Sedimentation velocity FDS studies of antibodies in pooled human serum.

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 so, while global direct boundary fitting with SEDANAL verifies the utility of a matrix of self- and cross-term phenomenological nonideality coefficients (ks and BM1) and the source of the JO effect. The best fits include weak reversible association (~ 4 × 103 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.

Simulation of Gilbert theory for self-association in sedimentation velocity experiments: a guide to evaluate best fitting models.

There is a long tradition in the Biophysics community of using simulations as a means to understand macromolecular behavior in various physicochemical methods. This allows a rigorous means to interpret observations in terms of fundamental principles, including chemical equilibrium, reaction kinetics, transport processes and thermodynamics. Here we simulate data for the Gilbert Theory for self-association, a fundamental analytical ultracentrifuge (AUC) technique to understand the shape of sedimentation velocity reaction boundaries that involve reversible monomer-Nmer interactions. Simulating monomer-dimer through monomer-hexamer systems as a function of concentration about the equilibrium constant allows a visual means to differentiate reaction stoichiometry by determining end points and inflection positions. Including intermediates (eg A1-A2-A3-A4-A5-A6) in the simulations reveals the smoothing of the reaction boundary and the removal of sharp inflections between monomers and polymers. The addition of cooperativity restores sharp boundaries or peaks to the observation and allows more discrimination in the selection of possible fitting models. Thermodynamic nonideality adds additional features when applied across wide ranges of concentration that might be appropriate for high-concentration therapeutic monoclonal antibody (mAb) solutions. This presentation serves as a tutorial for using modern AUC analysis software like SEDANAL for selecting potential fitting models.

Analysis of nonideality: insights from high concentration simulations of sedimentation velocity data.

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, ks, and thermodynamic, BM1, nonideality where concentration dependence is expressed as s = so/(1 + ksc) and D = Do(1 + 2BM1c)/(1 + ksc) and so and Do 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 ks (0-100 ml/g) and BM1 (0-100 ml/g). This provides a visual understanding of the separate and joint impact of ks and BM1 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 ks, BM1 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.

Detection of rabbit Haemorrhagic disease virus 2 during the wild rabbit (Oryctolagus cuniculus) eradication from the Berlengas archipelago, Portugal.

BACKGROUND: In the regular wildlife monitoring action carried out in the summer of the past few years at the Berlenga Island, wild rabbits (Oryctolagus cuniculus) have been repeatedly found dead. However, the origin of those deaths was never investigated. Our aim was to investigate the cause of death of 11 rabbits collected between April and May 2016. RESULTS: While screening samples from rabbit carcasses for the major viral rabbit pathogens, five tested positive to RHDV2 but all were negative for RHDV and myxoma virus (MYXV). For six RHDV2-negative specimens, emaciation and parasitism were considered the most probable cause of death. Lesions identified in the RHDV2-positive rabbits included non-suppurative diffuse hepatic necrosis and pulmonary lesions varying from congestion and oedema of the lungs to interstitial pneumonia. Sequencing analysis of the vp60 gene obtained from two specimens showed identical vp60 sequences. Comparison with other known RHDV2 strains from public databases through BLAST analysis revealed a closer similarity with strains from Alentejo collected during 2013. Maximum Likelihood and Bayesian phylogenetic analysis showed that the 2016 strains from the archipelago have a higher resemblance with a group of strains mostly collected in the South of Portugal between 2013 and 2014. CONCLUSION: The results suggest that RHDV2 may have been introduced on the Berlenga Island a few years ago, having evolved separately from mainland strains due to insularity.

Characterisation of CIME, an experimental chamber for simulating interactions between materials of the cultural heritage and the environment.

