DsRed, a brilliantly red fluorescent protein, was recently cloned from Discosoma coral by homology to the green fluorescent protein (GFP) from the jellyfish Aequorea. A core question in the biochemistry of DsRed is the mechanism by which the GFP-like 475-nm excitation and 500-nm emission maxima of immature DsRed are red-shifted to the 558-nm excitation and 583-nm emission maxima of mature DsRed. After digestion of mature DsRed with lysyl endopeptidase, high-resolution mass spectra of the purified chromophore-bearing peptide reveal that some of the molecules have lost 2 Da relative to the peptide analogously prepared from a mutant, K83R, that stays green. Tandem mass spectrometry indicates that the bond between the alpha-carbon and nitrogen of Gln-66 has been dehydrogenated in DsRed, extending the GFP chromophore by forming C==N==C==O at the 2-position of the imidazolidinone. This acylimine substituent quantitatively accounts for the red shift according to quantum mechanical calculations. Reversible hydration of the C==N bond in the acylimine would explain why denaturation shifts mature DsRed back to a GFP-like absorbance. The C==N bond hydrolyses upon boiling, explaining why DsRed shows two fragment bands on SDS/PAGE. This assay suggests that conversion from green to red chromophores remains incomplete even after prolonged aging.

Methylenetetrahydrofolate reductase (MTHFR) catalyses the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, a cofactor for homocysteine methylation to methionine. MTHFR deficiency, an autosomal recessive disorder, results in homocysteinemia. Using degenerate oligonucleotides based on porcine peptide sequence data, we isolated a 90-bp cDNA by PCR from pig liver RNA. This cDNA was used to isolate a human cDNA, the predicted amino acid sequence of which shows strong homology to porcine MTHFR and to bacterial metF genes. The human gene has been localized to chromosome 1p36.3. Two mutations were identified in MTHFR-deficient patients: a missense mutation (Arg to Gln), in a residue conserved in bacterial enzymes, and a nonsense mutation (Arg to Ter).

We have characterized a phosphoserine binding domain in the coactivator CREB-binding protein (CBP) which interacts with the protein kinase A-phosphorylated, and hence activated, form of the cyclic AMP-responsive factor CREB. The CREB binding domain, referred to as KIX, is alpha helical and binds to an unstructured kinase-inducible domain in CREB following phosphorylation of CREB at Ser-133. Phospho-Ser-133 forms direct contacts with residues in KIX, and these contacts are further stabilized by hydrophobic residues in the kinase-inducible domain which flank phospho-Ser-133. Like the src homology 2 (SH2) domains which bind phosphotyrosine-containing peptides, phosphoserine 133 appears to coordinate with a single arginine residue (Arg-600) in KIX which is conserved in the CBP-related protein P300. Since mutagenesis of Arg-600 to Gln severely reduces CREB-CBP complex formation, our results demonstrate that, as in the case of tyrosine kinase pathways, signal transduction through serine/threonine kinase pathways may also require protein interaction motifs which are capable of recognizing phosphorylated amino acids.

The posttranscriptional regulator (p27x-III) of human T cell leukemia virus type I (HTLV-I) is located predominantly in the cell nucleolus. A highly basic amino-terminal sequence (NH2-Met-Pro-Lys-Thr-Arg-Arg-Arg-Pro-Arg-Arg-Ser-Gln-Arg-Lys-Arg-Pro-Pro -Thr- Pro) in this protein, when fused to the amino termini of beta-galactosidase and p40x of HTLV-I, acts as an autonomous signal capable of directing the hybrid proteins to the cell nucleolus.

Thirteen mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa (ADRP) have been produced by transfection of cloned cDNA into tissue culture cells. Three mutants [class I: Phe-45----Leu, Gln-344----termination (deletion of C-terminal positions 344-348), and Pro-347----Leu] resemble wild-type rhodopsin in yield, regenerability with 11-cis-retinal, and plasma membrane localization. Ten mutants [class II: Thr-17----Met, Pro-23----His, Thr-58----Arg, Val-87----Asp, Gly-89----Asp, Gly-106----Trp, Arg-135----Leu, Arg-135----Trp, Tyr-178----Cys, and Asp-190----Gly] accumulate to significantly lower levels, regenerate with 11-cis-retinal variably or not at all, and are transported inefficiently to the plasma membrane, remaining primarily in the endoplasmic reticulum. These data suggest that there are at least two distinct biochemical defects associated with different rhodopsin mutants in ADRP.

