Identification of the primer binding domain in human immunodeficiency virus reverse transcriptase.

We have labeled the primer binding domain of HIV1-RT with 5'-32P-labeled (dT)15 primer using ultraviolet light energy. The specificity of the primer cross-linking to HIV1-RT was demonstrated by competition experiments. Both synthetic and natural primers, e.g., p(dA)15, p(dC)15, and tRNA(Lys), inhibit p(dT)15 binding and cross-linking to the enzyme. The observed binding and cross-linking of the primer to the enzyme were further shown to be functionally significant by the observation that tRNA(Lys) inhibits the polymerase activity on poly(rA).(dT)15 template-primer as well as the cross-linking of p(dT)15 to the enzyme to a similar extent. At an enzyme to p(dT)15 ratio of 1:3, about 15% of the enzyme can be cross-linked to the primer. To identify the domain cross-linked to (dT)15, tryptic peptides were generated and purified by a combination of HPLC on a C-18 reverse-phase column and DEAE-Sephadex chromatography. A single peptide cross-linked to p(dT)15 was identified. This peptide corresponded to amino acid residues 288-307 in the primary sequence of HIV1-RT as judged by amino acid composition and sequence analyses. Further, Leu(289)-Thr(290) and Leu(295)-Thr(296) of HIV1-RT appear to be the probable sites of cross-linking to the primer p(dT)15.     

Phenylalanines that are conserved among several RNA-binding proteins form part of a nucleic acid-binding pocket in the A1 heterogeneous nuclear ribonucleoprotein

We have studied the domain structure of the A1 heterogeneous nuclear ribonucleoprotein using both partial proteolysis and photochemical cross-linking to oligodeoxynucleotides. Both the intact A1 protein and its proteolytic fragment, the UP1 protein, can be cleaved by Staphylococcus aureus V-8 protease to produce two polypeptides of 92 amino acids. These two polypeptides correspond to the internal repeat sequence previously noted by us to occur in UP1. The two polypeptides can be purified via single-stranded DNA cellulose chromatography and independently cross-linked to [32P]p(dT)8, indicating that each domain can bind to single-stranded nucleic acids. Purification and sequencing of A1 tryptic peptides that had been cross-linked to oligothymidylic acid revealed that 4 phenylalanine residues, phenylalanines 16, 58, 107, and 149 are the sites of covalent adduct formation, with phenylalanine 16 being the major site of cross-linking. These phenylalanine residues are internally homologous when the repeat sequences in A1 are aligned, that is, phenylalanines 16 and 107 occupy analogous positions in the 91-residue repeat, as do phenylalanines 58 and 149. An examination of the primary structures of a variety of eucaryotic RNA-binding proteins with sequence homology to A1 reveals that the cross-linked phenylalanines in A1 are highly conserved among all of these proteins. Our results provide the first experimental evidence that conserved residues in the 90-amino acid repeating domains shared by A1 and other single-stranded nucleic acid binding-proteins form part of an RNA-binding pocket.     

Collagen cross-linking. Isolation of cross-linked peptides from collagen of chicken bone

Cross-linked peptides were isolated from chicken bone collagen that had been digested with CNBr or with bacterial collagenase. Analyses of (3)H radioactivity in disc electrophoretic profiles of the CNBr peptides from bone collagens that had been treated with NaB(3)H indicated that a major site of intermolecular cross-linking in chicken bone collagen is located between the carboxy-terminal region of an alpha1 chain and a small CNBr peptide, probably situated near the amino-terminus of an alpha1 or alpha2 chain in an adjacent collagen molecule. A small amount of this cross-linked CNBr peptide was isolated from a CNBr digest of chicken bone collagen by column chromatography. Amino acid analysis showed that the CNBr peptide, alpha1CB6B, the carboxy-terminal peptide of the alpha1 chain, was the major CNBr peptide in the preparation, and the reduced cross-linking components were identified as hydroxylysinohydroxynorleucine (HylOHNle), with a smaller amount of hydroxylysinonorleucine (HylNle). However, the composition and the low recovery of the cross-linking amino acids suggested that the preparation was a mixture of CNBr peptides alpha1CB6B and alpha1CB6B cross-linked to a small CNBr peptide whose identity could not be determined. A small cross-linked peptide was isolated from chicken bone collagen that had been reduced with NaB(3)H(4) and digested with bacterial collagenase. This peptide was the major cross-linked peptide in the digest and contained a stoicheiometric amount of the reduced cross-linking compounds. A peptide which had the same amino acid composition, but contained the cross-linking compounds in their reducible forms, was isolated from a collagenase digest of chicken bone collagen that had not been treated with NaBH(4). The absence of the reduced cross-links from this peptide indicates that, at least for the cross-linking site from which the peptide derives, natural reduction is not a significant pathway for biosynthesis of stable cross-links. However, most of the reducible cross-linking component in the peptide appeared to stabilize in the bone collagen by rearrangement from aldimine to ketoamine form.     