An approach consisting in combining in situ and laboratory experiments is often favoured for investigating the mechanisms involved in the weathering of the materials of the cultural heritage. However, the realistic simulation in the laboratory of the environmental conditions ruling the interactions of atmospheric compounds with materials is a very complex task. The aim of this work is to characterise CIME, a new chamber specially built to simulate the interactions between materials of the cultural heritage and the environment. The originality of this instrument is that beside the usual climatic parameters (temperature, relative humidity, solar radiation) and gaseous pollutants, it also allows the controlled injection of different types of particulate matter such as terrigenous, marine and anthropogenic. Therefore, varied realistic atmospheric environments (marine or urban) can be easily simulated within CIME. In addition to the technical description of CIME, this paper shows the first results obtained by the impact of gaseous pollutants on non-durable glass, bronze and limestone. The first experiments for the deposition of different particles (calcite, clays, soot and halite) are also presented.

Pacheco's parrot disease in macaws of the Lisbon's Zoological Garden. Description of an outbreak, diagnosis and management, including vaccination.

The Lisbon's Zoological Garden, Portugal, has maintained for many years a large collection of psittacine birds without any serious health problems. Unexpectedly, in April 1999, a total of nine macaws died after a short period of illness. Clinical signs consisted mainly of anorexia, ruffled feathers and yellowish droppings. A herpesvirus was isolated from brain, trachea, lung, liver, spleen, kidney and intestine of each of the examined dead birds, confirming that all animals succumbed during viraemia. Serotyping of the isolate in cross neutralization tests with reference sera prove that the outbreak was caused by serotype 3 of Pacheco's parrot disease herpesviruses. An autogenous, formalin-inactivated vaccine with adjuvant (aluminium hydroxid gel) was prepared from one of the isolates and injected intramuscularly 14 days and six weeks after the onset of mortality in an attempt to protect the remaining psittacine birds in the zoo from the disease. The autogenous vaccine was well tolerated and was able to rapidly stop virus spread and morbidity and mortality among the psittacine birds. Follow-up studies demonstrate that all nine blood samples from vaccinated birds obtained nine month' after the second vaccination contain neutralizing antibodies. Twenty five month' after vaccination two out of four serum samples were still antibody positive. No herpesvirus was isolated from faecal samples nine and twenty five months after the onset of the outbreak. These data prove that the autogenous vaccine played a major role in containing a severe outbreak of Pacheco's parrot disease in a large collection of psittacine birds.

A comparison of weight average and direct boundary fitting of sedimentation velocity data for indefinite polymerizing systems.

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.

Physiochemical aspects of tubulin-interacting antimitotic drugs.

A diverse group of natural biological compounds bind to microtubules and suppress microtubule dynamics. Here we review the mechanism of microtubule assembly and dynamics as well as structural features that are important for nucleotide binding, GTP hydrolysis and stabilization of longitudinal and lateral protofilament contacts. Specific emphasis is placed upon the polar structure of the microtubule, the exposure of the nucleotide hydrolysis site at the + end and the conformational and configurational plasticity of the microtubule lattice. These features have important implications for the mechanism of dynamic instability and the disruptive action of antimitotic drugs. We then discuss the various classes of tubulin binding drugs emphasizing their site and mode of binding as well as the structural and energetic basis for their effects on microtubule assembly and dynamics. A common feature of tubulin-interacting compounds is a linkage to assembly, either the stabilization of a microtubule lattice by compounds like taxol or epothilone A, or the preferential formation of alternate lattice contacts and polymers at microtubule ends by compounds like colchicine, vinca alkaloids and cryptophycin-52. Finally, we explore the likely possibility that these drugs also disrupt the regulation of microtubule dynamics. Future generations of these compounds may be selectively developed to directly target the proteins that regulate mitotic spindle dynamics.

The L3 loop and C-terminal phosphorylation jointly define Smad protein trimerization.

Smad proteins mediate the transforming growth factor beta responses. C-terminal phosphorylation of R-Smads leads to the recruitment of Smad4 and the formation of active signaling complexes. We investigated the mechanism of phosphorylation-induced Smad complex formation with an activating pseudo-phosphorylated Smad3. Pseudo-phosphorylated Smad3 has a greater propensity to homotrimerize, and recruits Smad4 to form a heterotrimer containing two Smad3 and one Smad4. The trimeric interaction is mediated through conserved interfaces to which tumorigenic mutations map. Furthermore, a conserved Arg residue within the L3 loop, located near the C-terminal phosphorylation sites of the neighboring subunit, is essential for trimerization. We propose that the phosphorylated C-terminal residues interact with the L3 loop of the neighboring subunit to stabilize the trimer interaction.

