Berry Birdsall

Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules

G-protein–coupled receptors (GPCRs) are the largest family of transmembrane signaling proteins in the human genome. Events in the GPCR signaling cascade have been well characterized, but the receptor composition and its membrane distribution are still generally unknown. Although there is evidence that some members of the GPCR superfamily exist as constitutive dimers or higher oligomers, interpretation of the results has been disputed, and recent studies indicate that monomeric GPCRs may also be functional. Because there is controversy within the field, to address the issue we have used total internal reflection fluorescence microscopy (TIRFM) in living cells to visualize thousands of individual molecules of a model GPCR, the M1 muscarinic acetylcholine receptor. By tracking the position of individual receptors over time, their mobility, clustering, and dimerization kinetics could be directly determined with a resolution of ~30 ms and ~20 nm. In isolated CHO cells, receptors are randomly distributed over the plasma membrane. At any given time, ~30% of the receptor molecules exist as dimers, and we found no evidence for higher oligomers. Two-color TIRFM established the dynamic nature of dimer formation with M1 receptors undergoing interconversion between monomers and dimers on the timescale of seconds.

Correction for Light Absorption in Fluorescence Studies of Protein-Ligand Interactions

It is shown that absorption of the excitation light can lead to substantial systematic errors in fluorescence measurements of equilibrium constants for formation of protein-ligand complexes. The assumptions about the optical arrangement of the fluorescence spectrometer involved in the calculation of the correction of this absorption are discussed. A general semiempirical correction procedure which can be used for (calculated) absorbance values as high as 5 is described. The importance of choosing the excitation wavelength so as to minimize the necessity for these corrections is emphasized.

The use of transferred nuclear Overhauser effects in the study of the conformations of small molecules bound to proteins

Abstract A novel method is proposed for the study of the conformation in solution of small molecules bound to proteins. In transfer of saturation experiments, irradiation at the frequency of a proton in the bound ligand can result in an intensity change in the signal from a different proton in the free excess ligand via a nuclear Overhauser effect between the two protons in the bound ligand. Approximatel calculations show that the observation of such effects depends upon the close spatial proximity (within about 4.0 A) of the two protons involved and thus gives useful conformational information. Two examples of this method are given, for the binding of trimethoprim and NADP + , respectively, to Lactobacillus casei dihydrofolate reductase.

HMQC-NOESY-HMQC, a three-dimensional NMR experiment which allows detection of nuclear overhauser effects between protons with overlapping signals

In recent years the structures of many small proteins have been determined by twodimensional NMR, using methods pioneered by Wiithrich et al. (I ). These methods depend exclusively on data from proton NMR experiments and despite their success it has become clear that they are more difficult to apply and less likely to work for proteins with molecular weights of more than about 15,000. The deleterious effects of greater molecular size arise both from the large number of signals and from the increase in the linr~tidths of the signals: among other things these effects result in spectra which have a much higher degree of overlap. This is particularly acute in proteins with a high helical content, in which, for example, the majority of the amide protons are expected to resonate within a range of just one part per million. Several different strategies for overcoming this problem have been proposed. Among the most promising are those that depend upon nonspecific near-complete isotopic substitution of 15N for 14N or 13C for “C, in conjunction with two-dimensional NMR experiments which exploit the shift range of the heteronuclei to separate the proton signals (2-8). An alternative and potentially very powerful strategy is the use of threedimensional NMR to alleviate spectral overlap (9-12). The combination of threedimensional NMR with heteronuclear labeling is proving particularly effective (1318), and an important example ofthis approach is the 3D NOESY-HMQC experiment, developed by Marion et ~11. (14) and Zuiderweg and Fesik (15) for use with “Nlabeled proteins. This experiment may be thought of as generating a 15N-edited NOESY spectrum, with signals spread out in a third dimension according to the lSN shift of the directly coupled amide nitrogens. More formally, a proton H, which has an NOE to an amide proton Hi, will generate a cross peak in the NOESY-HMQC experiment centered at frequency coordinates (h,, nb, hi,), where h, and hb are the chemical shifts of H, and Hh, respectively, and nb is the chemical shift of the “N nucleus that is directly coupled to Hi,. It is clear that by spreading the signals according to the nitrogen shifts this experiment would allow the Ha-HI, interaction to be identified even if there were one or more additional protons which had the same shift as Hi,. However, if H, and Hi, themselves overlap the NOESY-HMQC experiment does not allow an NOE interaction between them to be detected: in this case the presence of the characteristic cross peak at (h,, nb, hb) would be masked by the coincident and more intense “di-

1H nuclear magnetic resonance studies of the tyrosine residues of selectively deuterated Lactobacillus casei dihydrofolate reductase

