Patricia B. O'Hara

LANTHANIDE IONS AS LUMINESCENT PROBES OF BIOMOLECULAR STRUCTURE

Lanthanide luminescence provides a unique probe into questions of biological interest. This review article summarizes contributions to the literature in the last year in which light absorption or emission spectroscopy of the trivalent lanthanides [Ln(III)] was used as such a probe. Several optical properties make the Ln(II1)’s useful and convenient: their redshifted visible emission, their long luminescent lifetimes, the sensitivity of their absorption and emission to environment, the overlap of their excited state manifolds with the excited states of biological ligands and finally, the variety of their emission properties which make intra-Ln(II1) transfer and luminescence quenching possible. The long history of Ln(II1) substitution into biochemical environments and spectroscopic analysis of the product is best summarized in the review by Horrocks (1982). By far, the greatest amount of biological information has been obtained from Ln(II1) substitutions into Ca(I1) binding sites in proteins. The greatest similarity between Ca(I1) and the Ln(1II)’s is the ionic radius [Ca(II):1.06 A, Ln(1II)’s range from 1.06 A to 0.85 A]. The obvious difference, the trivalent charge, is accompanied by an increased differential binding entropy. Ln(II1)’s are much more highly solvated than Ca(II), and so must lose more of their solvation sphere to bind to the biological site. Thus there is a much greater increase in the entropy of the system when the Ln(II1) is bound which contributes to the generally larger binding constants. It must be remembered that the Ln(II1)’s are by no means an isomorphous replacement for Ca(I1).

Energy transfer as a probe of protein dynamics in the proteins transferrin and calmodulin

We have initiated an investigation into the usefulness of fluorescence energy transfer in probing protein dynamics. Our analysis involves measuring the energy transfer efficiency while perturbing the protein conformational equilibrium with heat. As the temperature increases, the amplitudes of vibrations increase, and fluorescence energy transfer should also increase if the donor and acceptor are in a flexible region of the protein. A theoretical analysis developed by Somogyi and co-workers for the temperature dependence of dipole-dipole energy transfer (Somogyi, B., J. Matko, S. Papp, J. Hevessey, G. R. Welch, and S. Damjanovich. 1984. Biochemistry. 23:3403-3411) was tested by the authors in one protein system. Energy transfer from tryptophan to a pyridoxamine derivatized side group in RNase increased 40% over 25 degrees C. Here we report further testing of this model in two additional protein systems: calmodulin, a calcium activated regulatory protein, and transferrin, a blood serum iron shuttle. Our studies show a slight differential sensitivity of the transfer efficiency to heat for the two systems. Normalized energy transfer over 6.5 A in calmodulin from a tyrosine donor to a Tb(III) acceptor increases 40% from 295 to 320 K. Normalized energy transfer over 42 A in transferrin from a Tb(III) donor to an Fe(III) acceptor increases 35% over the same temperature range. Whereas these results demonstrate that thermally induced fluctuations do increase energy transfer as predicted by Somogyi, they also appear rather insensitive to the nature of the protein host environment. In contrast to the Förster processes examined above, energy transfer over very short distances has shown an anomalously high temperature dependence.

Teaching structure: Student use of software tools for understanding macromolecular structure in an undergraduate biochemistry course

Because understanding the structure of biological macromolecules is critical to understanding their function, students of biochemistry should become familiar not only with viewing, but also with generating and manipulating structural representations. We report a strategy from a one‐semester undergraduate biochemistry course to integrate use of structural representation tools into both laboratory and homework activities. First, early in the course we introduce the use of readily available open‐source software for visualizing protein structure, coincident with modules on amino acid and peptide bond properties. Second, we use these same software tools in lectures and incorporate images and other structure representations in homework tasks. Third, we require a capstone project in which teams of students examine a protein‐nucleic acid complex and then use the software tools to illustrate for their classmates the salient features of the structure, relating how the structure helps explain biological function. To ensure engagement with a range of software and database features, we generated a detailed template file that can be used to explore any structure, and that guides students through specific applications of many of the software tools. In presentations, students demonstrate that they are successfully interpreting structural information, and using representations to illustrate particular points relevant to function. Thus, over the semester students integrate information about structural features of biological macromolecules into the larger discussion of the chemical basis of function. Together these assignments provide an accessible introduction to structural representation tools, allowing students to add these methods to their biochemical toolboxes early in their scientific development.