Here, we show that N-ethylmaleimide-sensitive fusion protein (NSF) interacts directly and selectively with the intracellular C-terminal domain of the GluR2 subunit of AMPA receptors. The interaction requires all three domains of NSF but occurs between residues Lys-844 and Gln-853 of rat GluR2, with Asn-851 playing a critical role. Loading of decapeptides corresponding to the NSF-binding domain of GluR2 into rat hippocampal CA1 pyramidal neurons results in a marked, progressive decrement of AMPA receptor-mediated synaptic transmission. This reduction in synaptic transmission was also observed when an anti-NSF monoclonal antibody (mAb) was loaded into CA1 neurons. These results demonstrate a previously unsuspected direct interaction in the postsynaptic neuron between two major proteins involved in synaptic transmission and suggest a rapid NSF-dependent modulation of AMPA receptor function.

Previously, we identified a novel rice gene, GROWTH-REGULATING FACTOR1 (OsGRF1), which encodes a putative transcription factor that appears to play a regulatory role in stem elongation. We now describe the GRF gene family of Arabidopsis thaliana (AtGRF), which comprises nine members. The deduced AtGRF proteins contain the same characteristic regions--the QLQ (Gln, Leu, Gln) and WRC (Trp, Arg, Cys) domains--as do OsGRF1 and related proteins in rice, as well as features indicating a function in transcriptional regulation. Most of the AtGRF genes are strongly expressed in actively growing and developing tissues, such as shoot tips, flower buds, and roots, but weakly in mature stem and leaf tissues. Overexpression of AtGRF1 and AtGRF2 resulted in larger leaves and cotyledons, as well as in delayed bolting of the inflorescence stem when compared to wild-type plants. In contrast, triple insertional null mutants of AtGRF1-AtGRF3 had smaller leaves and cotyledons, whereas single mutants displayed no changes in phenotype and double mutants displayed only minor ones. The alteration of leaf growth in overexpressors and triple mutants was based on an increase or decrease in cell size, respectively. These results indicate that AtGRF proteins play a role in the regulation of cell expansion in leaf and cotyledon tissues.

Hereditary genetic defects in DNA repair lead to increased risk of cancer. Polymorphisms in several DNA repair genes have been identified; however, the impact on repair phenotype has not been elucidated. We explored the relationship between polymorphisms in the DNA repair enzyme, XRCC1 (codons 194, 280, and 399), and genotoxic end points measured in two populations: (a) placental aflatoxin B1 DNA (AFB1-DNA) adducts in a group of Taiwanese maternity subjects (n = 120); and (b) somatic glycophorin A (GPA) variants in erythrocytes from a group of North Carolina smokers and nonsmokers (n = 59). AFB1-DNA adducts were measured by ELISA, and erythrocyte GPA variant frequency (NN and NO) was assessed in MN heterozygotes with a flow cytometric assay. XRCC1 genotypes were identified by PCR-RFLPs. The XRCC1 399Gln allele was significantly associated with higher levels of both AFB1-DNA adducts and GPA NN mutations. Individuals with the 399Gln allele were at risk for detectable adducts (odds ratio, 2.4; 95% confidence interval, 1.1-5.4; P = 0.03). GPA NN variant frequency was significantly higher in 399Gln homozygotes (19.6 x 10(-6)) than in Gln/Arg heterozygotes (11.4 x 10(-6); P < 0.05) or Arg/Arg homozygotes (10.1 x 10(-6); P = 0.01). No significant effects were observed for other XRCC1 polymorphisms. These results suggest that the Arg399Gln amino acid change may alter the phenotype of the XRCC1 protein, resulting in deficient DNA repair.

