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M A Weiss

Publications and source records attributed to M A Weiss.

At least 109 records · Page 6Linked to original sources
PubMed1991

Heteronuclear 2D NMR studies of an engineered insulin monomer: assignment and characterization of the receptor-binding surface by selective 2H and 13C labeling with application to protein design.

Insulin provides an important model for the application of genetic engineering to rational protein design and has been well characterized in the crystal state. However, self-association of insulin in solution has precluded complementary 2D NMR study under physiological conditions. We demonstrate here that such limitations may be circumvented by the use of a monomeric analogue that contains three amino acid substitutions on the protein surface (HisB10----Asp, ProB28----Lys, and LysB29----Pro); this analogue (designated DKP-insulin) retains native receptor-binding potency. Comparative 1H NMR studies of native human insulin and a series of three related analogues--(i) the singly substituted analogue [HisB10----Asp], (ii) the doubly substituted analogue [ProB28----Lys; LysB29----Pro], and (iii) DKP-insulin--demonstrate progressive reduction in concentration-dependent line-broadening in accord with the results of analytical ultracentrifugation. Extensive nonlocal interactions are observed in the NOESY spectrum of DKP-insulin, indicating that this analogue adopts a compact and stably folded structure as a monomer in overall accord with crystal models. Site-specific 2H and 13C isotopic labels are introduced by semisynthesis as probes for the structure and dynamics of the receptor-binding surface. These studies confirm and extend under physiological conditions the results of a previous 2D NMR analysis of native insulin in 20% acetic acid [Hua, Q. X., & Weiss, M. A. (1991) Biochemistry 30, 5505-5515]. Implications for the role of protein flexibility in receptor recognition are discussed with application to the design of novel insulin analogues.

Amino Acid Sequence↗
PubMed1991

Alternating zinc fingers in the human male associated protein ZFY: refinement of the NMR structure of an even finger by selective deuterium labeling and implications for DNA recognition.

ZFY, a male-associated Zn-finger protein encoded by the human Y chromosome, exhibits a distinctive two-finger repeat: whereas odd-numbered domains fit a general consensus, even-numbered domains exhibit systematic differences. Do these odd and even sequences encode structurally distinct surfaces for DNA recognition? As a first step toward answering this question, we have recently described the sequential 1H NMR assignment of a representative nonconsensus Zn finger (designated ZFY-6T) based on 2D NMR studies of a 30-residue peptide [Kochoyan, M., Havel, T.F., Nguyen, D.T., Dahl, C.E., Keutmann, H. T., & Weiss, M.A. (1991) Biochemistry 30, 3371-3386]. Initial structural modeling by distance geometry/simulated annealing (DG/SA) demonstrated that this peptide retained the N-terminal beta-hairpin and C-terminal alpha-helix (beta beta alpha motif) observed in consensus Zn fingers. However, the precision of this initial structure was limited by resonance overlap, which led to ambiguities in the assignment of key NOEs in the hydrophobic core. In this paper these ambiguities are resolved by selective deuterium labeling, enabling a refined structure to be calculated by DG/SA and restrained molecular dynamics. These calculations provide a detailed view of the hydrophobic core and protein surface, which are analyzed in reference to previously characterized Zn fingers. Variant (even) and consensus (odd) aromatic residues Y10 and F12, shown in an "aromatic swap" analogue to provide equivalent contributions to the hydrophobic core [Weiss, M.A., & Keutmann, H.T. (1990) Biochemistry 29, 9808-9813], nevertheless exhibit striking differences in packing interactions: Y10--but not F12--contributes to a contiguous region of the protein surface defined by putative specificity-determining residues. Alternating surface architectures may have implications for the mechanism of DNA recognition by the ZFY two-finger repeat.

Amino Acid Sequence↗
PubMed1991

DNA recognition by the helix-turn-helix motif: investigation by laser Raman spectroscopy of the phage lambda repressor and its interaction with operator sites OL1 and OR3.