Sedimentation studies reveal a direct role of phosphorylation in Smad3:Smad4 homo- and hetero-trimerization.

SMAD proteins are known to oligomerize and hetero-associate during their activation and translocation to the nucleus for transcriptional control. Analytical ultracentrifuge studies on Smad3 and Smad4 protein constructs are presented to clarify the model of homo- and hetero-oligomerization and the role of phosphorylation in the activation process. These constructs all exhibit a tendency to form disulfide cross-linked aggregates, primarily dimers, and a strong reducing agent, TCEP, was found to be required to determine the best estimates for reversible association models and equilibrium constants. A Smad4 construct, S4AF, consisting of the middle linker (L) domain and the C-terminal (C) domain, is shown to be a monomer, while a Smad3 construct, S3LC, consisting of the LC domains, is shown to form a trimer with an affinity K(3) = (1.2-3.1) x 10(9) M(-2). A Smad3 construct that mimics phosphorylation at the C-terminal target sequence, S3LC(3E), has 17--35-fold enhanced ability to form trimer over that of the wild-type construct, S3LC. S4AF associates with either S3LC or S3LC(3E) to form a hetero-trimer. In each case, the hetero-trimer is favored over the formation of the homo-trimer. Despite high sequence homology between Smad3 and Smad4, a chimeric Smad4 construct with an engineered Smad3 C-terminal pseudo-phosphorylation sequence, S4AF(3E), shows no tendency to form trimer. This suggests a Smad4-specific sequence insert inhibits homo-trimer formation, or other domains or sequences in S3LC are required in addition to the target sequence to mediate the formation of trimer. These results represent a direct molecular measure of the importance of hetero-trimerization and phosphorylation in the TGF-beta-activated Smad protein signal transduction process.

Structural basis of Smad1 activation by receptor kinase phosphorylation.

Phosphorylation of Smad1 at the conserved carboxyl terminal SVS sequence activates BMP signaling. Here we report the crystal structure of the Smad1 MH2 domain in a conformation that reveals the structural effects of phosphorylation and a molecular mechanism for activation. Within a trimeric subunit assembly, the SVS sequence docks near two putative phosphoserine binding pockets of the neighboring molecule, in a position ready to interact upon phosphorylation. The MH2 domain undergoes concerted conformational changes upon activation, which signal Smad1 dissociation from the receptor kinase for subsequent heteromeric assembly with Smad4. Biochemical and modeling studies reveal unique favorable interactions within the Smad1/Smad4 heteromeric interface, providing a structural basis for their association in signaling.

Vinca alkaloid-induced tubulin spiral formation correlates with cytotoxicity in the leukemic L1210 cell line.

The ability of a class of C-20' modified vinca alkaloid congeners to induce tubulin spiral formation was investigated relative to their ability to inhibit microtubule assembly, their cytotoxicity against a leukemic cell line, L1210, and their measured and calculated partition coefficients. These studies were prompted by the observation that the energetics of vinca alkaloid-induced tubulin spiral polymers, or spiraling potential, is inversely related to their clinical dosage and are aimed at the long-term goal of developing the ability to predict the cytotoxic and antineoplastic properties of antimitotic drugs. We demonstrate here that vinca-induced tubulin-spiraling potential is significantly correlated with cytotoxicity against L1210 cells. This is consistent with the size of spirals formed being proportional to the relaxation time for polymer redistribution, the lifetime of cell retention, and effects on microtubule ends and dynamics. Spiraling potential also correlates with calculated but not measured partition coefficients. Surprisingly, spiraling potential does not correlate with the ability to inhibit microtubule formation with purified tubulin or microtubule protein. For the set of C-20' modified compounds studied, the largest inhibitory effects on spiraling potential and cytotoxicity are caused by multiple sites of halogen (-F, -Cl) substitution with the introduction of increased rigidity in the ring. This suggests the C-20' position interacts with a hydrogen bond acceptor or an electrophilic region on the protein that electrostatically disfavors halogen substitutions. These studies are discussed in terms of the cellular mode of action of antimitotic drugs, particularly the importance of microtubule dynamics during mitosis and the factors that regulate those dynamics.

