How do proteins fold?

This article emphasizes the importance of getting students to understand the ways in which polypeptides fold to form protein molecules with complex higher-ordered structures. Modern views on how this folding occurs in vitro and in the cell are summarized and set within an appropriate biological context.     

How do bacteria resist human antimicrobial peptides?

Cationic antimicrobial peptides (CAMPs), such as defensins, cathelicidins and thrombocidins, are an important human defense mechanism, protecting skin and epithelia against invading microorganisms and assisting neutrophils and platelets. Staphylococcus aureus, Salmonella enterica and other bacterial pathogens have evolved countermeasures to limit the effectiveness of CAMPs, including the repulsion of CAMPs by reducing the net negative charge of the bacterial cell envelope through covalent modification of anionic molecules (e.g. teichoic acids, phospholipids and lipid A); expelling CAMPs through energy-dependent pumps; altering membrane fluidity; and cleaving CAMPs with proteases. Mutants susceptible to CAMPs are more efficiently inactivated by phagocytes and are virulence-attenuated, indicating that CAMP resistance plays a key role in bacterial infections.     

How do uncoupling proteins uncouple?

According to the proton buffering model, introduced by Klingenberg, UCP1 conducts protons through a hydrophilic pathway lined with fatty acid head groups that buffer the protons as they move across the membrane. According to the fatty acid protonophore model, introduced by Garlid, UCPs do not conduct protons at all. Rather, like all members of this gene family, they are anion carriers. A variety of anions are transported, but the physiological substrates are fatty acid (FA) anions. Because the carboxylate head group is translocated by UCP, and because the protonated FA rapidly diffuses across the membrane, this mechanism permits FA to behave as regulated cycling protonophores. Favoring the latter mechanism is the fact that the head group of long-chain alkylsulfonates, strong acid analogues of FA, is also translocated by UCP.     

How do membrane proteins sense water stress?

Maintenance of cell turgor is a prerequisite for almost any form of life as it provides a mechanical force for the expansion of the cell envelope. As changes in extracellular osmolality will have similar physicochemical effects on cells from all biological kingdoms, the responses to osmotic stress may be alike in all organisms. The primary response of bacteria to osmotic upshifts involves the activation of transporters, to effect the rapid accumulation of osmoprotectants, and sensor kinases, to increase the transport and/or biosynthetic capacity for these solutes. Upon osmotic downshift, the excess of cytoplasmic solutes is released via mechanosensitive channel proteins. A number of breakthroughs in the last one or two years have led to tremendous advances in our understanding of the molecular mechanisms of osmosensing in bacteria. The possible mechanisms of osmosensing, and the actual evidence for a particular mechanism, are presented for well studied, osmoregulated transport systems, sensor kinases and mechanosensitive channel proteins. The emerging picture is that intracellular ionic solutes (or ionic strength) serve as a signal for the activation of the upshift-activated transporters and sensor kinases. For at least one system, there is strong evidence that the signal is transduced to the protein complex via alterations in the protein-lipid interactions rather than direct sensing of ion concentration or ionic strength by the proteins. The osmotic downshift-activated mechanosensitive channels, on the other hand, sense tension in the membrane but other factors such as hydration state of the protein may affect the equilibrium between open and closed states of the proteins.     

How do Rab proteins function in membrane traffic?

The Rabs are a group of GTP-binding proteins implicated for some time in the targeting of different transport vesicles within the cell, but it has been unclear how they function, or how they relate to a second group of targeting proteins, the SNAREs. Recent work, discussed in this review, has used biophysical, biochemical and genetic approaches to begin to answer these questions for Rab3, Rab5 and the yeast protein Sec4p. However, the results from these three Rabs lead to a surprising conclusion: the different Rabs seem to function via highly diverse target proteins.     

How do site-specific DNA-binding proteins find their targets?