The lambda repressor provides a model system for biophysical studies of DNA recognition by the helix-turn-helix motif. We describe laser Raman studies of the lambda operator sites OL1 and OR3 and their interaction with the DNA-binding domain of lambda repressor (residues 1-102). Raman spectra of the two DNA sites exhibit significant differences attributable to interstrand purine-purine steps that differ in the two oligonucleotides. Remarkably, the conformation of each operator is significantly and specifically altered by repressor binding. Protein recognition, which involves hydrogen-bond formation and hydrophobic contacts in the major groove, induces subtle changes in DNA Raman bands of interacting groups. These include (i) site-specific perturbations to backbone phosphodiester geometry at AT-rich domains, (ii) hydrophobic interaction at thymine 5CH3 groups, (iii) hydrogen bonding to guanine 7N and 6C = O acceptors, and (iv) alterations in sugar pucker within the C2'-endo (B-DNA) family. These perturbations differ between aqueous OL1 and OR3 complexes of repressor, indicating that protein binding in solution determines the precise DNA conformation. The overall structure of the lambda domain is not greatly perturbed by binding to either OL1 or OR3, in accord with X-ray studies of other complexes. However, Raman markers indicate a change in hydrogen bonding of the OH group of tyrosine-22, which is a hydrogen-bond acceptor in the absence of DNA but a combined donor and acceptor in the OL1 complex; yet, Y22 hydrogen bonding is not altered in forming the OR3 complex. The present results demonstrate qualitatively different and distinguishable modes of interaction of the lambda repressor DNA-binding domain with operators OL1 and OR3 in solution. This application of laser Raman spectroscopy to a well-characterized system provides a prototype for future Raman studies of other DNA-binding motifs under physiological conditions.

Bacteriophage lambda↗
PubMed1991

Comparative 2D NMR studies of human insulin and des-pentapeptide insulin: sequential resonance assignment and implications for protein dynamics and receptor recognition.

The solution structure and dynamics of human insulin are investigated by 2D 1H NMR spectroscopy in reference to a previously analyzed analogue, des-pentapeptide(B26-B30) insulin (DPI; Hua, Q.X., & Weiss, M.A. (1990) Biochemistry 29, 10545-10555). This spectroscopic comparison is of interest since (i) the structure of the C-terminal region of the B-chain has not been determined in the monomeric state and (ii) the role of this region in binding to the insulin receptor has been the subject of long-standing speculation. The present NMR studies are conducted in the presence of an organic cosolvent (20% acetic acid), under which conditions both proteins are monomeric and stably folded. Complete sequential assignment of human insulin is obtained and leads to the following conclusions. (1) The secondary structure of the insulin monomer (three alpha-helices and B-chain beta-turn) is similar to that observed in the 2-Zn crystal state. (2) The folding of DPI is essentially the same as the corresponding portion of intact insulin, in accord with the similarities between their respective crystal structures. However, differences between insulin and DPI are observed in the extent of conformational broadening of amide resonances, indicating that the presence or absence of residues B26-B30 influences the overall dynamics of the protein on the millisecond time scale. (3) Residues B24-B28 adopt an extended configuration in the monomer and pack against the hydrophobic core as in crystallographic dimers; residues B29 and B30 are largely disordered. This configuration differs from that described in a more organic milieu (35% acetonitrile; Kline, A.D., & Justice, R.M., Jr. (1990) Biochemistry 29, 2906-2913), suggesting that the conformation of insulin in the latter study may have been influenced by solvent composition. (4) The insulin fold is shown to provide a model for collective motions in a protein with implications for the mechanism of protein-protein recognition. To our knowledge, this paper describes the first detailed analysis of a protein NMR spectrum under conditions of extensive conformational broadening. Such an analysis is made possible in the present case by comparative study of an analogue (DPI) with more tractable spectroscopic properties.

Amino Acid Sequence↗
PubMed1991

Two-dimensional NMR studies of Des-(B26-B30)-insulin: sequence-specific resonance assignments and effects of solvent composition.

