F. Luis González Flecha

Kinetics and thermodynamics of the interaction of 1-anilino-naphthalene-8-sulfonate with proteins

Although 1-anilino-naphthalene-8-sulfonate (ANS) has been widely used in protein folding and binding studies, the detailed mechanism of this interaction is not fully understood. In this work the binding of ANS was analyzed at pre-equilibrium and equilibrium conditions using bovine serum albumin (BSA) as model. We employed a combined approach including the analysis of fluorescence, near-UV circular dichroism and isothermal titration calorimetric data. Experiments at equilibrium with these techniques identify three ANS molecules bound at hydrophobic cavities in BSA. Pre-equilibrium fluorescence analysis unambiguously indicated that the binding of ANS at hydrophobic cavities of BSA occurs at two different and independent classes of sites with similar affinities and quantum yields, two features that are undetectable by the equilibrium analysis. The binding of ANS to the first site is thermodynamically favored by similar contributions of the enthalpic (DeltaH = -22 kJ/mol) and entropic terms (-TDeltaS = -17 kJ/mol), while the binding to the second site is enthalpically driven (DeltaH = -31 kJ/mol; -TDeltaS = -0.6 kJ/mol). Complementary information from molecular docking showed three ANS molecules bound at hydrophobic cavities in BSA subdomains IIA and IIIA with binding affinities in the order of those found experimentally and three additional ANS molecules bound at water exposed sites.

Determination of the molecular size of BSA by fluorescence anisotropy

This work describes a laboratory experiment to illustrate the usefulness of fluorescence anisotropy in the field of biophysics. Fluorescence anisotropy of dansyl‐labeled BSA was determined in media of increasing glycerol concentrations. The Perrin equation was fitted to the experimental data, obtaining the molecular volume of the protein. The simplicity of the experiment and data analysis helped the students to focus on the relationship between probe anisotropy and rotational diffusion. Additionally, this laboratory exercise has the advantage of using a protein and a probe that are inexpensive and very common in many laboratories.

Effects of phosphatidylethanolamine glycation on lipid-protein interactions and membrane protein thermal stability

Non-enzymatic glycation of biomolecules has been implicated in the pathophysiology of aging and diabetes. Among the potential targets for glycation are biological membranes, characterized by a complex organization of lipids and proteins interacting and forming domains of different size and stability. In the present study, we analyse the effects of glycation on the interactions between membrane proteins and lipids. The phospholipid affinity for the transmembrane surface of the PMCA (plasma-membrane Ca(2+)-ATPase) was determined after incubating the protein or the phospholipids with glucose. Results show that the affinity between PMCA and the surrounding phospholipids decreases significantly after phosphospholipid glycation, but remains unmodified after glycation of the protein. Furthermore, phosphatidylethanolamine glycation decreases by approximately 30% the stability of PMCA against thermal denaturation, suggesting that glycated aminophospholipids induce a structural rearrangement in the protein that makes it more sensitive to thermal unfolding. We also verified that lipid glycation decreases the affinity of lipids for two other membrane proteins, suggesting that this effect might be common to membrane proteins. Extending these results to the in vivo situation, we can hypothesize that, under hyperglycaemic conditions, glycation of membrane lipids may cause a significant change in the structure and stability of membrane proteins, which may affect the normal functioning of membranes and therefore of cells.