Essentially all the biological functions of DNA depend on site-specific DNA-binding proteins finding their targets, and therefore ‘searching’ through megabases of non-target DNA. In this article, we review current understanding of how this sequence searching is done. We review how simple diffusion through solution may be unable to account for the rapid rates of association observed in experiments on some model systems, primarily the Lac repressor. We then present a simplified version of the ‘facilitated diffusion’ model of Berg, Winter and von Hippel, showing how non-specific DNA–protein interactions may account for accelerated targeting, by permitting the protein to sample many binding sites per DNA encounter. We discuss the 1-dimensional ‘sliding’ motion of protein along non-specific DNA, often proposed to be the mechanism of this multiple site sampling, and we discuss the role of short-range diffusive ‘hopping’ motions. We then derive the optimal range of sliding for a few physical situations, including simple models of chromosomes in vivo, showing that a sliding range of ~100 bp before dissociation optimizes targeting in vivo. Going beyond first-order binding kinetics, we discuss how processivity, the interaction of a protein with two or more targets on the same DNA, can reveal the extent of sliding and we review recent experiments studying processivity using the restriction enzyme EcoRV. Finally, we discuss how single molecule techniques might be used to study the dynamics of DNA site-specific targeting of proteins.     

How do proteins avoid becoming too stable? Biophysical studies into metastable proteins

The vast majority of theoretical and experimental folding studies have shown that as a protein folds, it attempts to adopt a conformation that occurs at its lowest free energy minimum. However, studies on a small number of proteins have now shown that this is a generality. In this review we discuss recent data on how two proteins, -lytic protease and ₁-antitrypsin, successfully fold to their metastable native states, whilst avoiding more stable but inactive conformations.     

How do BCL-2 proteins induce mitochondrial outer membrane permeabilization?

The mitochondrial pathway of apoptosis proceeds when molecules sequestered between the outer and inner mitochondrial membranes are released to the cytosol by mitochondrial outer membrane permeabilization (MOMP). This process is controlled by the BCL-2 family, which is composed of both pro- and anti-apoptotic proteins. Although there is no disagreement that BCL-2 proteins regulate apoptosis, the mechanism leading to MOMP remains controversial. Current debate focuses on what interactions within the family are crucial to initiate MOMP. Specifically, do the BH3-only proteins directly engage BAX and/or BAK activation or do these proteins solely promote apoptosis by neutralization of anti-apoptotic BCL-2 proteins? We describe these models and contend that BH3-only proteins must perform both functions to efficiently engage MOMP and apoptosis.     

How are tonoplast proteins degraded?

Protein turnover is fundamental both for development and cellular homeostasis. The mechanisms responsible for the turnover of integral membrane proteins in plant cells are however still largely unknown. Recently, considerable attention has been devoted to the degradation of plasma membrane proteins. We have now studied the turnover of a tonoplast protein, the potassium channel TPK1, in fully differentiated Arabidopsis leaf cells and showed that its degradation occurs upon internalization into the vacuole. Here, we discuss the possible mechanisms and triggering events involved.     

How do chemical denaturants affect the mechanical folding and unfolding of proteins?

We present the first single-molecule atomic force microscopy study on the effect of chemical denaturants on the mechanical folding/unfolding kinetics of a small protein GB1 (the B1 immunoglobulin-binding domain of protein G from Streptococcus). Upon increasing the concentration of the chemical denaturant guanidinium chloride (GdmCl), we observed a systematic decrease in the mechanical stability of GB1, indicating the softening effect of the chemical denaturant on the mechanical stability of proteins. This mechanical softening effect originates from the reduced free-energy barrier between the folded state and the unfolding transition state, which decreases linearly as a function of the denaturant concentration. Chemical denaturants, however, do not alter the mechanical unfolding pathway or shift the position of the transition state for mechanical unfolding. We also found that the folding rate constant of GB1 is slowed down by GdmCl in mechanical folding experiments. By combining the mechanical folding/unfolding kinetics of GB1 in GdmCl solution, we developed the “mechanical chevron plot” as a general tool to understand how chemical denaturants influence the mechanical folding/unfolding kinetics and free-energy diagram in a quantitative fashion. This study demonstrates great potential in combining chemical denaturation with single-molecule atomic force microscopy techniques to reveal invaluable information on the energy landscape underlying protein folding/unfolding reactions.     

How do ALS-associated mutations in superoxide dismutase 1 promote aggregation of the protein?

More than 100 different mutations in the gene encoding copper-zinc superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS)--a fatal neurodegenerative disease in which aggregation of the SOD1 protein is considered to be the primary mode of pathogenesis. Recent results show that these mutations have remarkably diverse and unexpected effects on the structure, activity and native state stability of SOD1. Intriguingly, many mutations seem to have no measurable effect on the biophysical and biochemical properties of SOD1, except for decreasing the net charge of the protein. Thus, it seems likely that different ALS-associated mutations promote SOD1 aggregation by fundamentally distinct mechanisms. Understanding this complexity has implications for drug development and treatment of the disease.     