Extracts of the bovine tracheal mucosa have an abundant peptide with potent antimicrobial activity. The 38-amino acid peptide, which we have named tracheal antimicrobial peptide (TAP), was isolated by a sequential use of size-exclusion, ion-exchange, and reverse-phase chromatographic fractionations using antimicrobial activity as a functional assay. The yield was approximately 2 micrograms/g of wet mucosa. The complete peptide sequence was determined by a combination of peptide and cDNA analysis. The amino acid sequence of TAP is H-Asn-Pro-Val-Ser-Cys-Val-Arg-Asn-Lys-Gly-Ile-Cys-Val-Pro-Ile-Arg-Cys-Pr o- Gly-Ser-Met-Lys-Gln-Ile-Gly-Thr-Cys-Val-Gly-Arg-Ala-Val-Lys-Cys-Cys-Arg- Lys-Lys - OH. Mass spectral analysis of the isolated peptide was consistent with this sequence and indicated the participation of six cysteine residues in the formation of intramolecular disulfide bonds. The size, basic charge, and presence of three intramolecular disulfide bonds is similar to, but clearly distinct from, the defensins, a well-characterized class of antimicrobial peptides from mammalian circulating phagocytic cells. The putative TAP precursor is predicted to be relatively small (64 amino acids), and the mature peptide resides at the extreme carboxyl terminus and is bracketed by a short putative propeptide region and an inframe stop codon. The mRNA encoding this peptide is more abundant in the respiratory mucosa than in whole lung tissue. The purified peptide had antibacterial activity in vitro against Escherichia coli, Staphylococcus aureus, Klebsiella pneumonia, and Pseudomonas aeruginosa. In addition, the peptide was active against Candida albicans, indicating a broad spectrum of activity. This peptide appears to be, based on structure and activity, a member of a group of cysteine-rich, cationic, antimicrobial peptides found in animals, insects, and plants. The isolation of TAP from the mammalian respiratory mucosa may provide insight into our understanding of host defense of this vital tissue.

Amyloid fibrils were isolated from amyloid-laden tissue obtained from a chronic hemodialysis patient with carpal tunnel syndrome. After solubilization in guanidine HCl, a significant amount of the protein was located in a homogeneous low molecular weight fraction. The protein was found to be identical to beta 2-microglobulin, with regard to its molecular weight of 11,000, amino acid composition and 16 amino-terminal amino acids: Ile-Gln-Arg-Thr-Pro-Lys-Ile-Gln-Val-Tyr-Ser-Arg-His-Pro-Ala-Glu-. These results demonstrate that the amyloid associated with chronic hemodialysis contains as major component a new form of amyloid fibril protein that is homologous to beta 2-microglobulin.

Sequence variations have been identified in a number of DNA repair genes, including XPD, but the effect of these polymorphisms on DNA repair capacity (DRC) is uncertain. We therefore examined XPD polymorphisms at Lys751<em>Gln</em> and Asp312Asn in 341 white lung cancer cases and 360 age-, sex-, ethnicity-, and smoking-matched controls accrued in a hospital-based molecular epidemiological study of susceptibility markers for lung cancer. As previously reported, DRC was statistically significantly lower in the cases than in the controls (7.8% versus 9.5%; P < 0.001), which represents an average 18% reduction among the cases. The variant Lys751<em>Gln</em> and Asp312Asn allele frequencies were 0.36 and 0.29, respectively, for the cases and 0.33 and 0.27, respectively, for the controls. For subjects homozygous for the variant genotype at either locus, the adjusted odds ratio [95% confidence interval (CI)] was 1.84 (1.11-3.04; P = 0.018, for trend). Both cases and controls with the wild-type genotypes exhibited the most proficient DRC. The risk (95% CI) for suboptimal DRC (defined as less than the median DRC value among the controls) was 1.57 (0.74-3.35) for those with the <em>Gln</em>/<em>Gln</em>751 genotype. For cases with the Asn/Asn312 genotype, the risk (95% CI) was 3.50 (1.06-11.59). For cases who were homozygous at either locus, the risk was 2.29 (1.03-5.12; P = 0.048, for trend). The pattern was less evident among the controls, although there was a nonsignificant 41% increase in the risk of suboptimal DRC for controls who were homozygous at either locus. These results suggest that the two XPD polymorphisms have a modulating effect on DRC, especially in the cases.

