was confirmed by sedimentation data and the effects of ADP, calcium and ionic strength were in qualitative agreement. Thus scHMM exists in two conformations that can be distinguished in sedimentation profiles and by the rate of release of mant-ADP. Increasing salt concentrations biases the system toward the on state, suggesting a role for ionic interactions in stabilizing the off state.

IQ motifs are widespread in nature. Mlc1p is a calmodulin-like myosin light chain that binds to IQ motifs of a class V myosin, Myo2p, and an IQGAP-related protein, Iqg1p, playing a role in polarized growth and cytokinesis in Saccharomyces cerevisiae. The crystal structures of Mlc1p bound to IQ2 and IQ4 of Myo2p differ dramatically. When bound to IQ2, Mlc1p adopts a compact conformation in which both the N- and C-lobes interact with the IQ motif. However, in the complex with IQ4, the N-lobe no longer interacts with the IQ motif, resulting in an extended conformation of Mlc1p. The two light chain structures relate to two distinct subfamilies of IQ motifs, one of which does not interact with the N-lobes of calmodulin-like light chains. The correlation between light chain structure and IQ sequence is demonstrated further by sedimentation velocity analysis of complexes of Mlc1p with IQ motifs from Myo2p and Iqg1p. The resulting 'free' N-lobes of myosin light chains in the extended conformation could mediate the formation of ternary complexes during protein localization and/or partner recruitment.

KaiC from Synechococcus elongatus PCC 7942 (KaiC) is an essential circadian clock protein in cyanobacteria. Previous sequence analyses suggested its inclusion in the RecADnaB superfamily. A characteristic of the proteins of this superfamily is that they form homohexameric complexes that bind DNA. We show here that KaiC also forms ring complexes with a central pore that can be visualized by electron microscopy. A combination of analytical ultracentrifugation and chromatographic analyses demonstrates that these complexes are hexameric. The association of KaiC molecules into hexamers depends on the presence of ATP. The KaiC sequence does not include the obvious DNA-binding motifs found in RecA or DnaB. Nevertheless, KaiC binds forked DNA substrates. These data support the inclusion of KaiC into the RecADnaB superfamily and have important implications for enzymatic activity of KaiC in the circadian clock mechanism that regulates global changes in gene expression patterns.

Tapasin is a type I membrane glycoprotein involved with other accessory proteins in the assembly of class I MHC-beta(2)m-peptide complexes in the endoplasmic reticulum. We have probed the three-dimensional structure of the lumenal region of human tapasin (residues 1-392) tagged with a (His)(6) sequence at its C-terminus using biochemical and biophysical techniques. The far-UV circular dichroism spectrum revealed that tapasin possesses well-defined secondary structural elements corresponding predominantly to beta-sheets. A thermal denaturation curve recorded at 216 nm showed a midpoint transition centered at approximately 45 degrees C. Sedimentation analysis showed that tapasin is monomeric in solution with a sedimentation coefficient, S degrees (20,w), of 2.68 S. This value of S degrees (20,w) combined with the value of the molar mass obtained by MALDI mass spectrometry (44.2 kDa) yielded a frictional ratio, f/f(0), of 1.47. Assuming tapasin is a prolate ellipsoid, we calculated an apparent length of 22.5 nm and a diameter of 2.62 nm, consistent with an elongated molecular shape. Controlled proteolysis using various enzymes revealed that a narrow region of tapasin near residue 90 is highly susceptible to digestion, resulting in two fragments that are resistant to further cleavage. The identity of these fragments was determined by amino acid sequencing and MALDI mass spectrometry and revealed a 9 kDa N-terminal fragment and a 34 kDa C-terminal fragment. Collectively, these results suggest that tapasin is comprised of two core domains of different sizes loosely linked by a flexible region.

GrpE is the nucleotide exchange factor for the Escherichia coli molecular chaperone DnaK, the prokaryotic homologue of Hsp70. Thermodynamic properties of GrpE structural domains were characterized by examining a number of structural and point mutants using circular dichroism, differential scanning calorimetry and analytical ultracentrifugation. These structural domains are the long paired N-terminal helices, the central four-helix bundle, and the C-terminal compact beta-domains. We show that the central four-helix bundle (t(m) approximately 75 degrees C) provides a stable platform for the association of the long paired N-terminal helices (t(m) approximately 50 degrees C), which can then function as a temperature sensor. The stability of the N-terminal helices is linked to the presence of the C-terminal compact beta-domains of GrpE, providing a potential mechanism for coupling of DnaK-binding activity of GrpE with temperature. On the basis of our thermodynamic analysis of E.coli GrpE, we present a structure-based model for the melting properties of the nucleotide exchange factor, wherein the long paired helices function as a molecular thermocouple.

Calreticulin (CRT) is a soluble chaperone involved in the conformational maturation of glycoproteins in the endoplasmic reticulum. Using biochemical and biophysical techniques including circular dichroism, proteolysis, and analytical ultracentrifugation, we have determined the effects of calcium and zinc ions on the structural properties of human CRT. Circular dichroism analysis has shown that the binding of calcium and zinc ions to CRT induces no significant changes in the secondary structure of the protein but affects in very distinct ways the local tertiary packing of these elements. More specifically, these studies have revealed that CRT adopts a more rigid and thermally stable structure upon binding calcium ions and a more loosely packed and thermally destabilized structure upon binding zinc ions. Consistent with these results, proteolysis experiments demonstrated that the intrinsic conformational flexibility of CRT can be modulated toward either a decrease or an increase in susceptibility to cleavage by chymotrypsin upon binding calcium or zinc ions, respectively. Results from sedimentation analysis indicated that the global three-dimensional structure of CRT is essentially unchanged upon binding calcium ions. In marked contrast, CRT self-associates reversibly to form dimers upon binding zinc ions. Collectively, our results provide evidence that calcium and zinc ions induce strikingly different changes in the biochemical and structural properties of CRT.