Thermal profiles of förster energy transfer preliminary studies of luminescent probes of protein dynamics in transferrin and calmodulin

Abstract Fluorescence energy transfer, the transfer of energy from a donor to an acceptor via a dipole/induced dipole mechanism, has long been used to measure distances between donors and acceptors in proteins and other macromolecules. Because the transfer can occur over time scales larger than protein bending and breathing modes, multiple conformational states can be sampled. The analysis of these states is weighted by the donor-acceptor distance; shorter distances carry more weight, because the energy transfer depends on the inverse sixth power of the distance. The usefulness of fluorecence energy transfer in probing these large amplitude protein motions is studied here. The method involves measuring the nergy transfer efficiency while perturbing the protein conformation with heat. As the temperature increases, the amplitudes of vibrations increase, and fluorescence energy transfer should also increase if the donor and acceptor are in flexible region of the protein. This hypothesis was tested in two different protein systems; calmodulin, a calcium- activated regulatory protein, and transferrin, a blood serum iron shuttle. The preliminary studies show a differential sensitivity of the transfer efficiency to heat for the systems. Normalized energy transfer over 10 A in calmodiulin from a tyrosine donor to a Tb(III) acceptor increases 40% from 297 to 322 K. Normalized energy transfer over 42 A in transferrin from a Tb(III) donor to an Fe(III) acceptor increase 35% over the same temperature range. In marked contrast to these systems, energy transfer from tyrosine to a chelated Tb(III) shows anomalously high temperature- dependence.

Clarifying the Intersection Between Alpha-Crystallin B Function and Oligomerization by Altering Aggregation Conditions

Alpha-Crystallin-B (aXB) is a small heat shock protein found mainly in the lens of the eye. There it serves two purposes: acting as a chaperone to prevent the misfolding of the other protein and contributing to the high protein concentrations required to focus light. When aXB chaperone function fails cataract formation begins. Understanding the dependence of the mechanism of aXB chaperone function on its oligomerization state will aid in the delay or prevention of cataracts. Oligomerization of alpha-crystallinB is pH dependent. We present a mechanism for the chaperone function of aXB using insulin as a model system in which it is possible to induce aggregation, The relationship between oligomerization and chaperone function is tested by measuring the dependence of the onset of light scattering insulin aggregates on the oligomerization state of the aXB Different oligomerization states can be induced by pre-incubating aXB at a range of pHs. Attenuated chaperone function is observed for aXB pre-incubated at elevated pH even when the assay itself takes place at pH 7. These results are consistent with a model in which aXB is kinetically trapped into higher oligomer states at high pH and unable to return quickly to its functional equilibrium state upon dilution to neutral pH. We will also test the generality of this model by extending our studies to Gamma-Crystallin. Finally, we will measure the contribution of the C-terminal strand to chaperone function and oligomerization using fluorescence resonance energy transfer (FRET). Observation of pH dependent dynamics of the C-terminal strand will help to distinguish between stand-exchanged and dynamic states of the C terminus, allowing its contribution to oligomerization and chaperone function to be probed.

Agonist AMD Antagonist Activity in a GFP Yeast Based Estrogen Receptor Functional Assay

Estrogen receptors (ERα and ER β) are ligand-binding transcription factors activated by the hormone 17-β estradiol. Ligand binding triggers ER dimerization, translocation of the receptor from the cytoplasm into the nucleus and eventually activation of the genes under control of ER. Studies have revealed a role for estrogen receptors in male and female sexual development, reproductive functions, bone metabolism and regulation of neuroendocrine and cardiovascular systems. ER is also known to bind to other non-native ligands known in pharmacology as receptor agonists or antagonists. Agonists provoke a biological response when bound to the receptor; antagonists inhibit a biological response when bound. Our lab is interested in the promiscuous binding of the estrogen receptor and its ability to activate different genes in different tissues. Fluorescence Resonance Energy Transfer (FRET) assays have previously been performed using ER to study ligand binding affinities for the receptor. However, this technique is unable to determine whether these ligands are agonists or antagonists and allow ER dimerization and gene activation. To investigate these phenomena, an activity assay that measures ER controlled gene expression has been developed which provides the opportunity to gain further insight into the functional activity in living systems. Recombinant yeast cells that express ERα use the green fluorescent protein (GFP) reporter to determine whether ER α, in the presence of a particular ligand, has activated gene of expression. We have correlated the binding to agonist and antagonist behavior of several xeno and phyto estrgens.