A selectively deuterated dihydrofolate reductase, in which all the aromatic protons except the 2, 6-protons of the tyrosine residues have been replaced by deuterium, has been prepared from Lactobacillus casei grown on a mixture of normal and deuterated amino acids. The aromatic region of the 1H n. m. r. spectrum of this enzyme contains only resonances from the five tyrosine residues. For each tyrosine, the 2- and 6-protons have the same chemical shift, indicating rapid interconversion of the two conformers related by 180° rotation about the Cβ-Cγ bond. The effects of substrate, inhibitor and coenzyme binding on the tyrosine residues have been investigated; four of the five residues are affected by ligand binding. Using the weakly binding ligands 2, 4-diaminopyrimidine and p-nitrobenzoyl-l-glutamate to connect the spectra of the free enzyme with those of the complexes, it is possible to give a detailed description of the effects of ligand binding on individual residues. In the binary complexes, methotrexate affects three tyrosine residues, only one of which is affected by folate, indicating a significant difference in the mode of binding of substrates and inhibitors. The co-enzymes NADP+ and NADPH lead to broadly similar changes in the spectrum, except for one resonance which is shifted in opposite directions by the two co-enzymes. The oxidized and reduced coenzymes also differ in their effects on the changes produced by inhibitor binding; the spectrum of the enzyme-NADPH-methotrexate complex is similar to that of the enzyme-methotrexate complex, while that of the enzyme-NADP+-methotrexate complex is not. In contrast to the behaviour seen in the binary complexes, the spectrum of the enzyme-NADP+-folate complex is very similar to that of enzyme-NADP+-methotrexate. Evidence is presented that some, at least, of the changes in chemical shift of the tyrosine residues are due to ligand-induced conformational changes. The binding of p-nitrobenzoyl-l-glutamate to the enzyme-2, 4-diamino-pyrimidine complex is found to be tighter than that to the enzyme alone.

1H nuclear magnetic resonance studies of Lactobacillus casei dihydrofolate reductase: effects of substrate and inhibitor binding on the histidine residues.

The effects of the binding of substrates and inhibitors (folate, dihydrofolate, folinic acid, trimethoprim, methotrexate and aminopterin) to Lactobacillus casei dihydrofolate reductase on the histidine residues of the enzyme have been studied by 1H n. m. r. spectroscopy. The more weakly binding (rapidly exchanging) inhibitors 2, 4-diaminopyrimidine and p-aminobenzoyl-l-glutamate, which can be regarded as ‘fragments’ of methotrexate, have also been studied as an aid in interpreting the effects of the strongly-binding ligands. p-Aminobenzoyl-l-glutamate binds to two sites on the enzyme; binding to the stronger site is competitive with methotrexate, and affects three histidine residues, denoted HA, HE and HF. The second site is 30-fold weaker, is not competitive with methotrexate, and affects a single histidine residue (either HB or HC). The binding to the first site is 25-fold stronger in the presence of 2, 4-diaminopyrimidine, while binding to the second site is unaffected. Folate, dihydrofolate and folinic acid have identical effects on the histidine residues; the pK of HE is increased from 6.54 to 6.75, and that of HF from 6.54 to ca. 7.2, while the C2-H resonance of HA is shifted downfield. Methotrexate and aminopterin affect the same three histidine residues as does folate; for HA and HF the effects are the same as those produced by folate, while the pK of HE is decreased from 6.54 to 6.2. Trimethoprim and 2,4-diaminopyrimidine have effects very similar to those of methotrexate, with the exception that histidine HF is not affected by these compounds (which lack the p-amino-benzoyl-l-glutamate moiety).

Correlated bond rotations in interactions of arginine residues with ligand carboxylate groups in protein ligand complexes.

© 1997 Federation of European Biochemical Societies.

15N and 1H NMR evidence for multiple conformations of the complex of dihydrofolate reductase with its substrate, folate

The binding of folate to Lactobacillus casei dihydrofolate reductase in the presence and absence of NADP+ has been studied by 15N NMR, using [5‐15N]folate. In the presence of NADP+, three separate signals were observed for the single 15N atom, in agreement with our earlier evidence from 1H and 13C NMR for multiple conformations of this complex [(1982) Biochemistry 21, 5831–5838]. The 15N spectra of the binary enzymefolate complex provide evidence for the first time that this complex also exists in at least two conformational states. This is confirmed by the observation of two separate resonances for the 7‐proton of bound folate, located by two‐dimensional exchange spectroscopy.

Stereochemistry of reduction of folic acid using dihydrofolate reductase

The dihydrofolate reductase catalysed reduction of the vitamin folic acid (5) to the coenzyme 5,6,7,8-tetrahydrofolic acid (2) involves transfer of the 4-pro-R-hydrogen of NADPH to the si-face at C-7 of folic acid (5); the absolute stereochemistry at C-6 of 5,6,7,8-tetrahydrofolic acid (2) from the enzymic reduction has been correlated with that of biologically active folinic acid (3), and the reduction at both C-6 and C-7 therefore involves the same side of NADPH and the same face of folic acid.

Stereospecific assignments of the leucine methyl resonances in the 1H NMR spectrum of Lactobacillus casei dihydrofolate reductase

A general method is described for the stereospecific assignment of methyl resonances in protein NMR spectra based on selective deuteration procedures. A selectively deuterated dihydrofolate reductase from L. casei was prepared by incorporating stereoselectively deuterated l‐leucine, (2S,4R)[5,5,5‐2H3]leucine. By comparing the COSY spectra of the dihydrofolate reductase‐methotrexate complexes formed using deuterated and non‐deuterated enzyme the stereospecific assignments for resonances of all 13 leucine residues were obtained by noting the absence of cross‐peaks in spectra from the deuterated proteins.