Daniel P. Raleigh

Histone H2B ubiquitylation disrupts local and higher-order chromatin compaction

Regulation of chromatin structure involves histone post-translational modifications which can modulate intrinsic properties of the chromatin fiber to change the chromatin state. We used chemically defined nucleosome arrays to demonstrate that H2B ubiquitylation (uH2B), a modification associated with transcription, interferes with chromatin compaction and leads to an open and biochemically accessible fiber conformation. Importantly, these effects were specific for ubiquitin, as compaction of chromatin modified with a similar ubiquitin-sized protein, Hub1, was only weakly affected. Applying a fluorescence based method we found that uH2B acts through a mechanism distinct from H4 tail acetylation (acH4), a modification known to disrupt chromatin folding. Finally, incorporation of both uH2B and acH4 in nucleosomes resulted in synergistic inhibition of higher order chromatin structure formation, possibly a result of their distinct mode of action.

Protein folding : Defining a "standard" set of experimental conditions and a preliminary kinetic data set of two-state proteins

Recent years have seen the publication of both empirical and theoretical relationships predicting the rates with which proteins fold. Our ability to test and refine these relationships has been limited, however, by a variety of difficulties associated with the comparison of folding and unfolding rates, thermodynamics, and structure across diverse sets of proteins. These difficulties include the wide, potentially confounding range of experimental conditions and methods employed to date and the difficulty of obtaining correct and complete sequence and structural details for the characterized constructs. The lack of a single approach to data analysis and error estimation, or even of a common set of units and reporting standards, further hinders comparative studies of folding. In an effort to overcome these problems, we define here a “consensus” set of experimental conditions (25°C at pH 7.0, 50 mM buffer), data analysis methods, and data reporting standards that we hope will provide a benchmark for experimental studies. We take the first step in this initiative by describing the folding kinetics of 30 apparently two‐state proteins or protein domains under the consensus conditions. The goal of our efforts is to set uniform standards for the experimental community and to initiate an accumulating, self‐consistent data set that will aid ongoing efforts to understand the folding process.

De novo protein design: from molten globules to native-like states: Current opinion in structural biology 1993, 3:601–610

Abstract Recent advances in the field of de novo protein design are examined. The data are examined with respect to the hypothesis that most designed proteins exhibit characteristics similar to those of molten globules. Continuing efforts to produce novel proteins with the specific interactions that define natural proteins are assessed, and the impact that these efforts have had on our understanding of protein structure, stability, and folding is discussed.

Mechanism of IAPP amyloid fibril formation involves an intermediate with a transient β-sheet

Significance There is an enormous interest in the mechanism by which proteins misfold and aggregate into amyloid fibrils. Amyloid has been implicated in many human diseases, but the mechanism of aggregation is not understood. Intermediates have been postulated to play an important role in the process, but there have been very few direct measurements that provide specific structural details. The use of isotope labeling and 2D IR methods has allowed the characterization of a critical intermediate generated during amyloid formation by islet amyloid polypeptide, the peptide responsible for amyloid formation in type 2 diabetes. Identification of this intermediate provides a structural explanation for the lag phase and may explain why some species develop amyloid deposits of hIAPP while others do not. Amyloid formation is implicated in more than 20 human diseases, yet the mechanism by which fibrils form is not well understood. We use 2D infrared spectroscopy and isotope labeling to monitor the kinetics of fibril formation by human islet amyloid polypeptide (hIAPP or amylin) that is associated with type 2 diabetes. We find that an oligomeric intermediate forms during the lag phase with parallel β-sheet structure in a region that is ultimately a partially disordered loop in the fibril. We confirm the presence of this intermediate, using a set of homologous macrocyclic peptides designed to recognize β-sheets. Mutations and molecular dynamics simulations indicate that the intermediate is on pathway. Disrupting the oligomeric β-sheet to form the partially disordered loop of the fibrils creates a free energy barrier that is the origin of the lag phase during aggregation. These results help rationalize a wide range of previous fragment and mutation studies including mutations in other species that prevent the formation of amyloid plaques.

Low levels of asparagine deamidation can have a dramatic effect on aggregation of amyloidogenic peptides: Implications for the study of amyloid formation

The polypeptide hormone amylin forms amyloid deposits in Type 2 diabetes mellitus and a 10‐residue fragment of amylin (amylin20–29) is commonly used as a model system to study this process. Studies of amylin20–29 and several variant peptides revealed that low levels of deamidation can have a significant effect on the secondary structure and aggregation behavior of these molecules. Results obtained with a variant of amylin20–29, which has the primary sequence SNNFPAILSS, are highlighted. This peptide is particularly interesting from a technical standpoint. In the absence of impurities the peptide does not spontaneously aggregate and is not amyloidogenic. This peptide can spontaneously deamidate, and the presence of less than 5% of deamidation impurities leads to the formation of aggregates that have the hallmarks of amyloid. In addition, small amounts of deamidated material can induce amyloid formation by the purified peptide. These results have fundamental implications for the definition of an amyloidogenic sequence and for the standards of purity of peptides and proteins used for studies of amyloid formation.