Reversible Unfolding of a Thermophilic Membrane Protein in Phospholipid/Detergent Mixed Micelles

Folding mechanisms and stability of membrane proteins are poorly understood because of the known difficulties in finding experimental conditions under which reversible denaturation could be possible. In this work, we describe the equilibrium unfolding of Archaeoglobus fulgidus CopA, an 804-residue alpha-helical membrane protein that is involved in transporting Cu(+) throughout biological membranes. The incubation of CopA reconstituted in phospholipid/detergent mixed micelles with high concentrations of guanidinium hydrochloride induced a reversible decrease in fluorescence quantum yield, far-UV ellipticity, and loss of ATPase and phosphatase activities. Refolding of CopA from this unfolded state led to recovery of full biological activity and all the structural features of the native enzyme. CopA unfolding showed typical characteristics of a two-state process, with DeltaG(w) degrees =12.9 kJ mol(-)(1), m=4.1 kJ mol(-1) M(-1), C(m)=3 M, and DeltaCp(w) degrees =0.93 kJ mol(-1) K(-1). These results point out to a fine-tuning mechanism for improving protein stability. Circular dichroism spectroscopic analysis of the unfolded state shows that most of the secondary and tertiary structures were disrupted. The fraction of Trp fluorescence accessible to soluble quenchers shifted from 0.52 in the native state to 0.96 in the unfolded state, with a significant spectral redshift. Also, hydrophobic patches in CopA, mainly located in the transmembrane region, were disrupted as indicated by 1-anilino-naphtalene-8-sulfonate fluorescence. Nevertheless, the unfolded state had a small but detectable amount of residual structure, which might play a key role in both CopA folding and adaptation for working at high temperatures.

Imaging lipid lateral organization in membranes with C-laurdan in a confocal microscope

Lateral organization of biological membranes is frequently studied using fluorescence microscopy. One of the most widely used probes for these studies is 2-dimethylamino-6-lauroylnaphthalene (laurdan). The fluorescence of this probe is sensitive to the environment polarity, and thus laurdan reports the local penetration of water when inserted in membranes. Unfortunately, this probe can only be used under two-photon excitation due to its low photostability. This is a very important limitation, because there are not too many laboratories with capability for two-photon microscopy. In this work, we explored the performance of 6-dodecanoyl-2-[N-methyl-N-(carboxymethyl)amino]naphthalene (C-laurdan), a carboxyl-modified version of laurdan, for imaging biological membranes using a conventional confocal microscopy setup. We acquired generalized polarization (GP) images of C-laurdan inserted in giant unillamelar vesicles composed of binary mixtures of lipids and verified that the probe allows observing the coexistence of different phases. We also tested the performance of the probe for measurement with living cells and registered GP images of melanophore cells labeled with C-laurdan in which we could observe highly ordered regions such as filopodia. These findings show that C-laurdan can be successfully employed for studies of membrane lateral organization using a conventional confocal microscope and can open the possibility of studying a wide variety of membrane-related processes.

Oligomerization of the plasma membrane calcium pump involves two regions with different thermal stability

Ca2+ pump dimerization was studied by using a combined approach of thermal denaturation and fluorescence resonance energy transfer. The measurement of calcium pump ability to dimerize after the unfolding of individual functional domains of the enzyme demonstrated the existence of two different regions involved in the self‐association process. One of these regions is highly susceptible to thermal unfolding and was identified as the calmodulin (CaM)‐binding domain. The other region whose thermal stability is higher than those of the catalytic and CaM‐binding domains could be related with the previously found C28W‐binding regions.

Thermal stability of CopA, a polytopic membrane protein from the hyperthermophile Archaeoglobus fulgidus

Despite recent progress in understanding membrane protein folding, little is known about the mechanisms stabilizing these proteins. Here we characterize the kinetic thermal stability of CopA, a thermophilic P(IB)-type Cu+-ATPase from Archaeoglobus fulgidus. When heterologously expressed in Escherichia coli, purified and reconstituted in mixed micelles, CopA retained thermophilic characteristics with maximum activity at 75 degrees C. Incubation of CopA in the absence of substrates at temperatures in the 66-85 degrees C range led to an irreversible exponential decrease in enzyme activity suggesting a two-state process involving fully-active and inactive molecules. Although CopA inactivated much slower than mesophilic proteins, the activation energy was similar to that observed for mesophilic P-type ATPases. The inactivation process was found to be associated with the irreversible partial unfolding of the polypeptide chain, as assessed by Trp fluorescence, Phe UV spectroscopy, far UV circular dichroism, and 1-aniline-8-naphtalenesulfonate binding. However, the inactive thermally denatured protein still conserves large hydrophobic regions and considerable secondary structure.