Direct photoaffinity labeling of cysteine 211 or a nearby amino acid residue of beta-tubulin by guanosine 5'-diphosphate bound in the exchangeable site

Tubulin with [8-14C]GDP bound in the exchangeable site was exposed to ultraviolet light, and radiolabel was cross-linked to two peptide regions of the beta-subunit. Following enrichment for peptides cross-linked to guanosine by boronate chromatography, we confirmed that the cysteine 12 residue was the major site of cross-linking. However, significant radiolabel was also incorporated into a peptide containing amino acid residues 206 through 224. Although every amino acid in this peptide except cysteine 211 was identified by sequential Edman degradation, implying that this was the amino acid residue cross-linked to guanosine, radiolabel at C-8 was usually lost during peptide processing (probably during chromatography at pH 10). Consequently, the radiolabeled amino acid could not be unambiguously identified.     

Photochemical cross-linking of the Escherichia coli single-stranded DNA-binding protein to oligodeoxynucleotides. Identification of phenylalanine 60 as the site of cross-linking

The single-stranded DNA-binding proteins from bacteriophage T4, F plasmid, Escherichia coli, and calf thymus can all be covalently cross-linked in vitro to thymine oligonucleotides by irradiating the respective protein-oligonucleotide complexes with ultraviolet light. More extensive studies on the E. coli single-stranded DNA-binding protein (SSB) indicate that this reaction is dependent upon both the length of the oligonucleotide and the dose of ultraviolet irradiation. Using anion-exchange and reverse-phase ion-pairing high-performance liquid chromatography we have isolated a specific cross-linked tryptic peptide comprising residues 57-62 of the SSB protein with the sequence valine-valine-leucine-phenylalanine-glycine-lysine. Solid-phase sequence analysis of the covalent [32P] p(dT)8-peptide complex indicates that phenylalanine 60 is the site of cross-linking. This amino acid is located within the general region of SSB (residues 1-115) that has previously been shown to contain the DNA-binding site (Williams, K. R., Spicer, E. K., LoPresti, M. B., Guggenheimer, R. A., and Chase, J. W. (1983) J. Biol. Chem. 258, 3346-3355). The high-performance liquid chromatography purification procedure we have devised to isolate cross-linked peptide-oligonucleotide complexes should be of general applicability and should facilitate future structure/function studies on other nucleic acid-binding proteins.     

Characterization of bacteriophage T4 regA protein-nucleic acid interactions

The bacteriophage T4 regA protein is a translational repressor of a group of T4 early mRNAs. We have characterized the binding of regA protein to polynucleotides and to specific RNAs. Binding to nucleic acids was monitored by the quenching of the intrinsic tryptophan fluorescence of regA protein. regA protein exhibited differential affinities for the polynucleotides examined, with the order of affinity being poly(rU) greater than poly(dT) greater than poly(dU) = poly(rG) greater than poly(rC) = poly(rA). The binding site size calculated for regA protein binding to poly(rU) was n = 9 +/- 1 nucleotides. Cooperativity was observed in binding to multiple-site oligonucleotides, with a cooperativity parameter (omega) value of 10-22. To study the specific interaction between regA protein and T4 gene 44 mRNA, the affinity of regA protein for synthetic gene 44 RNA fragments was measured. The association constant (Ka) for regA protein binding to gene 44 RNA fragments was 100-fold higher than for binding to nontarget RNA. Study of variant gene 44 RNA fragments indicated that the nucleotides required for specific binding are contained within a 12-nucleotide sequence spanning -12 to -1, relative to the AUG codon. The bases of five nucleotides (indicated in upper case type) are critical for specific regA protein interaction with the gene 44 recognition element, 5'-aaUGAGgAaauu-3'. These studies further showed that formation of a regA protein-RNA complex involves a maximum of 2-3 ionic interactions and is primarily an enthalpy-driven process.     