Des-pentapeptide-insulin (DPI), a monomeric analogue which lacks the C-terminal five residues of the B-chain, provides a tractable model for 2D-NMR studies of insulin under a variety of solvent conditions. In this paper we present the sequential assignment of DPI at pH 1.8 and 25 degrees C in 10% deuterated DMSO/90% H2O; the chemical shifts are in general similar to those recently described in the absence of an organic cosolvent [1], in 20% acetic acid [2] and (for intact insulin) in 35% acetonitrile [3]. Under each of these solvent conditions qualitative analysis of the 2D-NMR data indicates that the major elements of secondary structure observed in the crystal state (three alpha-helices and B-chain beta-turn) are retained in solution. However, there is disagreement in the literature regarding the stability of the insulin fold, as monitored by amide-proton exchange rates and long-range nuclear Overhauser enhancements [1-3]. In contrast to a previous study [1], we observe slowly exchanging amide resonances (in freshly prepared D2O solutions) and nonlocal NOEs under each of the solvent conditions described, implying the existence of a stably folded secondary structure and hydrophobic core. The slowly-exchanging resonances are assigned to the central alpha-helix of the B-chain, the ends of the adjoining beta-turn, and the two A-chain alpha-helices. Qualitative analysis of long-range NOEs indicates that the major features of the crystal state are retained under these solvent conditions.

Amino Acid Sequence↗
PubMed1991

Design of the helix-turn-helix motif: nonlocal effects of quaternary structure in DNA recognition investigated by laser Raman spectroscopy.

The operator-binding domain of phage lambda repressor provides a model for DNA recognition by the helix-turn-helix (HTH) motif. In the wild-type protein, dimerization is mediated by hydrophobic packing (of the dyad-related helix 5), which serves as an indirect determinant of operator affinity. The mutant repressor, Tyr88----Cys, forms an intersubunit disulfide linkage and exhibits enhancement of both structural stability and operator affinity. Yet the dimer-specific operator affinity of the mutant is 10-fold weaker than that of the wild-type (noncovalent) dimer, suggesting nonlocal effects of the intersubunit disulfide bond on HTH recognition (Sauer et al., 1986). To explore such nonlocal effects, we describe laser Raman studies of the Cys88 mutant repressor and its interaction with operator sites OL1 and OR3. The following results have been obtained: (i) Wild-type and mutant dimers exhibit similar secondary structures, indicated by quantitative comparison of Raman amide I and amide III bands. (ii) The engineered disulfide of the mutant lacks rigorous symmetry; we observe mainly the gauche/gauche/trans CC-S-S-CC rotamer. (iii) Remarkably, distinctive local and nonlocal differences are observed in the mechanisms of DNA recognition by wild-type and mutant repressors. These differences involve specific hydrogen-bonding interactions between the protein and DNA, including guanine N7 sites in the major groove of DNA, and alterations in DNA phosphodiester conformation induced by protein binding. We analyze these differences in relation to crystal structures of the wild-type dimer with and without bound DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA↗
PubMed1991

Alternating zinc fingers in the human male associated protein ZFY: 2D NMR structure of an even finger and implications for "jumping-linker" DNA recognition.

ZFY, a sex-related Zn-finger protein encoded by the human Y chromosome, is distinguished from the general class of Zn-finger proteins by the presence of a two-finger repeat. Whereas odd-numbered domains and linkers fit a general consensus, even-numbered domains and linkers exhibit systematic differences. Because this alternation may have fundamental implications for the mechanism of protein-DNA recognition, we have undertaken biochemical and structural studies of fragments of ZFY. We describe here the solution structure of a representative nonconsensus (even-numbered) Zn finger based on 2D NMR studies of a 30-residue peptide. Structural modeling by distance geometry and simulated annealing (DG/SA) demonstrates that this peptide folds as a miniglobular domain containing a C-terminal beta--hairpin and N-terminal alpha-helix (beta beta alpha motif). These features are similar to (but not identical with) those previously described in consensus-type Zn fingers (derived from ADR1 and Xfin); the similarities suggest that even and odd ZFY domains bind DNA by a common mechanism. A model of the protein-DNA complex (designated the "jumping-linker" model) is presented and discussed in terms of the ZFY two-finger repeat. In this model every other linker is proposed to cross the minor groove by means of a putative finger/linker submotif HX4HX3-hydrophobic residue-X3. Analogous use of a hydrophobic residue in a linker that spans the minor groove has recently been described in crystallographic and 3D NMR studies of homeodomain-DNA complexes. The proposed model of ZFY is supported in part by the hydroxyl radical footprint of the TFIIIA-DNA complex [Churchill, M.E.A., Tullius, T.D., & Klug, A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5528-5532].