We examined a variety of strains of Sindbis virus for the genetic changes responsible for differences in neurovirulence in mice. SV1A (a low passage of the AR339 strain of Sindbis virus), a neuroadapted Sindbis virus (NSV), and two laboratory strains of Sindbis virus (HRSP and Toto1101) were examined. NSV causes severe encephalomyelitis with hind-limb paralysis and high mortality after intracerebral inoculation in weanling mice. In contrast, SV1A causes only mild, nonfatal disease in weanling mice; however, in suckling mice, SV1A causes a fatal encephalomyelitis after either intracerebral or subcutaneous inoculation. The two laboratory strains used have a greatly reduced neurovirulence for suckling mice and are avirulent for weanling mice. The nucleotide sequences and encoded amino acid sequences of the structural glycoproteins of these four strains were compared. Hybrid genomes were constructed by replacing restriction fragments in a full-length cDNA clone of Sindbis virus, from which infectious RNA can be transcribed in vitro, with fragments from cDNA clones of the various strains. These recombinant viruses allowed us to test the importance of each amino acid difference between the various strains for neurovirulence in weanling and suckling mice. Glycoproteins E2 and E1 were of paramount importance for neurovirulence in adult mice. Recombinant viruses containing the nonstructural protein region and the capsid protein region from an avirulent strain and the E1 and E2 glycoprotein regions from NSV were virulent, although they were less virulent than NSV. Furthermore, changes in either E2 (His-55 in NSV to Gln in SV1A) or E1 (Ala-72 in NSV to Val in SV1A and Asp-313 in NSV to Gly in SV1A) reduced virulence. For virulence in suckling mice, we found that a number of changes in E2 and E1 can lead to decreased virulence and that in fact, a gradient of virulence exists.

High resolution (1.43-1.8 A) crystal structures and the corresponding electron paramagnetic resonance (EPR) spectra were determined for T4 lysozyme derivatives with a disulfide-linked nitroxide side chain [-CH(2)-S-S-CH(2)-(3-[2,2,5,5-tetramethyl pyrroline-1-oxyl]) identical with R1] substituted at solvent-exposed helix surface sites (Lys65, Arg80, Arg119) or a tertiary contact site (Val75). In each case, electron density is clearly resolved for the disulfide group, revealing distinct rotamers of the side chain, defined by the dihedral angles X(1) and X(2). The electron density associated with the nitroxide ring in the different mutants is inversely correlated with its mobility determined from the EPR spectrum. Residue 80R1 assumes a single g(+)()g(+)() conformation (Chi(1) = 286, X(2) = 294). Residue 119R1 has two EPR spectral components, apparently corresponding to two rotamers, one similar to that for 80R1 and the other in a tg(-)() conformation (Chi(1) = 175, X(2) = 54). The latter state is apparently stabilized by interaction of the disulfide with a Gln at i + 4, a situation also observed at 65R1. R1 residues at helix surface site 65 and tertiary contact site 75 make intra- as well as intermolecular contacts in the crystal and serve to identify the kind of molecular interactions possible for the R1 side chain. A single conformation of the entire 75R1 side chain is stabilized by a variety of interactions with the nitroxide ring, including hydrophobic contacts and two unconventional C-H.O hydrogen bonds, one in which the nitroxide acts as a donor (with tyrosine) and the other in which it acts as an acceptor (with phenylalanine). The interactions revealed in these structures provide an important link between the dynamics of the R1 side chain, reflected in the EPR spectrum, and local protein structure. A library of such interactions will provide a basis for the quantitative interpretation of EPR spectra in terms of protein structure and dynamics.