Additivity of dilantin and vinblastine inhibitory effects on microtubule assembly.

Dilantin (phenytoin) is a commonly used antiepileptic agent that is known to decrease conductance of sodium and calcium ions and delay outward potassium currents. Separate from its antiseizure activity, dilantin interferes with microtubule protein polymerization. It induces metaphase arrest and potentiates the effects of the antimitotics vincristine and vinblastine in cell culture. We show here by fluorescence binding studies that dilantin interacts directly with tubulin at a low affinity site [Ka = 3.5 (+/- 2.5) x 10(3) M(-1); Kd = 286 microM]. We quantitatively examined the effect of dilantin on bulk microtubule formation and found that the drug raises the critical concentration for microtubule polymerization in 2 M glycerol identically in the presence or absence of vinblastine. The change in free energy for microtubule polymerization attributable to 400 microM dilantin [deltadelta G = 117 (+/- 28) cal/mol] is additive with vinblastine effects. Under the same conditions, mean microtubule lengths are 7.7 +/- 4.3 microm (n = 558) and 7.4 +/- 4.0 microm (n = 477) in the presence or absence of dilantin, respectively. Dilantin has no effect on vinblastine-induced tubulin spiral formation, as measured by sedimentation velocity. Our data suggest that the mechanism for the antimicrotubule effects of dilantin involves sequestration of tubulin heterodimers in 1:1 drug:tubulin complexes that do not participate in tubulin polymerization. The dilantin binding site is distinct from the Vinca binding site, and these independent binding modes account for the additive effects in vitro. The sequestration of tubulin heterodimers could explain the combined drug synergy in cell cultures if it disrupted interactions with proteins that regulate microtubule dynamics and/or cell cycle events.

Equilibrium binding studies of non-claret disjunctional protein (Ncd) reveal cooperative interactions between the motor domains.

Non-claret disjunctional protein (Ncd) is a minus end-directed microtubule motor required for normal spindle assembly and integrity during Drosophila oogenesis. We have pursued equilibrium binding experiments to examine the affinity of Ncd for microtubules in the presence of the ATP nonhydrolyzable analog 5'-adenylyl-beta, gamma-imidodiphosphate (AMP-PNP), ADP, or ADP + Pi using both dimeric (MC1) and monomeric (MC6) Ncd constructs expressed in Escherichia coli. Both MC1 and MC6 sediment with microtubules in the absence of added nucleotide as well as in the presence of either ADP or AMP-PNP. Yet, in the presence of ADP + Pi, there is a decrease in the affinity of both MC1 and MC6 for microtubules. The data for dimeric MC1 show that release of the dimer to the supernatant is sigmoidal with the apparent Kd(Pi) for the two phosphate sites at 23.3 and 1.9 mM, respectively. The results indicate that binding at the first phosphate site enhances binding at the second site, thus cooperatively stimulating release. Stopped-flow kinetics indicate that MgATP promotes dissociation of the Mt.MC1 complex at 14 s-1, yet AMP-PNP has no effect on the Mt.MC1 complex. These results are consistent with a model for the ATPase cycle in which ATP hydrolysis occurs on the microtubule followed by detachment as the Ncd.ADP.Pi intermediate.

A comparison of thermodynamic parameters for vinorelbine- and vinflunine-induced tubulin self-association by sedimentation velocity.

