Protein diffusion and macromolecular crowding in thylakoid membranes

The photosynthetic light reactions of green plants are mediated by chlorophyll-binding protein complexes located in the thylakoid membranes within the chloroplasts. Thylakoid membranes have a complex structure, with lateral segregation of protein complexes into distinct membrane regions known as the grana and the stroma lamellae. It has long been clear that some protein complexes can diffuse between the grana and the stroma lamellae, and that this movement is important for processes including membrane biogenesis, regulation of light harvesting, and turnover and repair of the photosynthetic complexes. In the grana membranes, diffusion may be problematic because the protein complexes are very densely packed (approximately 75% area occupation) and semicrystalline protein arrays are often observed. To date, direct measurements of protein diffusion in green plant thylakoids have been lacking. We have developed a form of fluorescence recovery after photobleaching that allows direct measurement of the diffusion of chlorophyll-protein complexes in isolated grana membranes from Spinacia oleracea. We show that about 75% of fluorophores are immobile within our measuring period of a few minutes. We suggest that this immobility is due to a protein network covering a whole grana disc. However, the remaining fraction is surprisingly mobile (diffusion coefficient 4.6 +/- 0.4 x 10(-11) cm(2) s(-1)), which suggests that it is associated with mobile proteins that exchange between the grana and stroma lamellae within a few seconds. Manipulation of the protein-lipid ratio and the ionic strength of the buffer reveals the roles of macromolecular crowding and protein-protein interactions in restricting the mobility of grana proteins.     

Self-association of polynucleosome chains by macromolecular crowding

The crowding of macromolecules in the cell nucleus, where their concentration is in the range of 100 mg/ml, is predicted to result in strong entropic forces between them. Here the effects of crowding on polynucleosome chains in vitro were studied to evaluate if these forces could contribute to the packing of chromatin in the nucleus in vivo. Soluble polynucleosomes approximately 20 nucleosomes in length formed fast-sedimenting complexes in the presence of inert, volume-occupying agents poly(ethylene glycol) (PEG) or dextran. This self-association was reversible and consistent with the effect of macromolecular crowding. In the presence of these crowding agents, polynucleosomes formed large assemblies as seen by fluorescence microscopy after labelling DNA with the fluorescent stain DAPI, and formed rods and sheets at a higher concentration of crowding agent. Self-association caused by crowding does not require exogenous cations. Single, approximately 800 nucleosome-long chains prepared in 100 muM Hepes buffer with no added cations, labelled with the fluorescent DNA stain YOYO-1, and spread on a polylysine-coated surface formed compact 3-D clusters in the presence of PEG or dextran. This reversible packing of polynucleosome chains by crowding may help to understand their compact conformations in the nucleus. These results, together with the known collapse of linear polymers in crowded milieux, suggest that entropic forces due to crowding, which have not been considered previously, may be an important factor in the packing of nucleosome chains in the nucleus.     

High frequency dynamics in hemoglobin measured by magnetic relaxation dispersion

The magnetic relaxation dispersion profiles for formate, acetate, and water protons are reported for aqueous solutions of hemoglobin singly and doubly labeled with a nitroxide and mercury(II) ion at cysteines at beta-93. Using two spin labels, one nuclear and one electron spin, a long intramolecular vector is defined between the two beta-93 positions in the protein. The paramagnetic contributions to the observed 1H spin-lattice relaxation rate constant are isolated from the magnetic relaxation dispersion profiles obtained on a dual-magnet apparatus that provides spectral density functions characterizing fluctuations sensed by intermoment dipolar interactions in the time range from the tens of microseconds to approximately 1 ps. Both formate and acetate ions are found to bind specifically within 5 angstroms of the beta-93 spin-label position and the relaxation dispersion has inflection points corresponding to correlation times of 30 ps and 4 ns for both ions. The 4-ns motion is identified with exchange of the anions from the site, whereas the 30-ps correlation time is identified with relative motions of the spin label and the bound anion in the protein environment close to beta-93. The magnetic field dependence of the paramagnetic contributions in both cases is well described by a simple Lorentzian spectral density function; no peaks in the spectral density function are observed. Therefore, the high frequency motions of the protein monitored by the intramolecular vector defined by the electron and nuclear spin are well characterized by a stationary random function of time. Attempts to examine long vector fluctuations by employing electron spin and nuclear spin double-labeling techniques did not yield unambiguous characterization of the high frequency motions of the vector between beta-93 positions on different chains.     