DNA repair genes have an important role in protecting individuals from cancer-causing agents. Polymorphisms in several DNA repair genes have been identified and individuals with non-dramatic reductions in the capacity to repair DNA damage are observed in the population, but the impact of specific genetic variants on repair phenotype and cancer risk has not yet been clarified. In 308 healthy Italian individuals belonging to the prospective European project EPIC, we have investigated the relationship between DNA damage, as measured by (32)P-DNA adduct levels, and three genetic polymorphisms in different repair genes: XRCC1-Arg399Gln (exon 10), XRCC3-Thr241Met (exon 7) and XPD-Lys751Gln (exon 23). DNA adduct levels were measured as relative adduct level (RAL) per 10(9) normal nucleotides by DNA (32)P-post-labelling assay in white blood cells from peripheral blood. Genotyping was performed by PCR-RFLP analysis. The XRCC3-241Met variant was significantly associated with higher DNA adduct levels, whereas XRCC1-399Gln and XPD-751Gln were associated with higher DNA adduct levels only in never-smokers. XRCC3-241Met homozygotes had an average DNA adduct level of 11.44 +/- 1.48 (+/-SE) compared with 7.69 +/- 0.88 in Thr/Met heterozygotes and 6.94 +/- 1.11 in Thr/Thr homozygotes (F = 3.206, P = 0.042). Never-smoking XRCC1-399Gln homozygotes had an average DNA adduct level of 15.60 +/- 5.42 compared with 6.16 +/- 0.97 in Gln/Arg heterozygotes and 6.78 +/- 1.10 in Arg/Arg homozygotes (F = 5.237, P = 0.007). A significant odds ratio (3.81, 95% CI 1.02-14.16) to have DNA adduct levels above median value was observed for XPD-751Gln versus XPD-751Lys never-smoking homozygotes after adjustment for several confounders. These data show that all the analysed polymorphisms could result in deficient DNA repair and suggest a need for further investigation into the possible interactions between these polymorphisms, smoking and other risk factors.

Chimeric mouse-human IgG was used to study the structural and functional roles of the carbohydrate present in the CH2 domain of human IgG molecules. To remove this N-linked carbohydrate, Asn-297, the oligosaccharide attachment residue, was changed to either Gln (a conservative replacement) or His for IgG1 or Lys for IgG3 (nonconservative replacements) by site-directed mutagenesis. Carbohydrate-deficient antibodies are properly assembled and secreted and bind Ag and protein A. However, aglycosylated IgG are more sensitive to most proteases than their corresponding wild-type IgG, indicating some conformational changes have occurred. Aglycosylated IgG do not bind to the human Fc gamma RI and do not activate C; depending on the isotype, C1q binding ability is either completely lost (IgG1) or dramatically decreased (IgG3). The serum half-life in mice of aglycosylated IgG1-Gln remains the same as wild-type IgG1, 6.5 +/- 0.5 days, whereas aglycosylated IgG3-Gln has a shorter half-life, 3.5 +/- 0.2 days, compared to that of wild-type IgG3, 5.1 +/- 0.4 days. These results indicate the carbohydrate interposed between CH2 domain of human IgG is necessary to maintain the appropriate structure for the maintenance of many of the effector functions dependent on the CH2 domain.

AMPA-receptor (AMPAR) transport to synapses plays a critical role in the modulation of synaptic strength. We show that the functionally critical GluR2 subunit stably resides in an intracellular pool in the endoplasmic reticulum (ER). GluR2 in this pool is extensively complexed with GluR3 but not with GluR1, which is mainly confined to the cell surface. Mutagenesis revealed that elements in the C terminus including the PDZ motif are required for GluR2 forward-transport from the ER. Surprisingly, ER retention of GluR2 is controlled by Arg607 at the Q/R-editing site. Reversion to Gln (R607Q) resulted in rapid release from the pool and elevated surface expression of GluR2 in neurons. Therefore, Arg607 is a central regulator. In addition to channel gating, it also controls ER exit and may thereby ensure the availability of GluR2 for assembly into AMPARs.