We present a comparison of the energetics of spiral formation for two vinca alkaloids: a novel difluorinated vinorelbine derivative 20',20'-difluoro-3',4'-dihydrovinorelbine (F12158, or vinflunine) and the parent compound, vinorelbine. Vinca alkaloids are antineoplastic agents that halt cell division at metaphase by inhibiting microtubule assembly and inducing tubulin self-association into spiral aggregates. The overall affinities for tubulin of vincristine, vinblastine, and vinorelbine seem to correlate with their clinical doses, where vincristine with the highest overall affinity is used at the lowest doses. Doses of chemotherapeutic agents, however, also are determined by toxicities. In the physicochemical study described here, we used sedimentation velocity to compare vinorelbine- and vinflunine-induced self-association of porcine brain tubulin in the presence of 50 micro M GDP or 50 micro M GTP. Vinflunine demonstrates 3-16-fold lower overall affinity for tubulin and induces smaller polymers compared with vinorelbine. Sedimentation velocity provides the only direct evidence to date that vinflunine is a tubulin-binding drug. Stopped-flow light scattering demonstrates the shortest relaxation times for polymer redistribution for vinflunine consistent with induction of the shortest spirals. Data collected at 5 degrees, 15 degrees, 25 degrees, and 37 degrees show increasing 20,w values with increasing temperature and are consistent with an entropically driven process. These data are entirely consistent with our hypothesis that vinflunine is likely to result in reduced clinical neurotoxicity relative to vinorelbine, vinblastine, and vincristine.

Energetics of vinca alkaloid interactions with tubulin isotypes: implications for drug efficacy and toxicity.

A number of vinca alkaloids, including vincristine, vinblastine, and vinorelbine, are currently used in cancer chemotherapy. These three vinca alkaloids interact differently with a range of solid and hematologic tumors. To test the possibility that the tubulin isotype composition is an important determinant in antineoplastic efficacy, we determined thermodynamic parameters for vinca alkaloid interactions with purified beta-tubulin isotypes, alphabetaII or alphabetaIII, as well as mixtures of alphabetaII and alphabetaIII, alphabetaII and alphabetaI&IV, or alphabetaIII and alphabetaI&IV (referred to as isotype-depleted tubulin) by quantitative sedimentation velocity. Vincristine-, vinblastine-, or vinorelbine-induced isotype self-association was studied at 25 degrees C in 10 mM Pipes, pH 6.9, 1 mM MgSO4, and 2 mM EGTA in the presence of 50 microM GTP or GDP. For all three drugs, we observed no significant differences in overall affinities, K1K2, or in GDP enhancement of purified isotypes compared to unfractionated tubulin, suggesting that differential antitumor efficacy observed clinically for these vinca alkaloids is not determined by tissue isotype composition. Small, but significant differences in the individual binding parameters, K1 and K2, are found in the vincristine data. In the presence of vincristine and GTP, K1, the affinity of drug for tubulin heterodimers, tends to be larger for purified alphabetaII- or alphabetaIII-tubulin compared to unfractionated tubulin. Furthermore, the apparent dimerization constant, K2app, at physiologically significant drug concentrations is larger for these purified isotypes. When alphabetaII- and alphabetaIII-tubulin are combined, the cooperativity between drug binding and spiral formation approaches that of unfractionated PC-tubulin. These differences are not observed in the presence of vinblastine or vinorelbine. The differences found with vincristine may be implicated in the dose-limiting neurotoxicity found with this drug, but not found with vinblastine or vinorelbine.

Role of guanine nucleotides in the vinblastine-induced self-association of tubulin: effects of guanosine alpha,beta-methylenetriphosphate and guanosine alpha,beta-methylenediphosphate.

