Lila M. Gierasch

Turns in Peptides and Proteins

Publisher Summary Turns are a fundamental class of polypeptide structure and are defined as sites where the peptide chain reverses its overall direction. In the past 20 years, the peptide field has witnessed major development, stimulated by the discovery of a host of bioactive peptides. Turn structures have been proposed and implicated in the bioactivity of several of these naturally occurring peptides. In addition, many structural details of turns have been derived from conformational studies of model peptides. During this same period, more than 100 complete protein structures have been elucidated in single-crystal X-ray studies. These examples document the rich diversity of structural patterns in the chain folds of native proteins. Turns are intrinsically polar structures with backbone groups that pack together closely and side chains that project outward. Such an array of atoms may constitute a site for molecular recognition, and indeed, the literature abounds with suggestions that turns serve as loci for receptor binding, antibody recognition, and post-translational modification. In peptides, turns are the conformations of choice for simultaneously optimizing both backbone–chain compactness (intramolecular nonbonded contacts) and side-chain clustering (to facilitate intermolecular recognition). Presence of turns in bioactive conformations may in fact also reflect the lack of alternative conformational possibilities. The aim of this chapter is to examine structural and functional roles of turns in peptides and proteins.

Sending Signals Dynamically

Proteins mediate transmission of signals along intercellular and intracellular pathways and between the exterior and the interior of a cell. The dynamic properties of signaling proteins are crucial to their functions. We discuss emerging paradigms for the role of protein dynamics in signaling. A central tenet is that proteins fluctuate among many states on evolutionarily selected energy landscapes. Upstream signals remodel this landscape, causing signaling proteins to transmit information to downstream partners. New methods provide insight into the dynamic properties of signaling proteins at the atomic scale. The next stages in the signaling hierarchy—how multiple signals are integrated and how cellular signaling pathways are organized in space and time—present exciting challenges for the future, requiring bold multidisciplinary approaches.

Inhibition of protein aggregation in vitro and in vivo by a natural osmoprotectant

Small organic molecules termed osmolytes are harnessed by a variety of cell types in a wide range of organisms to counter unfavorable physiological conditions that challenge protein stability and function. Using a well characterized reporter system that we developed to allow in vivo observations, we have explored how the osmolyte proline influences the stability and aggregation of a model aggregation-prone protein, P39A cellular retinoic acid-binding protein. Strikingly, we find that the natural osmolyte proline abrogates aggregation both in vitro and in vivo (in an Escherichia coli expression system). Importantly, proline also prevented aggregation of constructs containing exon 1 of huntingtin with extended polyglutamine tracts. Although compatible osmolytes are known to stabilize the native state, our results point to a destabilizing effect of proline on partially folded states and early aggregates and a solubilizing effect on the native state. Because proline is believed to act through a combination of solvophobic backbone interactions and favorable side-chain interactions that are not specific to a particular sequence or structure, the observed effect is likely to be general. Thus, the osmolyte proline may be protective against biomedically important protein aggregates that are hallmarks of several late-onset neurodegenerative diseases including Huntington’s, Alzheimer’s, and Parkinson’s. In addition, these results should be of practical importance because they may enable protein expression at higher efficiency under conditions where aggregation competes with proper folding.

The NPXY internalization signal of the LDL receptor adopts a reverse-turn conformation

Peptides corresponding to the proposed coated pit internalization signal of the native low density lipoprotein receptor, NPVY, take up in aqueous solution a reverse-turn conformation with the Asn in position i and the Tyr in position i + 3. By contrast, peptides derived from receptors that are defective for endocytosis do not adopt the reverse turn. These internalization-defective receptors include those with a nonaromatic residue substituted for the Tyr and those with Asn----Ala or Pro----Ala substitutions. While the tendency of an Asn-Pro sequence to induce a reverse turn was anticipated, the structural importance of an aromatic residue in position i + 3 was not. The sequences associated with the internalization signal thus appear to play a critical conformational role that is required for endocytosis, probably by enabling binding to adaptor molecules. With the results presented in the accompanying paper on lysosomal acid phosphatase, we now have direct evidence for previous proposals of a general correlation of internalization signals with a turn conformational motif.