In expanded CAG repeat diseases such as Huntington's disease, proteins containing polyglutamine (poly(Gln)) sequences with repeat lengths of about 37 residues or more are associated with development of both disease symptoms and neuronal intranuclear inclusions (NIIs). Disease physiology in animal and cellular models does not always correlate with NII formation, however, and the mechanism by which aggregate formation might lead to cytotoxicity is unknown. To help evaluate various possible mechanisms, we determined the biophysical properties of a series of simple poly(Gln) peptides. The circular dichroism spectra of poly(Gln) peptides with repeat lengths of five, 15, 28 and 44 residues are all nearly identical and are consistent with a high degree of random coil structure, suggesting that the length-dependence of disease is not related to a conformational change in the monomeric states of expanded poly(Gln) sequences. In contrast, there is a dramatic increase in both the kinetics and the thermodynamic favorability of the spontaneous formation of ordered, amyloid-like aggregates for poly(Gln) peptides with repeat lengths of greater than 37 residues. At the same time, poly(Gln) peptides with repeat lengths in the 15-20 residue range, despite their poor abilities to support spontaneous, self-nucleated aggregation, are capable of efficiently adding to an already-formed aggregate. We also find that morphologically small, finely divided aggregates are much more efficient at recruiting poly(Gln) peptides than are large aggregates, suggesting a possible explanation for why disease pathology does not always correlate with the observable NII burden. Together, these data are consistent with a model for disease pathology in which critical cellular proteins possessing poly(Gln) sequences of modest length become inactivated when they are recruited into aggregates of an expanded poly(Gln) protein.

The crystal structure of DsRed, a red fluorescent protein from a corallimorpharian, has been determined at 2.0-A resolution by multiple-wavelength anomalous dispersion and crystallographic refinement. Crystals of the selenomethionine-substituted protein have space group P2(1) and contain a tetramer with 222 noncrystallographic symmetry in the asymmetric unit. The refined model has satisfactory stereochemistry and a final crystallographic R factor of 0.162. The protein, which forms an obligatory tetramer in solution and in the crystal, is a squat rectangular prism comprising four protomers whose fold is extremely similar to that of the Aequorea victoria green fluorescent protein despite low ( approximately 23%) amino acid sequence homology. The monomer consists of an 11-stranded beta barrel with a coaxial helix. The chromophores, formed from the primary sequence -Gln-Tyr-Gly- (residues 66-68), are arranged in a approximately 27 x 34-A rectangular array in two approximately antiparallel pairs. The geometry at the alpha carbon of Gln-66 (refined without stereochemical restraints) is consistent with an sp(2) hybridized center, in accord with the proposal that red fluorescence is because of an additional oxidation step that forms an acylimine extension to the chromophore [Gross, L. A., Baird, G. S., Hoffman, R. C., Baldridge, K. K. & Tsien, R. Y. (2000) Proc. Natl. Acad. Sci. USA 87, 11990-11995]. The carbonyl oxygen of Phe-65 is almost 90 degrees out of the plane of the chromophore, consistent with theoretical calculations suggesting that this is the minimum energy conformation of this moiety despite the conjugation of this group with the rest of the chromophore.

Usual human livers contain two major aldehyde dehydrogenase [(ALDH) aldehyde:NAD+ oxidoreductase] isozymes--i.e., a cytosolic ALDH1 component and a mitochondrial ALDH2 component--whereas approximately equal to 50% of Orientals are "atypical" and have only the ALDH1 isozyme and are missing the ALDH2 isozyme. We previously demonstrated that atypical livers contain an enzymatically inactive but immunologically crossreactive material (CRM) corresponding to the ALDH2 component. The enzymatically active ALDH2 obtained from a usual liver and the CRM obtained from an atypical liver were reduced, S-carboxymethylated, and digested by trypsin. Separation of their digests by high-performance reverse-phase chromatography and by two-dimensional paper chromatography and electrophoresis revealed that ALDH2 contained a peptide sequence of -Glu-Leu-Gly-Glu-Ala-Gly-Leu-Gln-Ala-Asn-Val-Gln-Val-Lys- and that the glutamine adjacent to lysine was substituted by lysine in CRM. All other tryptic peptides, including eight peptides containing S-carboxymethylcysteine, were common in ALDH2 and CRM. It is concluded that a point mutation in the human ALDH2 locus produced the glutamine leads to lysine substitution and enzyme inactivation.