Amino Acid Sequence↗
PubMed1991

Disseminated histoplasmosis in a renal transplant patient: a cause of renal failure several years following transplantation.

A 49-year-old man developed disseminated histoplasmosis 6 1/2 years after transplantation. The organism was initially present in the urine and in a tongue lesion. Treatment with itraconazole was instituted. However, there was further dissemination of the disease and worsening of renal function. Allograft biopsy showed extensive involvement with the organism. Amphotericin B was started, resulting in a rapid resolution of the disease. However, renal function deteriorated, leading to permanent hemodialysis.

Biopsy↗
PubMed1991

The efficacy of OKT3 in vascular rejection.

Sixty-six consecutive biopsies of renal allograft recipients treated with OKT3 monoclonal antibody were reviewed and placed into one of two groups. Group I (29 patients) had evidence of acute vascular and cellular rejection, while group II (32 patients) had cellular rejection but no vascular rejection. In 5 cases, the sample was inadequate to determine if vascular rejection was present or not. The severity of the cellular rejection was graded histologically as mild, moderate, or severe. The severity was equivalent when comparing group I with group II (mild, 17% vs. 10%; moderate, 52% vs. 59%; and severe, 31% vs. 31%). There was no difference in the rejection reversal rate between the two groups (86% vs. 91%). However, at 6 and 12 months there was a higher graft loss in the group with vascular rejection (graft survival 64% vs. 81%, P = 0.13, and 58% vs. 75%, P = 0.08, respectively). The poorest outcome was in those patients with both severe acute cellular rejection and acute vascular rejection (4/9, or 44%). The serum creatinine level was higher both pre- and post-OKT3 therapy and at 1, 6, and 12 months in the group with vascular rejection. In conclusion, OKT3 was equally successful in reversing acute cellular rejection and acute vascular rejection. However, increased graft loss occurred at 6 and 12 months in the group with vascular rejection.

Adult↗
PubMed1990

Toward the solution structure of human insulin: sequential 2D 1H NMR assignment of a des-pentapeptide analogue and comparison with crystal structure.

2D 1H NMR studies are presented of des-pentapeptide-insulin, an analogue of human insulin lacking the C-terminal five residues of the B chain. Removal of these residues, which are not required for function, is shown to reduce conformational broadening previously described in the spectrum of intact insulin [Weiss et al. (1989) Biochemistry 28, 9855-9873]. This difference presumably reflects more rapid internal motions in the fragment, which lead to more complete averaging of chemical shifts on the NMR time scale. Sequential 1H NMR assignment and preliminary structural analysis demonstrate retention in solution of the three alpha-helices observed in the crystal state and the relative orientation of the receptor-binding surfaces. These studies provide a foundation for determining the solution structure of insulin.

Amino Acid Sequence↗
PubMed1990

Alternating zinc finger motifs in the male-associated protein ZFY: defining architectural rules by mutagenesis and design of an "aromatic swap" second-site revertant.

We describe spectroscopic and biochemical studies of native and mutant Zn finger peptides from ZFY, a putative transcription factor encoded by the sex-determining region of the human Y chromosome. The parent peptide, based on ZFY domain 6, exhibits metal-dependent helix formation within a rigid tertiary framework. Nonaromatic substitutions of the consensus aromatic group (Tyr 10----Ser or Lys) are surprisingly compatible with native architecture but result in loss of stability to pH or guanidine denaturation. Remarkably, these perturbations are reverted by a second-site mutation in which an alternative aromatic residue is introduced (Ser 12----Phe). Design of the second-site revertant ("aromatic swap") is based on the ZFY two-finger repeat, a conserved symmetry among the ZFY-related zinc finger proteins, and is in accord with recent 2D NMR structures of Zn finger peptides. These experiments suggest general rules for metal-dependent folding of the Zn finger motif.

Amino Acid Sequence↗
PubMed1990

Folding transition in the DNA-binding domain of GCN4 on specific binding to DNA.