Shuffling of amino acid sequence: an important control in synthetic peptide studies of nucleic acid-binding domains. Binding properties of fragments of a conserved eukaryotic RNA binding motif.

We have used synthetic peptides to study a conserved RNA binding motif in yeast poly(A)-binding protein. Two peptides, 45 and 44 amino acids in length, corresponding to amino and carboxyl halves of a 90-amino acid RNA-binding domain in the protein were synthesized. While the amino-terminal peptide had no significant affinity for nucleic acids, the carboxyl-terminal peptide-bound nucleic acids with similar characteristics to that for the entire 577 residue yeast poly(A)-binding protein. In 100 mM NaCl, the latter peptide retained over 50% of the intrinsic binding free energy of the protein, as well as, similar RNA versus DNA binding specificity. However, shuffling of the sequence of this 44 residue peptide had surprisingly little effect on its nucleic acid binding properties suggesting the overriding importance of amino acid composition as opposed to primary sequence. Deletion studies on the 44 residue peptide with the "correct" sequence succeeded in identifying amino acids important for conferring RNA specificity and for increasing our understanding of the molecular basis for nucleic acid binding by synthetic peptides. The shuffled peptide study, however, clearly indicates that considerable caution must be exercised before extrapolating results of structure/function studies on synthetic peptide analogues to the parent protein.     

Characterization of the nucleic acid binding region of adenovirus DNA binding protein by partial proteolysis and photochemical cross-linking.

The nucleic acid binding domain of the adenovirus type 2 (or type 5) DNA-binding protein (DBP) was characterized by using limited proteolysis and photochemical cross-linking. Three proteases were used to generate fragments of DBP which retained the ability to bind to single-stranded DNA. One fragment, a 35-kDa tryptic product, was partially sequenced and found to contain amino acid residues 153 to approximately 470. This fragment further defines the minimum region of the protein which is required for nucleic acid binding. The DNA binding pocket of DBP was defined by using ultraviolet irradiation to cross-link covalently the carboxyl-terminal portion of the protein to the oligonucleotide p(dT)14. Cross-linked complexes were digested with trypsin, and peptides which were associated with the oligonucleotide were isolated by anion-exchange and reverse-phase ion-pairing high performance liquid chromatography. Two DBP peptides comprised of residues 294-308 and 415-434 were isolated by this approach. Sequence analysis indicated that methionine 299 and phenylalanine 418 were probable sites of cross-linking between their respective peptides and the oligonucleotide; hence these residues may represent contact points between DBP and single-stranded DNA. Both residues are highly conserved and are near, but not identical to, regions of the protein implicated previously in DNA binding.     

Changes in the cross-linking of collagen from rat tail tendons due to diabetes

The acid solubility of Type I collagen from rat tail tendons decreases due to diabetes. This finding has been taken as evidence that collagen from diabetics may be more cross-linked than normal. We compared CNBr peptide maps prepared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for [3H] NaBH4-reduced tail tendons from streptozotocin-diabetic rats with maps from age-matched control rats. At least through 30 weeks of diabetes, the distribution of mass of both cross-linked and uncross-linked CNBr peptides was identical in diabetic and control tendons. Therefore, the number of cross-linked peptides did not increase due to diabetes. We analyzed the 3H-cross-linking compounds present on the CNBr peptides and found that the 3H content of peptides cross-linked in control tendons through the bivalent, reduced cross-links hydroxylysinonorleucine and lysinonorleucine was diminished on corresponding peptides from diabetic tendons as a function of duration of diabetes. The cross-linked peptides, however, persisted. Therefore, we conclude that a larger fraction of these bivalent cross-links is found in an unknown, non-reducible form in tendons from diabetic compared with control rats. This resembles a phenomenon normally associated with maturation and/or aging where the non-reducible form of the cross-links is acid-stable. An increase in the fraction of the cross-links that is non-reducible and acid-stable would explain, at least in part, the decrease in acid solubility of the collagen. Non-enzymatic glycation (NEG) was not very specific, since most CNBr peptides bound some glucose. However, peptides from the alpha 2-chain seemed to be preferential targets for NEG. While NEG clearly increased due to diabetes, we found no evidence that increased NEG led to an increased number of cross-links in tail tendon collagen from streptozotocin diabetic rats.     