The three genes, gatC, gatA, and gatB, which constitute the transcriptional unit of the Bacillus subtilis glutamyl-tRNAGln amidotransferase have been cloned. Expression of this transcriptional unit results in the production of a heterotrimeric protein that has been purified to homogeneity. The enzyme furnishes a means for formation of correctly charged Gln-tRNAGln through the transamidation of misacylated Glu-tRNAGln, functionally replacing the lack of glutaminyl-tRNA synthetase activity in Gram-positive eubacteria, cyanobacteria, Archaea, and organelles. Disruption of this operon is lethal. This demonstrates that transamidation is the only pathway to Gln-tRNAGln in B. subtilis and that glutamyl-tRNAGln amidotransferase is a novel and essential component of the translational apparatus.

With genome sequencing nearing completion for the model organisms used in biomedical research, there is a rapidly growing appreciation that proteomics, the study of covalent modification to proteins, and transcriptional regulation will likely dominate the research headlines in the next decade. Protein methylation plays a central role in both of these fields, as several different residues (Arg, Lys, Gln) are methylated in cells and methylation plays a central role in the "histone code" that regulates chromatin structure and impacts transcription. In some cases, a single lysine can be mono-, di-, or trimethylated, with different functional consequences for each of the three forms. This review describes structural aspects of methylation of histone lysine residues by two enzyme families with entirely different structural scaffolding (the SET proteins and Dot1p) and methylation of protein arginine residues by PRMTs.

Formation of a specific contact between two residues of a polypeptide chain is an important elementary process in protein folding. Here we describe a method for studying contact formation between tryptophan and cysteine based on measurements of the lifetime of the tryptophan triplet state. With tryptophan at one end of a flexible peptide and cysteine at the other, the triplet decay rate is identical to the rate of quenching by cysteine. We show that this rate is also close to the diffusion-limited rate of contact formation. The length dependence of this end-to-end contact rate was studied in a series of Cys-(Ala-Gly-Gln)(k)-Trp peptides, with k varying from 1 to 6. The rate decreases from approximately 1/(40 ns) for k = 1 to approximately 1/(140 ns) for k = 6, approaching the length dependence expected for a random coil (n(-3/2)) for the longest peptides.

The proposal that the absorption maximum of the visual pigments is governed by interaction of the 11-cis-retinal chromophore with charged carboxylic acid side chains in the membrane-embedded regions of the proteins has been tested by mutating five Asp and Glu residues thought to be buried in rhodopsin. Changing Glu113 to Gln causes a dramatic shift in the absorption maximum from 500 nanometers to 380 nanometers, a decrease in the pKa (acidity constant) of the protonated Schiff base of the chromophore to about 6, and a greatly increased reactivity with hydroxylamine. Thus Glu113 appears to be the counterion to the protonated Schiff base. Wavelength modulation in visual pigments apparently is not governed by electrostatic interaction with carboxylate residues, other than the counterion.

Calcium influx through the N-methyl-d-aspartate (NMDA)-type glutamate receptor and activation of calcium/calmodulin-dependent kinase II (CaMKII) are critical events in certain forms of synaptic plasticity. We have previously shown that autophosphorylation of CaMKII induces high-affinity binding to the NR2B subunit of the NMDA receptor (Strack, S., and Colbran, R. J. (1998) J. Biol. Chem. 273, 20689-20692). Here, we show that residues 1290-1309 in the cytosolic tail of NR2B are critical for CaMKII binding and identify by site-directed mutagenesis several key residues (Lys(1292), Leu(1298), Arg(1299), Arg(1300), Gln(1301), and Ser(1303)). Phosphorylation of NR2B at Ser(1303) by CaMKII inhibits binding and promotes slow dissociation of preformed CaMKII.NR2B complexes. Peptide competition studies imply a role for the CaMKII catalytic domain, but not the substrate-binding pocket, in the association with NR2B. However, analysis of monomeric CaMKII mutants indicates that the holoenzyme structure may also be important for stable association with NR2B. Residues 1260-1316 of NR2B are sufficient to direct the subcellular localization of CaMKII in intact cells and to confer dynamic regulation by calcium influx. Furthermore, mutation of residues in the CaMKII-binding domain in full-length NR2B bidirectionally modulates colocalization with CaMKII after NMDA receptor activation, suggesting a dynamic model for the translocation of CaMKII to postsynaptic targets.