Protein-DNA recognition is often mediated by a small domain containing a recognizable structural motif, such as the helix-turn-helix or the zinc-finger. These motifs are compact structures that dock against the DNA double helix. Another DNA recognition motif, found in a highly conserved family of eukaryotic transcription factors including C/EPB, Fos, Jun and CREB, consists of a coiled-coil dimerization element the leucine-zipper and an adjoining basic region which mediates DNA binding. Here we describe circular dichroism and 1H-NMR spectroscopic studies of another family member, the yeast transcriptional activator GCN4. The 58-residue DNA-binding domain of GCN4, GCN4-p, exhibits a concentration-dependent alpha-helical transition, in accord with previous studies of the dimerization properties of an isolated leucine-zipper peptide. The GCN4-p dimer is approximately 70% helical at 25 degrees C, implying that the basic region adjacent to the leucine zipper is largely unstructured in the absence of DNA. Strikingly, addition of DNA containing a GCN4 binding site (AP-1 site) increases the alpha-helix content of GNC4-p to at least 95%. Thus, the basic region acquires substantial alpha-helical structure when it binds to DNA. A similar folding transition is observed on GCN4-p binding to the related ATF/CREB site, which contains an additional central base pair. The accommodation of DNA target sites of different lengths clearly requires some flexibility in the GCN4 binding domain, despite its high alpha-helix content. Our results indicate that the GCN4 basic region is significantly unfolded at 25 degrees C and that its folded, alpha-helical conformation is stabilized by binding to DNA.

Activating Transcription Factors↗
PubMed1990

NMR and photo-CIDNP studies of human proinsulin and prohormone processing intermediates with application to endopeptidase recognition.

The proinsulin-insulin system provides a general model for the proteolytic processing of polypeptide hormones. Two proinsulin-specific endopeptidases have been defined, a type I activity that cleaves the B-chain/C-peptide junction (Arg31-Arg32) and a type II activity that cleaves the C-peptide/A-chain junction (Lys64-Arg65). These endopeptidases are specific for their respective dibasic target sites; not all such dibasic sites are cleaved, however, and studies of mutant proinsulins have demonstrated that additional sequence or structural features are involved in determining substrate specificity. To define structural elements required for endopeptidase recognition, we have undertaken comparative 1H NMR and photochemical dynamic nuclear polarization (photo-CIDNP) studies of human proinsulin, insulin, and split proinsulin analogues as models of prohormone processing intermediates. The overall conformation of proinsulin is observed to be similar to that of insulin, and the connecting peptide is largely unstructured. In the 1H NMR spectrum of proinsulin significant variation is observed in the line widths of insulin-specific amide resonances, reflecting exchange among conformational substates; similar exchange is observed in insulin and is not damped by the connecting peptide. The aromatic 1H NMR resonances of proinsulin are assigned by analogy to the spectrum of insulin, and assignments are verified by chemical modification. Unexpectedly, nonlocal perturbations are observed in the insulin moiety of proinsulin, as monitored by the resonances of internal aromatic groups. Remarkably, these perturbations are reverted by site-specific cleavage of the connecting peptide at the CA junction but not the BC junction. These results suggest that a stable local structure is formed at the CA junction, which influences insulin-specific packing interactions. We propose that this structure (designated the "CA knuckle") provides a recognition element for type II proinsulin endopeptidase.

Amino Acid Sequence↗
PubMed1990

Thermal unfolding studies of a leucine zipper domain and its specific DNA complex: implications for scissor's grip recognition.

A newly recognized class of eukaryotic transcription factors is characterized by a bipartite sequence motif, consisting of a C-terminal dimerization region (the leucine zipper) and an N-terminal basic region (which mediates DNA binding). In studies of isolated leucine zipper peptides, the dimerization region has been characterized as a coiled coil of parallel alpha-helices. To extend these studies to a functional DNA-binding domain, we describe CD studies of the thermal unfolding and refolding of a 58-residue fragment of GCN4, the yeast homologue of the c-Jun protooncoprotein. This fragment, which contains the complete leucine zipper and basic region, retains the DNA-binding properties of the intact protein. The GCN4 DNA-binding domain exhibits two independent helix-coil unfolding transitions. The major transition (midpoint 65 degrees C) is due to dissociation of the dimer in accord with previous studies of an isolated leucine zipper. A novel pretransition in the temperature range 0-40 degrees C is also observed, which reflects partial stabilization of the nascent helix in the basic region. Remarkably, complete folding of the basic region as an alpha-helix requires specific DNA binding, and the protein-DNA complex exhibits a single cooperative unfolding transition. These results support a major feature of the recently proposed "scissor's grip" model of DNA recognition, in which the basic regions extend from the leucine zipper as bifurcating alpha-helical arms.