Bovine Peripheral Nervous System Myelin P2 Protein: Chemical and Immunological Characterization of the Cyanogen Bromide Peptides

Cleavage of bovine P2 protein by cyanogen bromide (CNBr) produced peptide fractions CN1, CN2, and CN3 which were isolated by gel filtration chromatography. CN2 was found to contain two NH2-terminals (lysine and valine) and accounted for both of the cysteine residues of P2. When reduced carboxymethylated P2 (RCM-P2) was digested with CNBr, peptides CN1 and CN3 were obtained as were (1) a peptide with NH2-terminal lysine (Lys) that contained no homoserine and only one cysteine residue and (2) a peptide with NH2-terminal valine (Val) that was co-eluted with CN3. These data and the chemical characterization of all the CNBr peptides obtained from P2 and RCM-P2 suggest that isolated P2 protein has a structure composed of the CNBr peptides in the order CN3-CN1-CN2(Val)-CN2(Lys) with an intrachain disulfide bond between the cysteine residues located in the two constituent peptides of CN2, CN2(Lys) and CN2(Val). To locate the neuritogenic region(s) within the P2 protein structure, CN1, CN2, and CN3 were tested for the ability to induced experimental allergic neuritis (EAN) in Lewis rats. The disease-inducing sites of P2 protein were found only in CN1; neither CN2 nor CN3 produced disease. EAN induced by CN1 was comparable to that induced with P2 protein as determined by disease onset, clinical symptoms, and histologic lesions.     

Biochemistry of terminal deoxynucleotidyltransferase. Affinity labeling and identification of the deoxynucleoside triphosphate binding domain of terminal deoxynucleotidyltransferase

Using the technique of UV-mediated cross-linking of nucleotides to their acceptor sites (Modak, M. J., and Gillerman-Cox, E. (1982) J. Biol. Chem. 257, 15105-15109), we have labeled calf terminal deoxynucleotidyltransferase (TdT) with [32P]dTTP. The specificity of dTTP cross-linking at the substrate binding site in TdT is demonstrated by the competitive inhibition of the cross-linking reaction by other deoxynucleoside triphosphates, and ATP and its analogues, requiring concentrations consistent with their kinetic constants. Tryptic peptide mapping of the [32P]dTTP-labeled enzyme showed the presence of a single radioactive peptide fraction that contained the site of dTTP cross-linking. The amino acid composition and sequence analysis of the radioactive peptide fraction revealed it to contain two tryptic peptides, spanning residues 221-231 and 234-249. Since these two peptides were covalently linked to dTTP, the region encompassed by them constitutes a substrate binding domain in TdT. Further proteolytic digestion of the tryptic peptide-dTTP complex, using V8 protease, yielded a smaller peptide, and its analysis narrowed the substrate binding domain to 14 amino acids corresponding to residues 224-237 in the primary amino acid sequence of TdT. Furthermore, 2 cysteine residues, Cys-227 and Cys-234, within this domain were found to be involved in the cross-linking of dTTP, suggesting their participation in the process of substrate binding in TdT.     