The Protein Folding Transition State: What Are φ-Values Really Telling Us?

Protein engineering-based studies of the folding transition state have accelerated significantly in the last decade, and more than a half dozen proteins have been subjected to extensive Phi-value analysis. A general picture is emerging from these studies of a transition state in which the large majority of experimentally characterized side chains participate in relatively homogeneous and energetically weak interactions playing only a relatively small role in defining relative folding rates.

The Fluorescent Amino Acid p-Cyanophenylalanine Provides an Intrinsic Probe of Amyloid Formation

Amyloid formation has been implicated in more than fifteen different human diseases including Alzheimer’s disease, Parkinson’s disease, prion-based diseases, and type 2 diabetes. 2] The kinetics of amyloid formation are complex, and typically consist of a lag phase during which little fibrillar material is produced followed by a rapid growth phase. Characterization of the kinetics of amyloid formation and the nature of any intermediates that are formed have emerged as critical topics in the field since there is growing evidence that prefibrillar intermediate structures might be the toxic species. Unfortunately, a limited set of low resolution spectroscopic methods can be applied to study the kinetics of amyloid formation and residue-specific information is generally not obtainable. Amyloid formation, in vitro, is traditionally followed by fluorescence detection in thioflavin T-binding experiments. The fluorescence of the dye significantly increases upon binding to the amyloid fibril. The assay is simple to execute, however, it does suffer from some noticeable drawbacks. First, the exact mechanism for the fluorescence enhancement is not completely understood, hence, it is not completely clear what the dye binding probes. Second, the dye does not bind to prefibrillar intermediates and thus cannot be used to follow their formation. A third extremely important but somewhat subtle issue involves the study of inhibitors. Some compounds can bind to amyloid fibrils and displace bound thioflavin T without inhibiting amyloid formation. In these cases thioflavin T assays lead to the incorrect conclusion that the compound is an amyloid inhibitor. Fourth, the dye is an extrinsic probe and there is always the risk that the kinetics of assembly could be affected since the assay is conducted by adding the dye to the peptide solution and it binds to the fibrils as they are being formed. In principle, intrinsic protein fluorescence could be used to follow amyloid formation since Trp fluorescence is sensitive to the local environment. However, a surprising number of important amyloidogenic polypeptides lack Trp including Ab, a-synuclein, and IAPP (amylin), the causative agents of amyloid formation in Alzheimer’s disease, Parkinson’s disease, and type 2 diabetes, respectively. Furthermore, the addition of Trp by mutagenesis often represents a nonconserved mutation. Tyr fluorescence might be useful, but it is less sensitive than Trp fluorescence and its interpretation is much less straightforward. It would clearly be desirable to have access to another fluorescent amino acid that could be used as a probe of amyloid formation. An ideal amino-acid analogue should exhibit a large, easily interpretable change in fluorescence during the process of amyloid formation, but represent only a small perturbation on the structure and hydrophobicity of one or more of the twenty genetically encoded residues, and thus allow for conservative substitution. p-Cyanophenyalanine (p-cyanoPhe) appears to meet all of these requirements. Its fluorescence quantum yield is very sensitive to solvent interactions and is decreased significantly in a hydrophobic environment compared to its value in water ; this makes it a sensitive probe of the local environment. Importantly, it has a blue-shifted absorption band, which allows its fluorescence to be selectively excited in the presence of Tyr or Trp. The cyano group is a hydrogen-bond acceptor, but it has the very desirable feature that it is readily accommodated in the hydrophobic core of proteins since its polarity is intermediate between that of an amide and a methylene. It is also considerably smaller than Trp, which makes it a very conservative replacement for either Phe or Tyr. In the present work we demonstrate the use of p-cyanoPhe fluorescence to probe amyloid formation using islet amyloid polypeptide (IAPP, amylin) as a test case. IAPP is responsible for the formation of pancreatic islet amyloid in type 2 diabetes. Islet amyloid formation plays a role in the pathology of the disease by killing pancreatic b cells, and contributing to the loss of b cell mass and the decline in insulin secretion. IAPP is 37 residues in length, contains a disulfide bond that links residues 2 and 7, and has an amidated C terminus. It does not contain Trp, but does have two Phe residues at positions 15 and 23 and a single Tyr at its C terminus. We replaced Tyr37 with p-cyanoPhe. The peptide is denoted hIAPP-Y37FC N. The sequence of the wild-type human peptide—denoted here hIAPP—is: KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY

Crystallization of a designed peptide from a molten globule ensemble

Backgound. The design of amino acid sequences that adopt a desired three-dimensional fold has been of keen interest over the past decade. However, the design of proteins that adopt unique conformations is still a considerable problem. Until very recently, all of the designed proteins that have been extensively characterized possess the hallmarks of the molten globular state. Molten globular intermediates have been observed in both equilibrium and kinetic protein folding/stability studies, and understanding the forces that determine compact non-native states is critical for a comprehensive understanding of proteins. This paper describes the solution and early solid state characterization of peptides that form molten globular ensembles. Results. Crystals diffracting to 3.5Å resolution have been grown of a 16-residue peptide (alpha1A) designed to form a tetramer of alpha-helices. In addition, a closely related peptide, alpha1, has previously been shown to yield crystals that diffract to 1.2Å resolution. The solution properties of these two peptides were examined to determine whether their well defined crystalline conformations were retained in solution. On the basis of an examination of their NMR spectra, sedimentation equilibria, thermal unfolding, and ANS binding, it is concluded that the peptides form alpha-helical aggregates with properties similar to those of the molten globule state. Thus, for these peptides, the process of crystallization bears many similarities to models of protein folding. Upon dissolution, the peptides rapidly assume compact molten globular states similar to the molten globule like intermediates that are formed at short times after refolding is initiated. Following a rate-determining nucleation step, the peptides crystallize into a single or a small number of conformations in a process that mimics the formation of native structure in proteins.

A peptide model for proline isomerism in the unfolded state of staphylococcal nuclease.

Nuclear magnetic resonance spectroscopy has been used to investigate a synthetic peptide (YVYKPNNTHE) corresponding to residues 113 to 122 of staphylococcal nuclease. In the major folded state of the protein this region forms a type VIa beta-turn containing a cis Lys116-Pro117 peptide bond. There is, however, no evidence for any significant population of such a turn in the peptide in aqueous solution and the X-Pro bond is predominantly in the trans configuration. The peptide exhibits several well-resolved minor resonances due to the presence of a small fraction (4 +/- 2%) of the cis-proline isomer. The ratio of cis to trans isomer populations was found to be independent of temperature between 5 degrees C and 70 degrees C, indicating that delta H for the isomerism is close to zero. Using magnetization transfer techniques the rate of trans to cis interconversion was found to be 0.025(+/- 0.013) s-1 at 50 degrees C. The thermodynamics and kinetics of isomerism in the peptide are very similar to those estimated for the Lys116-Pro117 peptide bond in unfolded nuclease, suggesting that the cis-trans equilibrium in the unfolded protein is largely determined by the residues adjacent to Pro117 in the sequence. These results are consistent with previous suggestions that the cis-proline bond is stabilized late in the folding process and that the predominance of the cis form in folded nuclease is due to stabilizing interactions within the protein that give rise to a favorable enthalpy term.

Neprilysin Impedes Islet Amyloid Formation by Inhibition of Fibril Formation Rather Than Peptide Degradation

Deposition of islet amyloid polypeptide (IAPP) as islet amyloid in type 2 diabetes contributes to loss of β-cell function and mass, yet the mechanism for its occurrence is unclear. Neprilysin is a metallopeptidase known to degrade amyloid in Alzheimer disease. We previously demonstrated neprilysin to be present in pancreatic islets and now sought to determine whether it plays a role in degrading islet amyloid. We used an in vitro model where cultured human IAPP (hIAPP) transgenic mouse islets develop amyloid and thereby have increased β-cell apoptosis. Islet neprilysin activity was inhibited or up-regulated using a specific inhibitor or adenovirus encoding neprilysin, respectively. Following neprilysin inhibition, islet amyloid deposition and β-cell apoptosis increased by 54 and 75%, respectively, whereas when neprilysin was up-regulated islet amyloid deposition and β-cell apoptosis both decreased by 79%. To determine if neprilysin modulated amyloid deposition by cleaving hIAPP, analysis of hIAPP incubated with neprilysin was performed by mass spectrometry, which failed to demonstrate neprilysin-induced cleavage. Rather, neprilysin may act by reducing hIAPP fibrillogenesis, which we showed to be the case by fluorescence-based thioflavin T binding studies and electron microscopy. In summary, neprilysin decreases islet amyloid deposition by inhibiting hIAPP fibril formation, rather than degrading hIAPP. These findings suggest that targeting the role of neprilysin in IAPP fibril assembly, in addition to IAPP cleavage by other peptidases, may provide a novel approach to reduce and/or prevent islet amyloid deposition in type 2 diabetes.