Amino Acid Sequence↗
PubMed1990

Alternating zinc-finger motifs in the human male-associated protein ZFY.

ZFY, a putative transcription factor encoded by the human Y chromosome, contains a distinctive two-finger repeat: odd-numbered and even-numbered CC/HH metal-binding motifs exhibit systematic alternation in sequence pattern. Such alternation, which is not generally observed in zinc-finger proteins, has also been described in an extensive family of Kruppel-like genes in Xenopus laevis and in the AIDS-associated human DNA-binding protein HIV-EP1. The strict conservation of a two-finger repeat among ZFY-, Kruppel- and HIV-related zinc-finger proteins suggests distinct mechanisms of protein-nucleic acid recognition. To test whether this sequence pattern reflects an underlying alternation in domain structure, we have synthesized and characterized single-finger peptides from the human ZFY gene. Remarkably, systematic differences in metal-dependent folding are observed in the circular dichroism spectra of even- and odd-numbered domains. Our results suggest the existence of distinct CC/HH finger submotifs, which may play different roles in nucleic acid recognition.

Amino Acid Sequence↗
PubMed1990

Resolution of pain associated with varicose and telangiectatic leg veins after compression sclerotherapy.

Many patients presenting for treatment of superficial varicosities and telangiectasias on the legs are seeking relief of pain or discomfort in addition to cosmetic improvement. Of 215 sclerotherapy patients, 114 (53%) complained of various painful symptoms involving areas of telangiectasias (vessels 0.1-1 mm in diameter) and varicosities (vessels greater than 1 mm in diameter). In order to determine whether sclerotherapy is effective in reducing leg pain and discomfort, hundreds of patients receiving two or more treatment sessions of compression sclerotherapy with hypertonic saline were surveyed for the pretreatment and post-treatment presence of numerous symptoms associated with telangiectasias and varicosities of the leg. For all symptoms combined, the overall reported relief of pain was 85% (p less than 0.001). Side effects from the treatment were minimal. These results indicate that sclerotherapy for telangiectasias and varicosities is not only a cosmetic procedure but a safe and valuable medical therapeutic modality, highly effective in relieving leg pain and discomfort.

Adolescent↗
PubMed1990

Incidence of side effects in the treatment of telangiectasias by compression sclerotherapy: hypertonic saline vs. polidocanol.

Two of the most common side effects of sclerotherapy of leg veins are telangiectatic matting (TM), and postsclerosis hyperpigmentation. A random sample of 113 female patients with similar treatment sites of telangiectasias (less than 1 mm in diameter) and venulectasias of 1-2 mm diameter on the legs were treated by sclerotherapy and assessed at 1 and 6 months later for the presence of TM and hyperpigmentation. Patients received treatment with either 23.4% hypertonic saline (HS) alone (N = 74), 1% polidocanol (POL) alone (N = 21), or a comparison treatment with both HS and 1% POL (N = 18). A subgroup of patients received both 1% POL and 0.5% POL (N = 10). The incidence of hyperpigmentation was related both to vessel size and to sclerosing solution; only one patient showed hyperpigmentation in a vessel less than 1 mm, while in 1-2 mm vessels the incidence was 10.9% for HS and 30.7% for 1% POL (p less than .004) at 1 month. At 6 months postsclerotherapy, the incidence of pigmentation was 2% for HS and 7.5% for 1% POL. Both the type and concentration of sclerosing solution affected the incidence of TM; the incidence of TM was 33% for 1% POL, compared to only 13% for HS (p less than .004). At 6 months follow-up, the incidence of TM decreased to 2% for HS and 9% for 1% POL. Ten patients who developed TM with 1% POL were treated subsequently with 0.5% POL and demonstrated no matting. These data indicate that the incidence of side effects was affected by both the concentration and type of sclerosant.

Female↗