Stoichiometry of lipid–protein interaction assessed by hydrophobic photolabeling

Here we undertook a comparative study of the composition of the lipid annulus of three ATPases pertaining to the P‐type family: plasma membrane calcium pump (PMCA), sarcoplasmic reticulum calcium pump (SERCA) and Na,K‐ATPase. The photoactivatable phosphatidylcholine analogue [125I]TID‐PC/16 was incorporated into mixtures of dimyristoyl phosphatidylcholine (DMPC) and each enzyme with the aid of the nonionic detergent C12E10. After photolysis, the extent of the labeling reaction was assessed to determine the lipid:protein stoichiometry: 17 for PMCA, 18 for SERCA, 24 for the Na,K‐ATPase (α‐subunit) and 5.6 mol PC/mol protein for the Na,K‐ATPase (β‐subunit).

PIP Water Transport and Its pH Dependence Are Regulated by Tetramer Stoichiometry

Many plasma membrane channels form oligomeric assemblies, and heterooligomerization has been described as a distinctive feature of some protein families. In the particular case of plant plasma membrane aquaporins (PIPs), PIP1 and PIP2 monomers interact to form heterotetramers. However, the biological properties of the different heterotetrameric configurations formed by PIP1 and PIP2 subunits have not been addressed yet. Upon coexpression of tandem PIP2-PIP1 dimers in Xenopus oocytes, we can address, for the first time to our knowledge, the functional properties of single heterotetrameric species having 2:2 stoichiometry. We have also coexpressed PIP2-PIP1 dimers with PIP1 and PIP2 monomers to experimentally investigate the localization and biological activity of each tetrameric assembly. Our results show that PIP2-PIP1 heterotetramers can assemble with 3:1, 1:3, or 2:2 stoichiometry, depending on PIP1 and PIP2 relative expression in the cell. All PIP2-PIP1 heterotetrameric species localize at the plasma membrane and present the same water transport capacity. Furthermore, the contribution of any heterotetrameric assembly to the total water transport through the plasma membrane doubles the contribution of PIP2 homotetramers. Our results also indicate that plasma membrane water transport can be modulated by the coexistence of different tetrameric species and by intracellular pH. Moreover, all the tetrameric species present similar cooperativity behavior for proton sensing. These findings throw light on the functional properties of PIP tetramers, showing that they have flexible stoichiometry dependent on the quantity of PIP1 and PIP2 molecules available. This represents, to our knowledge, a novel regulatory mechanism to adjust water transport across the plasma membrane.

Kinetics and Thermodynamics of Membrane Protein Folding

Understanding protein folding has been one of the great challenges in biochemistry and molecular biophysics. Over the past 50 years, many thermodynamic and kinetic studies have been performed addressing the stability of globular proteins. In comparison, advances in the membrane protein folding field lag far behind. Although membrane proteins constitute about a third of the proteins encoded in known genomes, stability studies on membrane proteins have been impaired due to experimental limitations. Furthermore, no systematic experimental strategies are available for folding these biomolecules in vitro. Common denaturing agents such as chaotropes usually do not work on helical membrane proteins, and ionic detergents have been successful denaturants only in few cases. Refolding a membrane protein seems to be a craftsman work, which is relatively straightforward for transmembrane β-barrel proteins but challenging for α-helical membrane proteins. Additional complexities emerge in multidomain membrane proteins, data interpretation being one of the most critical. In this review, we will describe some recent efforts in understanding the folding mechanism of membrane proteins that have been reversibly refolded allowing both thermodynamic and kinetic analysis. This information will be discussed in the context of current paradigms in the protein folding field.