Where do all the proteins go?

The subcellular localization of a protein can be very informative in identifying its function and in understanding the regulatory mechanisms by which it is controlled. Past efforts to define protein localization have typically entailed methods of immunological and fluorescence-based detection applied to a limited number of gene products. Several current studies are shifting this paradigm – utilizing traditional and novel approaches in molecular biology, proteomics, histochemistry and bioinformatics to define protein localization on a proteome-wide scale. Selected studies highlighting each of these approaches are presented here as an overview of the diverse avenues by which protein localization may be investigated for the identification of new drug targets.     

POPP the question: what do LEA proteins do?

Late embryogenesis abundant (LEA) proteins are produced in maturing seeds and anhydrobiotic plants, animals and microorganisms, in which their expression correlates with desiccation tolerance. However, their function has remained obscure for 20 years. We argue that novel computational tools devised for non-globular proteins might now overcome this problem. Predictions arising from bioinformatics fit well with recent data on Group 3 proteins, which potentially form cytoskeletal filaments, and suggest experimentally testable functions for these and other LEA protein groups.     

How do spores germinate?

Spore germination, as defined as those events that result in the loss of the spore-specific properties, is an essentially biophysical process. It occurs without any need for new macromolecular synthesis, so the apparatus required is already present in the mature dormant spore. Germination in response to specific chemical nutrients requires specific receptor proteins, located at the inner membrane of the spore. After penetrating the outer layers of spore coat and cortex, germinant interacts with its receptor: one early consequence of this binding is the movement of monovalent cations from the spore core, followed by Ca2(+) and dipicolinic acid (DPA). In some species, an ion transport protein is also required for these early stages. Early events - including loss of heat resistance, ion movements and partial rehydration of the spore core - can occur without cortex hydrolysis, although the latter is required for complete core rehydration and colony formation from a spore. In Bacillus subtilis two crucial cortex lytic enzymes have been identified: one is CwlJ, which is DPA-responsive and is located at the cortex-coat junction. The second, SleB, is present both in outer layers and at the inner spore membrane, and is more resistant to wet heat than is CwlJ. Cortex hydrolysis leads to the complete rehydration of the spore core, and then enzyme activity within the spore protoplast resumes. We do not yet know what activates SleB activity in the spore, and neither do we have any information at all on how the spore coat is degraded.     

How many membrane proteins are there?

One of the basic issues that arises in functional genomics is the ability to predict the subcellular location of proteins that are deduced from gene and genome sequencing. In particular, one would like to be able to readily specify those proteins that are soluble and those that are inserted in a membrane. Traditional methods of distinguishing between these two locations have relied on extensive, time-consuming biochemical studies. The alternative approach has been to make inferences based on a visual search of the amino acid sequences of presumed gene products for stretches of hydrophobic amino acids. This numerical, sequence-based approach is usually seen as a first approximation pending more reliable biochemical data. The recent availability of large and complete sequence data sets for several organisms allows us to determine just how accurate such a numerical approach could be, and to attempt to minimize and quantify the error involved. We have optimized a statistical approach to protein location determination. Using our approach, we have determined that surprisingly few proteins are misallocated using the numerical method. We also examine the biological implications of the success of this technique.     

How do communication systems emerge?

Communication involves a pair of behaviours—a signal and a response—that are functionally interdependent. Consequently, the emergence of communication involves a chicken-and-egg problem: if signals and responses are dependent on one another, then how does such a relationship emerge in the first place? The empirical literature suggests two solutions to this problem: ritualization and sensory manipulation; and instances of ritualization appear to be more common. However, it is not clear from a theoretical perspective why this should be the case, nor if there are any other routes to communication. Here, we develop an analytical model to examine how communication can emerge. We show that: (i) a state of non-interaction is evolutionarily stable, and so communication will not necessarily emerge even when it is in both parties'' interest; (ii) the conditions for sensory manipulation are more stringent than for ritualization, and hence ritualization is likely to be more common; and (iii) communication can arise by a third route, when the intention to communicate can itself be communicated, but this may be limited to humans. More generally, our results demonstrate the utility of a functional approach to communication.