Molecular Mechanisms of Protein Secretion: The Role of the Signal Sequence

Publisher Summary All cells make proteins that are destined for non-cytoplasmic locations, such as the extracellular fluid, the lumina of organelles, or the cell membranes. These proteins are almost invariably synthesized in the cytoplasm. Thus, such a protein must cross or enter one or more membranes in order to reach its destination. Furthermore, this process is specific in a manner that a particular protein travels only to its proper location, and only proteins destined for a particular location are transported there. The molecular mechanism of these processes has been the subject of intense investigation in the last decade. Most knowledge concerning the secretory process has come from genetic and biochemical studies. More recently, however, biophysical techniques have been used to investigate properties of the secretory apparatus and the signal sequence. The aim of this chapter is to describe the requirement for protein secretion and also to evaluate hypotheses of the ways in which secretion occurs. Particular emphasis is given to the role of the signal sequence.

Structural insights into substrate binding by the molecular chaperone DnaK

How substrate affinity is modulated by nucleotide binding remains a fundamental, unanswered question in the study of 70 kDa heat shock protein (Hsp70) molecular chaperones. We find here that the Escherichia coli Hsp70, DnaK, lacking the entire α-helical domain, DnaK(1–507), retains the ability to support λ phage replication in vivo and to pass information from the nucleotide binding domain to the substrate binding domain, and vice versa, in vitro. We determined the NMR solution structure of the corresponding substrate binding domain, DnaK(393–507), without substrate, and assessed the impact of substrate binding. Without bound substrate, loop L3,4 and strand β3 are in significantly different conformations than observed in previous structures of the bound DnaK substrate binding domain, leading to occlusion of the substrate binding site. Upon substrate binding, the β-domain shifts towards the structure seen in earlier X-ray and NMR structures. Taken together, our results suggest that conformational changes in the β-domain itself contribute to the mechanism by which nucleotide binding modulates substrate binding affinity.

Physicochemical Properties of Cells and Their Effects on Intrinsically Disordered Proteins (IDPs)

It has long been axiomatic that a protein’s structure determines its function. Intrinsically disordered proteins (IDPs) and disordered protein regions (IDRs) defy this structure–function paradigm. They do not exhibit stable secondary and/or tertiary structures and exist as dynamic ensembles of interconverting conformers with preferred, nonrandom orientations.1−4 The concept of IDPs and IDRs as functional biological units was initially met with skepticism. For a long time, disorder, intuitively implying chaos, had no place in our perception of orchestrated molecular events controlling cell biology. Over the past years, however, this notion has changed. Aided by findings that structural disorder constitutes an ubiquitous and abundant biological phenomenon in organisms of all phyla,5−7 and that it is often synonymous with function,8−11 disorder has become an integral part of modern protein biochemistry. Disorder thrives in eukaryotic signaling pathways12 and functions as a prominent player in many regulatory processes.13−15 Disordered proteins and protein regions determine the underlying causes of many neurodegenerative disorders and constitute the main components of amyloid fibrils.16 They further contribute to many forms of cancer, diabetes and to cardiovascular and metabolic diseases.17,18 Research into disordered proteins produced significant findings and established important new concepts. On the structural side, novel experimental and computational approaches identified and described disordered protein ensembles3,19,20 and led to terms such as secondary structure propensities, residual structural features, and transient long-range contacts.1,21 The discovery of coupled folding-and-binding reactions defined the paradigm of disorder-to-order transitions22 and high-resolution insights into the architectures of amyloid fibrils were obtained.23,24 On the biological side, we learned about the unexpected intracellular stability of disordered proteins, their roles in integrating post-translational protein modifications in cell signaling and about their functions in regulatory processes ranging from transcription to cell fate decisions.15,25,26 One open question remaining to be addressed is how these in vitro structural insights relate to biological in vivo effects. How do complex intracellular environments modulate the in vivo properties of disordered proteins and what are the implications for their biological functions (Figure ​(Figure11)?27−29 Figure 1 Intracellular complexity. (A) Left: Cryo-electron tomography slice of a mammalian cell. Middle: Close-up view of cellular structures colored according to their identities: Right: Three-dimensional surface representation of the same region. Yellow, endoplasmic ...