Laser cross-linking of nucleic acids to proteins. Methodology and first applications to the phage T4 DNA replication system

Single-pulse (approximately 8 ns) ultraviolet laser excitation of protein-nucleic acid complexes can result in efficient and rapid covalent cross-linking of proteins to nucleic acids. The reaction produces no nucleic acid-nucleic acid or protein-protein cross-links, and no nucleic acid degradation. The efficiency of cross-linking is dependent on the wavelength of the exciting radiation, on the nucleotide composition of the nucleic acid, and on the total photon flux. The yield of cross-links/laser pulse is largest between 245 and 280 nm; cross-links are obtained with far UV photons (200-240 nm) as well, but in this range appreciable protein degradation is also observed. The method has been calibrated using the phage T4-coded gene 32 (single-stranded DNA-binding) protein interaction with oligonucleotides, for which binding constants have been measured previously by standard physical chemical methods (Kowalczykowski, S. C., Lonberg, N., Newport, J. W., and von Hippel, P. H. (1981) J. Mol. Biol. 145, 75-104). Photoactivation occurs primarily through the nucleotide residues of DNA and RNA at excitation wavelengths greater than 245 nm, with reaction through thymidine being greatly favored. The nucleotide residues may be ranked in order of decreasing photoreactivity as: dT much greater than dC greater than rU greater than rC, dA, dG. Cross-linking appears to be a single-photon process and occurs through single nucleotide (dT) residues; pyrimidine dimer formation is not involved. Preliminary studies of the individual proteins of the five-protein T4 DNA replication complex show that gene 43 protein (polymerase), gene 32 protein, and gene 44 and 45 (polymerase accessory) proteins all make contact with DNA, and can be cross-linked to it, whereas gene 62 (polymerase accessory) protein cannot. A survey of other nucleic acid-binding proteins has shown that E. coli RNA polymerase, DNA polymerase I, and rho protein can all be cross-linked to various nucleic acids by the laser technique. The potential uses of this procedure in probing protein-nucleic acid interactions are discussed.     

Cross-linking of rabbit skeletal muscle troponin subunits: labeling of cysteine-98 of troponin C with 4-maleimidobenzophenone and analysis of products formed in the binary complex with troponin T and the ternary complex with troponins I and T

The sulfhydryl-specific, heterobifunctional, photoactivatable cross-linker 4-maleimidobenzophenone (BPMal) was used to study the interaction of rabbit skeletal muscle troponin subunits TnC, TnT, and TnI. TnC was labeled at Cys-98 by the maleimide moiety of BPMal and then mixed with either TnT alone or TnI plus TnT, in the presence of Ca2+. Upon photolysis, TnI and/or TnT formed covalent cross-links with TnC. The cross-linked TnC-TnT heterodimer obtained from the binary complex was digested into progressively smaller cross-linked peptides that were purified by HPLC and then characterized by amino acid analysis and sequencing. An initial cross-linked CNBr fraction contained the expected peptide CB9 (residues 84-135) of TnC, plus CNBr peptides spanning residues 152-230 of TnT. Results from a peptic digest of the CNBr cross-linked fraction permitted the identification of residues 159-197 as the most highly cross-linked region in TnT. A final subtilisin digest yielded a heterogeneous cross-linked fraction, which suggested that an especially high degree of cross-links was formed in the vicinity of residues 175-178 (Met-Lys-Lys-Lys) of TnT. Although this region of TnT had previously been implicated in binding, we show here for the first time that it is close to Cys-98 of TnC. In an analogous study on the binary complex of TnC and TnI [Leszyk, J., Collins, J. H., Leavis, P. C., & Tao, T. (1987) Biochemistry 26, 7042-7047], we previously showed that Cys-98 of TnC was cross-linked mainly to CN4, the "inhibitory region", of TnI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photoaffinity labeling of the thymidine triphosphate binding domain in Escherichia coli DNA polymerase I: identification of histidine-881 as the site of cross-linking

Using the technique of ultraviolet-mediated cross-linking of substrate deoxynucleoside triphosphates (dNTPs) to their acceptor site [Abraham, K. I., & Modak, M. J. (1984) Biochemistry 23, 1176-1182], we have labeled the Klenow fragment of Escherichia coli DNA polymerase I (Pol I) with [alpha-32P]dTTP. Covalent cross-linking of [alpha-32P]dTTP to the Klenow fragment is shown to be at the substrate binding site by the following criteria: (a) the cross-linking reaction requires dTTP in its metal chelate form; (b) dTTP is readily competed out by other dNTPs as well as by substrate binding site directed reagents; (c) labeling with dTTP occurs at a single site as judged by peptide mapping. Under optimal conditions, a modification of approximately 20% of the enzyme was achieved. Following tryptic digestion of the [alpha-32P]dTTP-labeled Klenow fragment, reverse-phase high-performance liquid chromatography demonstrated that 80% of the radioactivity was contained within a single peptide. The amino acid composition and sequence of this peptide identified it as the peptide spanning amino acid residues 876-890 in the primary sequence of E. coli Pol I. Chymotrypsin and Staphylococcus aureus V8 protease digestion of the labeled tryptic peptide in each case yielded a single smaller fragment that was radioactive. Amino acid analysis and sequencing of these smaller peptides further narrowed the dTTP cross-linking site to within the region spanning residues 876-883. We concluded that histidine-881 is the primary attachment site for dTTP in E. coli DNA Pol I, since during amino acid sequencing analysis of all three radioactive peptides loss of the histidine residue at the expected cycle is observed.     