Elucidating Molecular Constraints that Effect Alpha Crystallin Oligomerization, Stability, and Chaperone Function

Alpha Crystallin is the major protein component of the human lens and plays an important role in the prevention of cataracts. α-Crystallin (αX) oligomers consist of two isoforms, αX-A and αX-B which share high sequence similarity and define the common α-Crystallin fold found in many small heat shock proteins (sHSPs). αX-A and αX-B are hypothesized to play two important roles within the lens. First, αX-A and αX-B belong to a group of proteins called Crystallins (α, β, and γ) that are very stable proteins that play a role in preserving a uniform density within the lens, which allows it to focus light. The Crystallin proteins’ ability to form diverse and stable oligomers results in a glass-like rather than crystalline organization to the lens protein material, which also aids in the long-term stability of this high-density protein organ. Second, αX-A and αX-B both function as sHSPs that bind to misfolded proteins, preventing formation of large, insoluble protein aggregates (the beginning of cataracts). Our lab is investigating the molecular interactions between αX-A and αX-B that result in its stability, diverse oligomerization, and chaperone function. To this end we are using a model, inducible misfolding protein (insulin B-chain) to study chaperone function by light scatter under various conditions. We are also using random and targeted modification of αX-A and αX-B to simulate long-term protein damage and degradation observed in aged lenses. Focus on the C-terminal strand exchange observed in recent crystal structures and proposed to aid in αX-A and αX-B polydisperse oligomerization is additionally aiding experimental design. We hope to identify specific molecular interactions that result in αX-A and αX-B’s chaperone function, and determine how those interactions relate to stability and self-oligomerization.

Selectivity of Binding To Estrogen Receptors Alpha and Beta As Determined by Fluorescence Polarization

Estrogens role in cell growth and proliferation has long been appreciated both in the normal development of secondary sexual characteristics and in diseased states in cancers of the breast, ovaries and uterus. We are beginning to appreciate estrogens expanded role in maintaining such diverse functions as the skins elasticity, the health of the central nervous system, bone density and cardiac health. Estrogen plays out its roles in varied tissues by binding to two major ligand activated nuclear receptors, estrogen receptor alpha (ER-α) and estrogen receptor beta (ER-β). The interrelationship of the two receptors plays a role in the responsiveness of certain breast cancers to drug treatment. Though the ligand binding sites of the two receptors differ by only two amino acids, the overall degree of homology between ER-α and ER-β is low. The body uses the receptor selectivity to its advantage by dispersing the receptors in varying ratios to different tissues. Of these actions ER-α is thought to be responsible for the majority with ER-β playing a minor role in all and having more significance in the cardiovascular and skeletal systems. Small molecules have been identified which bind to one or the other receptors with differing binding affinities. These selective estrogen receptor modulators (SERMs) hold the potential to be pharmacologically effective in treating diseases specific to one type of estrogen receptor while not affecting the other. For example, ER-a and ER-b are both present in breast tissue, and the ratio of beta to alpha is being examined as one indicator in determining the likelihood of successful treatment of breast cancer by certain drugs. Here we use changes in the fluorescence polarization to calculate binding affinities for each of several small molecules to ER-α and ER-β.

Chemistry and Luminescence (the author replies)

Thanks Dr. Cintas for highlighting yet another process by which materials can be induced to emit light.

Target-peptide-induced changes in the structure and dynamics of calmodulin as probed by frequency domain fluorimetry of bound Tb(III)

Tb(III) luminescence is used to probe the conformational change induced in the calcium regulatory protein calmodulin upon binding to a target peptide. The luminescence lifetime for Tb(III) measured by frequency domain fluorimetry increases from 1278 microseconds for the calmodulin complex to 1496 microsecond for the complex of calmodulin and M13, a peptide derived from the calmodulin target protein myosin light chain kinase. The intensity of the Tb(III) emission increases over the solution value by a factor of 726 and 891 when bound to calmodulin and to the complex of calmodulin and M13 respectively. The sensitivity of the Tb(III) decay rate to deuterated solvent was also measured and is consistent with a single water molecule bound to the metal in both the calmodulin and calmodulin-M13 complex. The most dramatic change induced by M13 is the threefold reduction in the width of the Tb(III) lifetime distribution, which is interpreted to be a target-peptide-induced annealing of the previously flexible metal-binding site.