Protein Folding in the Cell: Challenges and Progress

It is hard to imagine a more extreme contrast than that between the dilute solutions used for in vitro studies of protein folding and the crowded, compartmentalized, sticky, spatially inhomogeneous interior of a cell. This review highlights recent research exploring protein folding in the cell with a focus on issues that are generally not relevant to in vitro studies of protein folding, such as macromolecular crowding, hindered diffusion, cotranslational folding, molecular chaperones, and evolutionary pressures. The technical obstacles that must be overcome to characterize protein folding in the cell are driving methodological advances, and we draw attention to several examples, such as fluorescence imaging of folding in cells and genetic screens for in-cell stability.

Macromolecular crowding remodels the energy landscape of a protein by favoring a more compact unfolded state

The interior of cells is highly crowded with macromolecules, which impacts all physiological processes. To explore how macromolecular crowding may influence cellular protein folding, we interrogated the folding landscape of a model beta-rich protein, cellular retinoic acid-binding protein I (CRABP I), in the presence of an inert crowding agent (Ficoll 70). Urea titrations revealed a crowding-induced change in the water-accessible polar amide surface of its denatured state, based on an observed ca. 15% decrease in the change in unfolding free energy with respect to urea concentration (the m-value), and the effect of crowding on the equilibrium stability of CRABP I was less than our experimental error (i.e., < or = 1.2 kcal/mol). Consequently, we directly probed the effect of crowding on the denatured state of CRABP I by measuring side-chain accessibility using iodide quenching of tryptophan fluorescence and chemical modification of cysteines. We observed that the urea-denatured state is more compact under crowded conditions, and the observed extent of reduction of the m-value by crowding agent is fully consistent with the extent of reduction of the accessibility of the Trp and Cys probes, suggesting a random and nonspecific compaction of the unfolded state. The thermodynamic consequences of crowding-induced compaction are discussed. In addition, over a wide range of Ficoll concentration, crowding significantly retarded the unfolding kinetics of CRABP I without influencing the urea dependence of the unfolding rate, arguing for no appreciable change in the nature of the transition state. Our results demonstrate how macromolecular crowding may influence protein folding by effects on both the unfolded state ensemble and unfolding kinetics.

How Hsp70 Molecular Machines Interact with Their Substrates to Mediate Diverse Physiological Functions

Hsp70 molecular chaperones are implicated in a wide variety of cellular processes, including protein biogenesis, protection of the proteome from stress, recovery of proteins from aggregates, facilitation of protein translocation across membranes, and more specialized roles such as disassembly of particular protein complexes. It is a fascinating question to ask how the mechanism of these deceptively simple molecular machines is matched to their roles in these wide-ranging processes. The key is a combination of the nature of the recognition and binding of Hsp70 substrates and the impact of Hsp70 action on their substrates. In many cases, the binding, which relies on interaction with an extended, accessible short hydrophobic sequence, favors more unfolded states of client proteins. The ATP-mediated dissociation of the substrate thus releases it in a relatively less folded state for downstream folding, membrane translocation, or hand-off to another chaperone. There are cases, such as regulation of the heat shock response or disassembly of clathrin coats, however, where binding of a short hydrophobic sequence selects conformational states of clients to favor their productive participation in a subsequent step. This Perspective discusses current understanding of how Hsp70 molecular chaperones recognize and act on their substrates and the relationships between these fundamental processes and the functional roles played by these molecular machines.