Cross-linking site in fibrinogen for alpha 2-plasmin inhibitor

A plasma proteinase inhibitor, alpha 2-plasmin inhibitor (alpha 2PI), is cross-linked with alpha chain of fibrin(ogen) by activated coagulation Factor XIII (plasma transglutaminase). alpha 2PI serves only as a glutamine substrate (amine acceptor) for activated Factor XIII in the cross-linking reaction, and the cross-linking occurs between Gln-2 of the alpha 2PI molecule and a lysine residue (amine donor) of fibrin(ogen) alpha chain, whose position was investigated. alpha 2PI and fibrinogen were reacted by activated Factor XIII. The resulting alpha 2PI fibrinogen A alpha chain complex was separated and subjected to two cycles of Edman degradation using phenyl isothiocyanate for the first cycle and dimethylaminoazobenzene-isothiocyanate for the second cycle. The aqueous phase after the cleavage stage of the second cycle, containing dimethylaminoazobenzene-thiohydantoin-Gln cross-linked with A alpha chain, was subjected to CNBr fragmentation and tryptic digestion. Only one of the peptides was found to have the peak of absorbance at 420 nm, indicating the presence of dimethylaminoazobenzene-thiohydantoin-Gln in that peptide. The peptide was identified as corresponding to residues Asn-290-Arg-348 of A alpha chain by analyses of the NH2-terminal amino acid sequence and amino acid composition. The peptide contains a single lysine at position 303, indicating that Lys-303 of fibrinogen A alpha chain is the lysine residue that forms a cross-link with Gln-2 of alpha 2PI.     

Lys-65 and Glu-168 are the residues for carbodiimide-catalyzed cross-linking between the two heads of rigor smooth muscle heavy meromyosin

We have previously demonstrated that the two heads of chicken gizzard heavy meromyosin (HMM) in a rigor complex with rabbit skeletal F-actin could be cross-linked by the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Here, we report the location of the cross-linked sites in the amino acid sequence of the HMM heavy chain. One of the cross-linked residues was identified as Glu-168 by sequencing the CN1.CN6 cross-linked peptide containing residues 1-77 (CN1) and 164-203 (CN6). This site is located close to the ATP-binding site of HMM. Since the other site was further into the amino acid sequence of CN1, another cross-linked peptide corresponding to residues 53-66 and 145-182 was isolated from the 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide-treated acto-tryptic gizzard HMM digested further by other proteolytic enzymes. The amino acid sequence of this peptide and its cyanogen bromide fragment indicated that the cross-linking occurred between Glu-168 and Lys-65. Our results suggests that these two amino acid side chains are in contact with each other in the acto-gizzard HMM rigor complex and participate in the electrostatic interaction between the two HMM heads bound to F-actin. Based on the head-to-head contact, we propose a three-dimensional model for the attachment of gizzard HMM heads to F-actin.     