It is now well established that guanine nucleotides are allosteric effectors of the vinca alkaloid-induced self-association of tubulin. GDP enhances self-association for vinblastine-, vincristine- and vinorelbine-induced spiral assembly relative to GTP by 0.90 +/- 0.17 kcal/mol [Lobert et al. (1996) Biochemistry 35, 6806-6814]. Since chemical modifications of the vinca alkaloid structure are known to modulate the overall affinity of drug binding, it is very likely that, by Wyman linkage, chemical modifications of guanine nucleotide allosteric effectors also modulate drug binding. Here we compare the effects of the GTP and GDP alpha,beta-methylene analogues GMPCPP and GMPCP on vinblastine-induced tubulin association in 10 and 100 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (Pipes), 1 mM MgSO4, and 2 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), pH 6. 9, at different temperatures. We found that GMPCPP perfectly mimics GTP in its effect on spiral assembly under all ionic strength and temperature conditions. However, GMPCP in 10 mM Pipes behaves not as a GDP analogue, but as a GTP analogue. In 100 mM Pipes, GMPCP has characteristics that are intermediate between GDP and GTP. These data suggest that the alpha,beta methylene group in GMPCP and GMPCPP is sufficient to produce a GTP-like effect on vinblastine-induced tubulin self-assembly. This is consistent with previous observations that GMPCP-tubulin will assemble into microtubules in a 2 M glycerol and 100 mM Pipes buffer [Vulevic & Correia (1997) Biophys. J. 72, 1357-1375]. Our results demonstrate that an alpha,beta methylene modification of the guanine nucleotide phosphate moiety can induce a salt-dependent conformational change in the tubulin heterodimer that favors the GTP-tubulin structure. This has important implications for understanding allosteric interactions that occur in the binding of guanine nucleotides to tubulin.

Thermodynamic and structural analysis of microtubule assembly: the role of GTP hydrolysis.

Different models have been proposed that link the tubulin heterodimer nucleotide content and the role of GTP hydrolysis with microtubule assembly and dynamics. Here we compare the thermodynamics of microtubule assembly as a function of nucleotide content by van't Hoff analysis. The thermodynamic parameters of tubulin assembly in 30-100 mM piperazine-N,N'-bis(2-ethanesulfonic acid), 1 mM MgSO4, 2 mM EGTA, pH 6.9, in the presence of a weakly hydrolyzable analog, GMPCPP, the dinucleotide analog GMPCP plus 2 M glycerol, and GTP plus 2 M glycerol were obtained together with data for taxol-GTP/GDP tubulin assembly (GMPCPP and GMPCP are the GTP and GDP nucleotide analogs where the alpha beta oxygen has been replaced by a methylene, -CH2-). All of the processes studied are characterized by a positive enthalpy, a positive entropy, and a large, negative heat capacity change. GMPCP-induced assembly has the largest negative heat capacity change and GMPCPP has the second largest, whereas GTP/2 M glycerol- and taxol-induced assembly have more positive values, respectively. A large, negative heat capacity is most consistent with the burial of water-accessible hydrophobic surface area, which gives rise to the release of bound water. The heat capacity changes observed with GTP/2 M glycerol-induced and with taxol-induced assembly are very similar, -790 +/- 190 cal/mol/k, and correspond to the burial of 3330 +/- 820 A2 of nonpolar surface area. This value is shown to be very similar to an estimate of the buried nonpolar surface in a reconstructed microtubule lattice. Polymerization data from GMPCP- and GMPCPP-induced assembly are consistent with buried nonpolar surface areas that are 3 and 6 times larger. A linear enthalpy-entropy and enthalpy-free energy plot for tubulin polymerization reactions verifies that enthalpy-entropy compensation for this system is based upon true biochemical correlation, most likely corresponding to a dominant hydrophobic effect. Entropy analysis suggests that assembly with GTP/2 M glycerol and with taxol is consistent with conformational rearrangements in 3-6% of the total amino acids in the heterodimer. In addition, taxol binding contributes to the thermodynamics of the overall process by reducing the delta H degree and delta S degree for microtubule assembly. In the presence of GMPCPP or GMPCP, tubulin subunits associate with extensive conformational rearrangement, corresponding to 10% and 26% of the total amino acids in the heterodimer, respectively, which gives rise to a large loss of configurational entropy. An alternative, and probably preferable, interpretation of these data is that, especially with GMPCP-tubulin, additional isomerization or protonation events are induced by the presence of the methylene moiety and linked to microtubule assembly. Structural analysis shows that GTP hydrolysis is not required for sheet closure into a microtubule cylinder, but only increases the probability of this event occurring. Sheet extensions and sheet polymers appear to have a similar average length under various conditions, suggesting that the minimum cooperative unit for closure of sheets into a microtubule cylinder is approximately 400 nm long. Because of their low level of occurrence, sheets are not expected to significantly affect the thermodynamics of assembly.