We expressed the small subunit of smooth muscle myosin light chain phosphatase (MPs) in Escherichia coli, and have studied its molecular properties as well as its interaction with the targeting subunit (MPt). MPs (M(r) = 18,500) has an anomalously low electrophoretic mobility, running with an apparent M(r) of approximately 21,000 in sodium dodecyl sulfate-gel electrophoresis. CD spectroscopy shows that it is approximately 45% alpha-helix and undergoes a cooperative temperature-induced unfolding with a transition midpoint of 73 degrees C. Limited proteolysis rapidly degrades MPs to a stable C-terminal fragment (M(r) = 10,000) that retains most of the helical content. Rotary shadowing electron microscopy reveals that it is an elongated protein with two domains. Sedimentation velocity measurements show that recombinant MPt (M(r) = 107,000), intact MPs, and the 10-kDa MPs fragment are all dimeric, and that MPs and MPt form a complex with a molar mass consistent with a 1:1 heterodimer. Sequence analysis predicts that regions in the C-terminal portions of both MPs and MPt have high probabilities for coiled coil formation. A synthetic peptide from a region of MPs encompassing residues 77-116 was found to be 100% alpha-helical, dimeric, and formed a complex with MPt with a molecular mass corresponding to a heterodimer. Based on these results, we propose that MPs is an elongated molecule with an N-terminal head and a C-terminal stalk domain. It dimerizes via a coiled coil interaction in the stalk domain, and interacts with MPt via heterodimeric coiled coil formation. Since other proteins with known regulatory function toward MP also have predicted coiled coil regions, our results suggest that these regulatory proteins target MP via the same coiled coil strand exchange mechanism with MPt.

The mechanism of calcium regulation of scallop myosin is not understood, although it is known that both myosin heads are required. We have explored possible interactions between the heads of heavy meromyosin (HMM) in the presence and absence of calcium and nucleotides by sedimentation and electron microscope studies. The ATPase activity of the HMM preparation was activated over tenfold by calcium, indicating that the preparation contained mostly regulated molecules. In the presence of ADP or ATP analogs, calcium increased the asymmetry of the HMM molecule as judged by its slower sedimentation velocity compared with that in EGTA. In the absence of nucleotide the asymmetry was high even in EGTA. The shift in sedimentation occurred with a sharp midpoint at a calcium level of about 0.5 microM. Sedimentation of subfragment 1 was not dependent on calcium or on nucleotides. Modeling accounted for the observed sedimentation behavior by assuming that both HMM heads bent toward the tail in the absence of calcium, while in its presence the heads had random positions. The sedimentation pattern showed a single peak at all calcium concentrations, indicating equilibration between the two forms with a t(1/2) less than 70 seconds. Electron micrographs of crosslinked, rotary shadowed specimens indicated that 81 % of HMM molecules in the presence of nucleotide had both heads pointing back towards the tail in the absence of calcium, as compared with 41 % in its presence. This is consistent with the sedimentation data. We conclude that in the "off" state, scallop myosin heads interact with each other, forming a rigid structure with low ATPase activity. When molecules are switched "on" by binding of calcium, communication between the heads is lost, allowing them to flex randomly about the junction with the tail; this could facilitate their interaction with actin in contracting muscle., (Copyright 2001 Academic Press.)

Calreticulin (CRT) is an abundant soluble protein of the endoplasmic reticulum lumen that functions as a molecular chaperone for nascent glycoproteins. We have probed the three-dimensional structure of human CRT using a series of biochemical and biophysical approaches in an effort to understand the molecular basis of its chaperone function. Sedimentation analysis and chemical cross-linking experiments showed that CRT is monodisperse and monomeric in solution with a molecular mass (MW) of 46 +/- 1 kDa. This MW value together with a sedimentation coefficient, s(o)(20,w), of 2.71 S yielded a frictional ratio, f/f(0), of 1.65. Assuming CRT to be a prolate ellipsoid, we calculated an apparent length of 29.8 nm and diameter of 2.44 nm consistent with an asymmetric elongated molecule. These hydrodynamic dimensions account for the apparent anomalous elution position of CRT on gel filtration columns. Far-UV circular dichroism experiments showed that CRT has a cooperative thermal denaturation transition with a midpoint temperature of 42.5 degrees C suggesting a marginally stable structure. Proteolysis experiments showed that the highly acidic segment at the C-terminus of CRT is most susceptible to digest, consistent with the absence of a well-defined polypeptide backbone structure in this region of the protein. Temperature-dependent proteolysis with thermolysin revealed a stable core region within the N- and P-domains. A stable fragment encompassing most of the P-domain was also identified in the thermolytic mixture. Collectively, our results suggest that CRT is likely to be a flexible molecule in solution which may be important for its chaperone function.

This chapter has described a bioenergetic analysis of the interaction of sCD4 with an IgG1 and two IgG4 derivatives of an anti-sCD4 MAb. The MAbs have identical VH and VL domains but differ markedly in their CH and CL domains, raising the question of whether their antigen-binding chemistries are altered. We find the sCD4-binding kinetics and thermodynamics of the MAbs are indistinguishable, which indicates rigorously that the molecular details of the binding interactions are the same. We also showed the importance of using multiple biophysical methods to define the binding model before the bioenergetics can be appropriately interpreted. Analysis of the binding thermodynamics and kinetics suggests conformational changes that might be coupled to sCD4 binding by these MAbs are small or absent.