Cross-linking of rabbit skeletal muscle troponin with the photoactive reagent 4-maleimidobenzophenone: identification of residues in troponin I that are close to cysteine-98 of troponin C

We have used the sulfhydryl-specific, heterobifunctional, photoactivatable cross-linker 4-maleimidobenzophenone (BPMal) to study the interaction of rabbit skeletal muscle troponin C (TnC) and troponin I (TnI). TnC was specifically labeled at Cys-98 by the maleimide moiety of BPMal, and a binary complex was formed with TnI in the presence of Ca2+. Upon photolysis, covalent cross-links were formed between TnC and TnI [Tao, T., Scheiner, C.J., & Lamkin, M. (1986) Biochemistry 25, 7633-7639]. The cross-linked heterodimer was digested with cyanogen bromide, pepsin, and chymotrypsin into progressively smaller cross-linked peptides, which were purified by HPLC and then characterized by amino acid analysis and sequencing. We obtained a fraction from the initial CNBr digest that contained the expected peptide CB9 (residues 84-135) of TnC, cross-linked mainly to CN4 (residues 96-116), the "inhibitory region" of TnI. The peptides CN1 and CN3 of TnI were also detected in this fraction, but their molar ratios (compared to CB9) were only about 0.15 each, compared to 0.60 for CN4. Sequence analyses of fractions obtained after peptic and chymotryptic digests of the cross-linked CNBr fraction confirmed that CB9 and CN4 were the major cross-linked species. Quantitative analysis of sequencer results indicated that the residues in TnI that appeared to be most highly cross-linked to Cys-98 of TnC were Arg-108 and Pro-110, and to a lesser extent Arg-103 and Lys-107. These findings are consistent with previous studies on interactions between TnI and TnC and provide, for the first time, direct information on the identities of proximate amino acids in the two proteins.     

Biochemistry of terminal deoxynucleotidyltransferase. Identification and unity of ribo- and deoxyribonucleoside triphosphate binding site in terminal deoxynucleotidyltransferase

Terminal deoxynucleotidyltransferase is the only DNA polymerase that is strongly inhibited in the presence of ATP. We have labeled calf terminal deoxynucleotidyltransferase with [32P]ATP in order to identify its binding site in terminal deoxynucleotidyltransferase. The specificity of ATP cross-linking to terminal deoxynucleotidyltransferase is shown by the competitive inhibition of the overall cross-linking reaction by deoxynucleoside triphosphates, as well as the ATP analogs Ap4A and Ap5A. Tryptic peptide mapping of [32P]ATP-labeled enzyme revealed a peptide fraction that contained the majority of cross-linked ATP. The properties, chromatographic characteristics, amino acid composition, and sequence analysis of this peptide fraction were identical with those found associated with dTTP cross-linked terminal deoxynucleotidyl-transferase peptide (Pandey, V. N., and Modak, M. J. (1988a). J. Biol. Chem. 263, 3744-3751). The involvement of the same 2 cysteine residues in the crosslinking of both nucleotides further confirmed the unity of the ATP and dTTP binding domain that contains residues 224-237 in the primary amino acid sequence of calf terminal deoxynucleotidyltransferase.     

Identification of the amino acid residues of the amino terminus of vimentin responsible for DNA binding by enzymatic and chemical sequencing and analysis by MALDI-TOF

The amino acid residues responsible for stable binding of nucleic acids by the intermediate filament (IF) subunit protein vimentin were identified by a combination of enyzmatic and chemical ladder sequencing of photo-cross-linked vimentin-oligodeoxyribonucleotide complexes and analysis by MALDI-TOF mass spectrometry. Three tryptic peptides of vimentin (vim(28)(-)(35), vim(36)(-)(49), and vim(50)(-)(63)) were found to be cross-linked to oligo(dG.BrdU)(12). dG.3'-FITC. From a methodological standpoint, it was necessary to remove the bulk of the bound oligonucleotide by digestion with nuclease P1 to get reproducible spectra for most of the peptides studied. Additionally, removal of the phosphate group of the residually bound dUMP or modification of the amino terminus of the peptide-oligonucleotide complexes with dimethylaminoazobenzene isothiocyanate dramatically improved the quality of the MALDI-TOF spectra obtained, particularly for the vim(28)(-)(35) peptide. A single Tyr residue within each of these peptides (Tyr(29), Tyr(37), and Tyr(52)) was unequivocally demonstrated to be the unique site of cross-linking in each peptide. These three Tyr residues are contained within the two beta-ladder DNA-binding wings proposed for the middle of the vimentin non-alpha-helical head domain. The experimental approach described should be generally applicable to the study of protein-nucleic acid interactions and is currently being employed to characterize the DNA-binding sites of several other IF subunit proteins.