Nuclear magnetic relaxation dispersion in monoclinic lysozyme crystals

Nuclear magnetic relaxation measurements are reported as a function of field strength corresponding to the frequency range from 0.01 to 20 MHz for water protons in monoclinic lysozyme crystals at 278 and 298 K. Though the instrumentation used selects only a portion of the total magnetization to sample, the data clearly indicate a field dependence of the relaxation rate that signals the presence of slow motions characterized by time constants in the range of tenths of microseconds and slower. The data support, but do not uniquely prove, the hypothesis that this time scale is that appropriate to the isotropic averaging of locally anisotropic water molecule motion at the protein surface.     

Stabilization of T4 polynucleotide kinase by macromolecular crowding.

T4 polynucleotide kinase rapidly loses activity during its reaction on duplex DNA termini. Addition of high concentrations of nonspecific polymers reverses or prevents this inactivation. In contrast, additions of related materials of lower molecular weight are relatively ineffective in stabilizing the kinase. Such a pattern suggests that the stabilizing effects of polymers on kinase activity are due to macromolecular crowding. An effect of crowding on the known tendency of the kinase to undergo oligomerization reactions is consistent with our observations.     

Structure of metaphase chromosomes: a role for effects of macromolecular crowding

In metaphase chromosomes, chromatin is compacted to a concentration of several hundred mg/ml by mechanisms which remain elusive. Effects mediated by the ionic environment are considered most frequently because mono- and di-valent cations cause polynucleosome chains to form compact ~30-nm diameter fibres in vitro, but this conformation is not detected in chromosomes in situ. A further unconsidered factor is predicted to influence the compaction of chromosomes, namely the forces which arise from crowding by macromolecules in the surrounding cytoplasm whose measured concentration is 100-200 mg/ml. To mimic these conditions, chromosomes were released from mitotic CHO cells in solutions containing an inert volume-occupying macromolecule (8 kDa polyethylene glycol, 10.5 kDa dextran, or 70 kDa Ficoll) in 100 μM K-Hepes buffer, with contaminating cations at only low micromolar concentrations. Optical and electron microscopy showed that these chromosomes conserved their characteristic structure and compaction, and their volume varied inversely with the concentration of a crowding macromolecule. They showed a canonical nucleosomal structure and contained the characteristic proteins topoisomerase IIα and the condensin subunit SMC2. These observations, together with evidence that the cytoplasm is crowded in vivo, suggest that macromolecular crowding effects should be considered a significant and perhaps major factor in compacting chromosomes. This model may explain why ~30-nm fibres characteristic of cation-mediated compaction are not seen in chromosomes in situ. Considering that crowding by cytoplasmic macromolecules maintains the compaction of bacterial chromosomes and has been proposed to form the liquid crystalline chromosomes of dinoflagellates, a crowded environment may be an essential characteristic of all genomes.     

Proton magnetic relaxation dispersion in human fluoromethaemoglobin solutions.

The solvent proton spin-lattice relaxation time of high spin Fe3+ (S=5/2) human A fluoromethaemoglobin aqueous solutions was measured at 14 Larmor frequencies in the range from 2.2 to 96 MHz. The observed paramagnetic relaxation rates are analysed in terms of the Solomon-Bloembergen theory, with the g-tensor value of 2 based on the consideration of the protein tertiary structure. From the H2O (pH 6) haemoprotein solution relaxation data, tau(c) =(9.3+/-0.3) X 10(-10) sec. If the total relaxation rates are corrected for the "outer-sphere" paramagnetic contribution, tau(c)=(6.5+/-0.4) X 10(-10) sec. The latter correction is obtained from the p.m.r. of the non-exchangeable aliphatic protons of C2H4(OD)2 added to the D2O-solution of fluoromethaemoglobin. Assuming that single proton transfer is taking place through the protein channel along the axis normal to the haem (g=2), the protein "binding" site is at a distance of 3.93 to 3.98 A from the haem Fe3+ ion.     

Effects of macromolecular crowding on biochemical reaction equilibria: a molecular thermodynamic perspective

A molecular thermodynamic model is developed to investigate the effects of macromolecular crowding on biochemical reactions. Three types of reactions, representing protein folding/conformational isomerization, coagulation/coalescence, and polymerization/association, are considered. The reactants, products, and crowders are modeled as coarse-grained spherical particles or as polymer chains, interacting through hard-sphere interactions with or without nonbonded square-well interactions, and the effects of crowder size and chain length as well as product size are examined. The results predicted by this model are consistent with experimentally observed crowding effects based on preferential binding or preferential exclusion of the crowders. Although simple hard-core excluded-volume arguments do in general predict the qualitative aspects of the crowding effects, the results show that other intermolecular interactions can substantially alter the extent of enhancement or reduction of the equilibrium and can even change the direction of the shift. An advantage of the approach presented here is that competing reactions can be incorporated within the model.     

