Current Protein and Peptide Science - Volume 6, Issue 4, 2005

The existence of surface guided electromagnetic waves has been theoretically predicted from Maxwell's equations and investigated during the first decades of the 20th century. However, it is only since the late 1960's that they have attracted the interest of surface physicists and earned the moniker of “surface plasmon”. With the advent of commercially available instruments and well established theories, the technique has been used to study a wide variety of biochemical and biotechnological phenomena. Spectral response of the resonance condition serves as a sensitive indicator of the optical properties of thin films immobilized within a wavelength of the surface. This enhanced surface sensitivity has provided a boon to the surface sciences, and fosters collaboration between surface chemistry, physics and the ongoing biological and biotechnological revolution. Since then, techniques based on surface plasmons such as Surface Plasmon Resonance (SPR), SPR Imaging, Plasmon Waveguide Resonance (PWR) and others, have been increasingly used to determine the affinity and kinetics of a wide variety of real time molecular interactions such as protein-protein, lipid-protein and ligand-protein, without the need for a molecular tag or label. The physical-chemical methodologies used to immobilize membranes at the surface of these optical devices are reviewed, pointing out advantages and limitations of each method. The paper serves to summarize both historical and more recent developments of these technologies for investigating structure-function aspects of these molecular interactions, and regulation of specific events in signal transduction by Gprotein coupled receptors (GPCRs).

The Serratia marcescens hemolysin represents the prototype of a growing family of pore forming toxins. The available bacterial genome sequences reveal Serratia hemolysin homologues in additional species. However, only S. marcescens hemolysin has been studied in great molecular detail. This family of toxins has nothing in common with the pore forming toxins of E. coli type (RTX toxins), the Staphylococcus aureus α-toxin or the thiol activated toxin of group A β-hemolytic streptococci (Streptolysin O). Studies on erythrocytes, eukaryotic cells and artificial black lipid membranes, have shown that the mechanism of pore formation of ShlA is different form other pore forming toxins. The S. marcescens hemolysin proteins ShlB and ShlA, exhibit protein sequence homologues in Proteus mirabilis, Haemophilus ducreyi, Yersinia pestis, Yersinia enterocolitica, Edwardsiella tarda, Photorhabdus luminescens and Xylella fastidiosa . The family of Serratia type pore forming toxins show a unique secretory mechanism which has been described as a two partner secretion system (TPSS) or type V-secretion system. Not only Serratia type pore forming toxins are secreted via TPSS but also adhesins from Bordetella pertussis, Erwinia chrysanthemi and Haemophilus influenzae. The uniqueness of the Serratia family is underlined by the fact that activation of ShlA by ShlB strictly requires phosphatidylethanolamine as a cofactor. And, quite unusual, ShlA undergoes a conformational change during activation. ShlA pore formation in erythrocytes and in nucleated eukaryotic cells results in cell lysis. In sub-lytic doses, as will normally be the situation in vivo, ShlA exerts additionally effects like vacuolization, cytoskeleton rearrangements and apoptosis. The knowledge of the structure, activation, secretion and mode of action of S. marcescens hemolysin has implications for proteins, related in sequence or in mode of secretion and activation.

Regulation of energy balance consists of two intertwined circuitries: food intake - metabolic rate - body weight, vs. metabolic rate - heat loss - body temperature. Metabolic rate serves interaction between the two. Some peptides influence individual components of energy homeostasis, without having coordinated anabolic or catabolic properties. Anabolic and catabolic peptides function with redundancy, and also show specific features. They all influence ingestive behavior vs. metabolic rate and temperature, but do not necessarily act directly at central thermoregulatory pathways. Most of them alter metabolic rate (but not heat loss) through the ventromedial nucleus, while consequent moderate changes in thermal signals can influence function of the preoptic/anterior hypothalamic region and initiate compensating regulatory steps to restore temperature. Thus, besides ingestion, these peptides influence metabolic rate, whereas the passive temperature changes will only be obvious as long as environmental circumstances allow. Other substances cause coordinated central regulatory changes resembling fever (e.g. cholecystokinin), anapyrexia, or cold-defense: they primarily affect body temperature, and then the temperature-dependent changes in catabolic/anabolic peptide functions alter feeding behavior. Such arrangement can secure relative independence of the two regulatory circles, allowing for minimization of depression in metabolic rate and body temperature during starvation (despite elevated anabolic activity), or for increased food intake with lack of hypothermia in cold adaptation (despite high anabolic activity), or for normal body temperature in overfed states (despite enhanced catabolic activity), etc. However, the independence is relative since the two systems interact in the overall regulation of energy homeostasis: neuropeptides influence body temperature and temperature modifies peptide actions.

The brain renin-angiotensin system enables the formation of different biological active forms of angiotensins within the brain. All enzymes and peptides necessary for the biosynthesis of these angiotensins have been recognized within the central nervous system. Since there are considerable mismatches concerning the localization of the different enzymes, this system is not fully understood. Moreover, since alternative pathways of the angiotensin biosynthesis exists, localization and generation, especially of the short forms of biologically active angiotensins, are largely enigmatic. The brain renin-angiotensin system mediates several classic physiological effects including body water balance, maintenance of blood pressure, sexual behaviors, and regulation of pituitary gland hormones. Beside these classic functions, the brain renin-angiotensin system has more subtle functions involving complex mechanisms such as learning and memory. The mechanisms of action seem to differ depending on the utilized different bioactive angiotensin fragments, which are formed by the action of a variety of enzymes. This phenomenon appears to represent an important mechanism for neuromodulation. Moreover, there is evidence to suggest that the renin-angiotensin system is involved in neurological disorders, as e.g. Alzheimer's or Parkinson's disease.

Secretoneurin (SN) represents a 33 amino acid neuropeptide, which is highly conserved between mammals, reptiles, birds, amphibians and fish. It is specifically expressed in endocrine, neuroendocrine and neuronal tissues. In brain, the pattern of SN expression is widespread and unique, partially overlapping with established neurotransmitters. ProSN, the precursor protein, also named secretogranin II, belongs to a class of proteins collectively called chromogranins. Changes in ProSN mRNA, which is significantly regulated by cell depolarisation, represent a useful marker for both rapid and long-lasting changes (positive and negative) of neuronal activity. Under pathophysiological conditions, especially following cellular hypoxia, SN expression can be induced in non-endocrine tissues like muscle cells, pneumocytes or tumor epithelial cells. Several biological effects were attributed to SN. SN releases dopamine from rat striatal slices in a dose dependent manner and influences neurite outgrowth in the developing cerebellum. It potently and specifically attracts monocytes, eosinophils and endothelial cells towards a concentration gradient and acts as an angiogenic cytokine comparable in potency to VEGF. Thus, SN contributes to neurogenic inflammation and might play a role in the (hypoxia-driven) induction of neo-vascularisation in ischemic diseases like peripheral or coronary artery disease, diabetic retinopathia, cerebral ischemia or in solid tumors. The signalling pathways of various biological effects have not been identified in detail, but most data point to a G-protein coupled receptor structure with respective associated intracellular events.

Atomic force microscopy (AFM) has become a common tool for biophysical studies of proteins; mainly due its property to perform characterizations near physiological conditions. The tertiary and quaternary structures, forces driving folding-unfolding processes, and secondary structure elements can be studied in their native environments allowing high resolution level associated with small distortions. This review outlines the operational principles and applications of AFM for protein biophysics.