A quantitative analysis of cardiac myocyte relaxation: a simulation study

The determinants of relaxation in cardiac muscle are poorly understood, yet compromised relaxation accompanies various pathologies and impaired pump function. In this study, we develop a model of active contraction to elucidate the relative importance of the [Ca2+]i transient magnitude, the unbinding of Ca2+ from troponin C (TnC), and the length-dependence of tension and Ca2+ sensitivity on relaxation. Using the framework proposed by one of our researchers, we extensively reviewed experimental literature, to quantitatively characterize the binding of Ca2+ to TnC, the kinetics of tropomyosin, the availability of binding sites, and the kinetics of crossbridge binding after perturbations in sarcomere length. Model parameters were determined from multiple experimental results and modalities (skinned and intact preparations) and model results were validated against data from length step, caged Ca2+, isometric twitches, and the half-time to relaxation with increasing sarcomere length experiments. A factorial analysis found that the [Ca2+]i transient and the unbinding of Ca2+ from TnC were the primary determinants of relaxation, with a fivefold greater effect than that of length-dependent maximum tension and twice the effect of tension-dependent binding of Ca2+ to TnC and length-dependent Ca2+ sensitivity. The affects of the [Ca2+]i transient and the unbinding rate of Ca2+ from TnC were tightly coupled with the effect of increasing either factor, depending on the reference [Ca2+]i transient and unbinding rate.     

Assessment by sedimentation equilibrium analysis of a heterologous macromolecular interaction in the presence of self-association: interaction of S5 with S8

The proteins S5 and S8 from the Escherichia coli 30S ribosomal subunit have been examined by sedimentation equilibrium methods for behavior in solution as isolated components and in mixtures. The means of resolving two simultaneous associations in this system is discussed, and the energy of association of S5 and S8 is reported. It was found that protein S5 from the MRE 600 strain tends to self-associate weakly at 4 degree C in a manner that can be described as an isodesmic self-association with an association constant and corresponding standard Gibbs free energy equal to (7.7 +/- 0.7) X 10(3) M-1 and -4.9 +/- 0.1 kcal/mol, respectively. Protein S8 was found to have a molecular weight of 15800 and was monomeric in a pure state. Mixtures of S5 and S8 clearly demonstrated the presence of an S5-S8 complex in addition to the self-association of S5. The equilibrium constant of association for the formation of a simple S5-S8 complex at 4 degree C and the corresponding standard Gibbs free energy were found to be (5.5 +/- 1.0) X 10(4) M-1 and -6.0 +/- 0.1 kcal/mol, respectively.     

Metabolic buffering exerted by macromolecular crowding on DNA-DNA interactions: origin and physiological significance

Crowding, which characterizes the interior of all living cells, has been shown to dramatically affect biochemical processes, leading to stabilization of compact morphologies, enhanced macromolecular associations, and altered reaction rates. Due to the crowding-mediated shift in binding equilibria toward association, crowding agents were proposed to act as a metabolic buffer, significantly extending the range of intracellular conditions under which interactions occur. Crowding may, however, impose a liability because, by greatly and generally enhancing macromolecular association, it can lead to irreversible interactions. To better understand the physical determinants and physiological consequences of crowding-mediated buffering, we studied the effects of crowding, or excluded volume, on DNA structures. Results obtained from isothermal titration calorimetry (ITC) and UV melting experiments indicate that crowding-induced effects are marginal under conditions that a priori favor association of DNA strands but become progressively larger when conditions deteriorate. As such, crowding exerts "genuine" buffering activity. Unexpectedly, crowding-mediated effects are found to include enthalpy terms that favorably contribute to association processes. We propose that these enthalpy terms and preferential stabilization derive from a reconfiguration of DNA hydration that occurs in dense DNA-rich phases obtained in crowded environments.     

Efficient analysis of macromolecular rotational diffusion from heteronuclear relaxation data

A novel program has been developed for the interpretation of ¹⁵N relaxation rates in terms of macromolecular anisotropic rotational diffusion. The program is based on a highly efficient simulated annealing/minimization algorithm, designed specifically to search the parametric space described by the isotropic, axially symmetric and fully anisotropic rotational diffusion tensor models. The high efficiency of this algorithm allows extensive noise-based Monte Carlo error analysis. Relevant statistical tests are systematically applied to provide confidence limits for the proposed tensorial models. The program is illustrated here using the example of the cytochrome c from Rhodobacter capsulatus, a four-helix bundle heme protein, for which data at three different field strengths were independently analysed and compared.     

Effects of macromolecular crowding on intracellular diffusion from a single particle perspective

Compared to biochemical reactions taking place in relatively well-defined aqueous solutions in vitro, the corresponding reactions happening in vivo occur in extremely complex environments containing only 60-70% water by volume, with the remainder consisting of an undefined array of bio-molecules. In a biological setting, such extremely complex and volume-occupied solution environments are termed 'crowded'. Through a range of intermolecular forces and pseudo-forces, this complex background environment may cause biochemical reactions to behave differently to their in vitro counterparts. In this review, we seek to highlight how the complex background environment of the cell can affect the diffusion of substances within it. Engaging the subject from the perspective of a single particle's motion, we place the focus of our review on two areas: (1) experimental procedures for conducting single particle tracking experiments within cells along with methods for extracting information from these experiments; (2) theoretical factors affecting the translational diffusion of single molecules within crowded two-dimensional membrane and three-dimensional solution environments. We conclude by discussing a number of recent publications relating to intracellular diffusion in light of the reviewed material.     

Manganese-deoxyribonucleic acid binding modes. Nuclear magnetic relaxation dispersion results.

Ion-DNA interactions are discussed and the applied magnetic field strength dependence of water proton spin-lattice relaxation rates is used to study the Mn(II)-DNA interaction both qualitatively and quantitatively. Associations in which the manganese II (Mn(II)) ion is completely immobilized on the DNA are identified as well as a range of associations in which the ion is only partially reorientationally restricted. Quantitative analysis of the strength of the association in which manganese is immobilized is carried out both with and without a counter-ion condensation correction for electrostatic attraction of the mobile ions. From competition experiments with manganese the relative strengths of the interactions of magnesium and calcium with DNA are found to be identical but less than that of manganese with DNA and the affinity of lithium for DNA is found to be slightly higher than that of sodium. The data demonstrate that the reduced mobility of nonsite-bound ions may have a significant effect on DNA-ion binding analyses performed using magnetic resonance and relaxation methods.     

DNA condensation in bacteria: Interplay between macromolecular crowding and nucleoid proteins

The volume of a typical Eschericia coli nucleoid is roughly 10⁴ times smaller than the volume of a freely coiling linear DNA molecule with the same length as the E. coli genome. We review the main forces that have been suggested to contribute to this compaction factor: macromolecular crowding (that “pushes” the DNA together), DNA charge neutralization by various polycationic species (that “glues” the DNA together), and finally, DNA deformations due to DNA supercoiling and nucleoid proteins. The direct contributions of DNA supercoiling and nucleoid proteins to the total compaction factor are probably small. Instead, we argue that the formation of the bacterial nucleoid can be described as a consequence of the influence of macromolecular crowding on thick, supercoiled protein-DNA fibers, that have been partly charge neutralized by small multivalent cations.     

A quantitative analysis of astigmatism induced by pterygium

An analytical model has been developed for the localized corneal deformation produced in the region of the head of a pterygia. Astigmatism results when this localized deformation enters the central region or optical zone of the cornea. The amount and direction of the pterygia-induced astigmatism may be predicted from the values of the corneal curvature within the optical zone. The analytical solution of Lur'e based on the Papkovich-Neuber theory was applied to the anatomical and mechanical conditions affecting the cornea and conjunctiva. The force exerted by the head of a pterygia was measured experimentally for the first time. This force is of the order of that produced by the extraocular muscles in primary gaze. Using this model it is possible to predict that 2.35 diopters of the pterygia-induced astigmatism would result from a pterygia exerting 5 g of force along a meridian passing through the center of the cornea, and whose head is located 2.38 millimeters from the optical center of the cornea of an eye having a 4 mm pupillary diameter.     

Protein reorientation and bound water molecules measured by 1H magnetic spin-lattice relaxation

The water-proton spin-lattice relaxation rate constant, 1/T(1), was measured as a function of magnetic field strength for several dilute protein solutions. By separating the intermolecular contributions from the intramolecular contributions to the water-proton spin-lattice relaxation, the number of water molecules that bind to the protein for a time long compared with the rotational correlation time may be measured. We find a good correlation between the number of long-lived water molecules and the predictions based on available free volume in the proteins studied. The rotational correlation times of these proteins are larger than predicted by the Stokes-Einstein-Debye (SED) model for a sphere reorienting in a viscous liquid. The discrepancy between experiment and theory is usually attributed to hydration effects increasing the effective radius of the particle. However, the average lifetime of water molecules at the protein interface is far too short to justify such a picture. We suggest that surface roughness may be responsible for the retardation of rotational mobility and find that the SED model provides a reasonable representation of experiment if the radius assumed for the reorienting particle is the arithmetic mean of the crystallographic packing radius and the radius deduced from the effective surface area of the protein.